Microdialysis in Nonhuman Primates

Richard C. Saunders1, Bhaskar S. Kolachana1, Daniel R. Weinberger1

1 NIMH/Clinical Brain Disorders Branch, Bethesda, Maryland
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 7.3
DOI:  10.1002/0471142301.ns0703s00
Online Posting Date:  May, 2001
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Abstract

This unit presents a technique that allows for routine repeated microdialysis experiments in the monkey using a sedated preparation or, with further minor modification, in the awake behaving animal. Protocols are provided for construction and in vitro calibration of dialysis probes, obtaining magnetic resonance imaging (MRI) scans of the monkey brain, surgically attaching a guide holder to the skull, and performing postoperative MRI scanning, in vivo dialysis sample collection, anatomical verification of the probes, and neurochemical analysis. An alternate protocol describes microdialysis in awake, behaving monkeys; this requires substantial preparatory work in training the monkeys to sit quietly in a restraining chair and/or to perform a series of behavioral tasks.

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of Dialysis Probe
  • Basic Protocol 2: Attachment of Guide Assembly to the Monkey Skull
  • Support Protocol 1: Determination of Skull Coordinates by MRI
  • Support Protocol 2: Postoperative Verification of Guide Assembly Position by MRI
  • Basic Protocol 3: In Vivo Microdialysis
  • Support Protocol 3: Anatomical Verification of the Probes
  • Alternate Protocol 1: Modifications for Using In Vivo Microdialysis in the Awake Monkey
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Dialysis Probe

  Materials
  • Epoxy
  • Superglue
  • 5% to 10% methanol
  • Small bags of CaCl 2
  • Monkey artificial cerebrospinal fluid (aCSF; appendix 2A), prepared fresh for experiment and supplemented immediately before use (step ) with neurotransmitter standards (see recipe), 37°C
  • Monkey aCSF without neurotransmitter standards
  • 3% to 4% formalin (optional)
  • MRI scan of monkey brain (see protocol 3)
  • Fused silica tubing (o.d. 425 µm, i.d. 320 µm for outer barrel of probe; o.d. 166, i.d. 101 µm for inner barrel of probe)
  • Polyethylene (PE) 10 and 50 tubing
  • 25‐G needle, blunt
  • 50‐ml syringe
  • 35° and 37°C ovens
  • Dialysis membrane tubing (e.g., Filtral 20, AN 69, Hospal Medica)
  • Dissecting microscope
  • 1‐ml Hamilton syringe; gas‐tight
  • Microinfusion pump
  • Blue connectors (Bioanalytical Systems)
  • 4‐O suture thread (optional)
  • 700‐µl (7 × 40–mm) conical‐bottom amber glass vials
  • Additional reagents and equipment for analyzing dialysate by HPLC (unit 7.4)

Basic Protocol 2: Attachment of Guide Assembly to the Monkey Skull

  Materials
  • 6‐ to 12.5‐kg rhesus monkey (Macaca mulatta)
  • Ketamine
  • Isoflurane
  • Ringer's solution
  • Bone cement
  • Guide holder
  • Intubation tube
  • Venous catheter
  • i.v. setup
  • Vital sign monitor and pads
  • MRI‐compatible stereotaxic instrument, equipped with manipulators
  • 37°C heating blanket
  • Surgical instruments for aseptic neurosurgery
  • Guide block, plastic cap, and screws
  • Marking pen, sterile
  • Drill, hand‐held or dental, 14 to 20 mm × 7 to 9 mm
  • Rongeurs (optional)
  • Plastic I‐shaped anchors
  • Additional reagents and equipment for determining skull coordinates by MRI (see protocol 3)

Support Protocol 1: Determination of Skull Coordinates by MRI

  • Xylazine
  • Container for transporting monkey
  • GE/Signa 1.5 T MRI scanner or equivalent

Support Protocol 2: Postoperative Verification of Guide Assembly Position by MRI

  • Vitamin E capsules
  • Soft clay
  • Fused silica tubing (o.d. 425 µm, i.d. 320 µm)

Basic Protocol 3: In Vivo Microdialysis

  Materials
  • Monkey artificial cerebrospinal fluid (aCSF; see appendix 2A)
  • Ketamine hydrochloride
  • Monkey with attached dialysis probe guide assembly (see protocol 2)
  • Isoflurane
  • Ophthalmic antibiotic ointment
  • Dialysis probes (see protocol 1)
  • 700‐µl (7 × 40–mm) conical‐bottom amber glass vials
  • 1‐ml Hamilton syringe, gas‐tight
  • Microinfusion pump
  • MRI‐compatible stereotaxic instrument
  • 25‐G needle
  • 25‐G stainless steel tubing (o.d. 0.020 in., i.d. 0.010 in.; Hypo tube, Small Parts)
  • Blue connectors (Bioanalytical Systems)
  • Polyethylene (PE) 10 tubing
  • 4‐O suture thread (optional)
  • Additional reagents and equipment for performing aseptic neurosurgery (see protocol 2) and conducting pharmacological manipulation (unit 7.1)

Support Protocol 3: Anatomical Verification of the Probes

  • Primate chair
  • Stainless steel post
  • Steel straps
  • Bone cement
  • No. 8 screws
  • Steel rod
  • Crossing rod
  • 4 × 8–in. thermoplastic sheet
  • Nuts
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Figures

Videos

Literature Cited

Literature Cited
   Kolachana, B.S., Saunders, R.C., and Weinberger, D.R. 1994. An improved methodology for routine in vivo microdialysis in nonhuman primates. J. Neurosci. Methods 55:1‐6.
   Kolachana, B.S., Saunders, R.C., and Weinberger, D.R. 1996. In vivo characterization of extracellular GABA release in the caudate nucleus and prefrontal cortex of the rhesus monkey. Synapse 25:1‐8.
   NIH (National Institutes of Health). 1985. Guide for the Care and Use of Laboratory Animals. NIH Publication no. 85‐23. NIH, Bethesda, Md.
   Saunders, R.C., Aigner, T.G., and Frank, J.A. 1990 Magnetic resonance imaging of the rhesus monkey brain: Use for stereotactic neurosurgery. Exp. Brain Res. 81:443‐446.
   Saunders, R.C., Kolachana, B.S., and Weinberger, D.R. 1993. Local pharmacological manipulation of dopamine in prefrontal cortex and caudate in the rhesus monkey: An in vivo microdialysis study. Exp. Brain Res. 98:44‐52.
   Wang, J., Skirboll, S., Aigner, T.G., Saunders, R.C., Hsiao, J., and Bankiewicz, K.S. 1990. Methodology of microdialysis of neostriatum in hemiparkinsonian nonhuman primates. Exp. Neurol. 110:181‐186.
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