Quantified Assessment of Terminal Density and Innervation

David I. Finkelstein1, Davor Stanic1, Clare L. Parish1, John Drago1, Malcolm K. Horne1

1 Howard Florey Institute, University of Melbourne, Parkville, Victoria
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 1.13
DOI:  10.1002/0471142301.ns0113s27
Online Posting Date:  September, 2004
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Abstract

Stereological methods allow for the determination of cell numbers, terminal densities, and, subsequently, the estimation of terminal arbor size within a given brain nucleus. This unit provides an explanation for determining the terminal arbor size of dopaminergic neurons of the nigrostriatal pathway in rodents. In contrast to previously used single‚Äźaxon reconstructions, these stereological methods allow for quick and easy determination of terminal arbor size.

Keywords: Stereology; fractionator; terminal density; terminal arbor; immunohistochemistry

     
 
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Table of Contents

  • Basic Protocol 1: Tissue Preparation and Histology for Determining Innervation Density and Terminal Arbor Size in Rats and Mice
  • Basic Protocol 2: Neuroanatomy and Guidelines for Identifying Dopaminergic Varicosities and Dopaminergic Cell Bodies
  • Basic Protocol 3: Stereological Fractionator Design for Estimating Terminal Tree Size of Neurons
  • Basic Protocol 4: Estimating an Index of Terminal Tree Size
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Tissue Preparation and Histology for Determining Innervation Density and Terminal Arbor Size in Rats and Mice

  Materials
  • 1 U/ml heparin in 0.1 M PBS (see recipe for PBS), 37°C
  • Mouse (∼24 g) or rat (∼ 300 g)
  • Sodium pentobarbitone, such as Lethabarb (VIRBAC) or SP5 (CII) EUTHANASIA 5 GRAIN (Vedco)
  • Paraformaldehyde fixative (see recipe), 4°C
  • 30% (w/v) sucrose in 0.1 M PBS
  • Mounting medium for cryostat sectioning (e.g., OCT; Miles Labs)
  • Gelatinized slides (see recipe)
  • 24‐well tissue culture plates containing cryoprotectant solution (see recipe)
  • 10% neutral buffered formalin
  • 0.1 M PBS (see recipe)
  • DAT blocking solution: 0.3% (v/v) Triton X‐100 (Sigma)/5.0% (v/v) normal rabbit serum (Chemicon International) in 0.1 M PBS
  • Goat anti‐rat dopamine transporter (DAT) antibody (Chemicon International), diluted 1:3000 in DAT diluting solution (0.3% Triton X‐100/1.0% normal rabbit serum in 0.1 M PBS)
  • Rabbit anti‐rat biotinylated secondary antibody (Vector Laboratories), diluted 1:300 in DAT diluting solution (see above)
  • Avidin peroxidase solution (see recipe)
  • Cobalt‐nickel DAB (see recipe)
  • 30% (v/v) hydrogen peroxide
  • 50%, 70%, 90%, and 100% (v/v) ethanol
  • Clearing agent: citrus‐based xylene replacement (e.g., Histoclear; Raymond A. Lamb or xylene substitute; Sigma)
  • Polystyrene mounting medium
  • TH blocking solution: 0.3% (v/v) Triton X‐100/10.0% (v/v) normal goat serum (Chemicon International) in 0.1 M PBS
  • Mouse anti‐tyrosine‐hydroxylase (TH; Boehringer Mannheim), diluted 1:3000 in TH diluting solution (0.3% Triton X‐100/1% normal goat serum in 0.1 M PBS)
  • Goat anti‐mouse biotinylated secondary antibody (Sigma), diluted 1:300 in TH diluting solution (see above)
  • 1% (w/v) gelatin
  • 1% neutral red solution (see recipe)
  • Peristaltic perfusion pump (e.g., Masterflex with variable‐speed standard drives; Cole‐Parmer) and appropriate flexible silicon tubing
  • 23‐G needle (for mouse) or blunt drawing‐up 18‐G needle (for rat)
  • 37°C water bath or hot plate
  • Surgical instruments, including:
    • Scalpel
    • Large‐toothed forceps
    • Large scissors
    • Small sharp scissors
    • Artery forceps
    • Rongeur
    • Spatula
  • Cryostat microtome with appropriate chucks
  • Hydrophobic barrier pen (e.g., PAP pen; Vector Laboratories)
  • Humidified chamber
  • Staining racks and dishes for microscope slides
  • Coverslips
  • Soft paintbrush or fine sieve for 24‐well plate
  • Microscope slides
  • Fine paintbrush

Basic Protocol 2: Neuroanatomy and Guidelines for Identifying Dopaminergic Varicosities and Dopaminergic Cell Bodies

  Materials
  • Mouse or rat brain sections stained for dopamine transporter (DAT) and tyrosine hydroxylase (TH) immunoreactivity with defined substantia nigra pars compacta (SNpc) and dorsal caudate putamen (CPu) boundaries (see protocol 2)
  • Microscope equipped with computer‐controlled stage (e.g., Leica DML) and 100× numerical aperture 1.30 oil immersion objective (e.g., PL Fluotar; Leica Microsystems), connected to computer with appropriate stereological software (Table 1.13.1)
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Figures

Videos

Literature Cited

Literature Cited
   Adams, J.C. 1981. Heavy metal intensification of DAB‐based HRP reaction product. J. Histochem. Cytochem. 29:775.
  The authors have been supported by grants from the Australian National Health and Medical Research Council. John Drago is an Australian National Health and Medical Research practitioner fellow.
   Braendgaard, H., Evans, S.M., Howard, C.V., and Gundersen, H.J. 1990. The total number of neurons in the human neocortex unbiasedly estimated using optical disectors. J. Microsc. 157:285‐304.
   Fallon, J.H. and Moore, R.Y. 1978. Catecholamine innervation of the basal forebrain. IV. Topography of the dopamine projection to the basal forebrain and neostriatum. J. Comp. Neurol. 180:545‐580.
   Finkelstein, D.I., Stanic, D., Parish, C.L., Tomas, D., Dickson, K., and Horne, M.K. 2000. Axonal sprouting following lesions of the rat substantia nigra. Neuroscience 97:99‐112.
   Gerfen, C.R., Herkenham, M., and Thibault, J. 1987. The neostriatal mosaic: II. Patch‐ and matrix‐directed mesostriatal dopaminergic and non‐dopaminergic systems. J. Neurosci. 7:3915‐3934.
   Gundersen, H.J., Bagger, P., Bendtsen, T.F., Evans, S.M., Korbo, L., Marcussen, N., Moller, A., Nielsen, K., Nyengaard, J.R., Pakkenberg, B., Sorensen, F.B., Vesterby, A., and West, M.J. 1988. The new stereological tools: Disector, fractionator, nucleator and point sampled intercepts and their use in pathological research and diagnosis. APMIS 96:857‐881.
   Nelson, E.L., Liang, C.L., Sinton, C.M., and German, D.C. 1996. Midbrain dopaminergic neurons in the mouse: Computer‐assisted mapping. J. Comp. Neurol. 369:361‐371.
   Parish, C.L., Finkelstein, D.I., Drago, J., Borrelli, E., and Horne, M.K. 2001. The role of dopamine receptors in regulating the size of axonal arbors. J. Neurosci. 21:5147‐5157.
   West, M.J. and Gundersen, H.J. 1990. Unbiased stereological estimation of the number of neurons in the human hippocampus. J. Comp. Neurol. 296:1‐22.
   West, M.J., Slomianka, L., and Gundersen, H.J. 1991. Unbiased stereological estimation of the total number of neurons in the subdivisions of the rat hippocampus using the optical fractionator. Anat. Rec. 231:482‐497.
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