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Quantified Assessment of Terminal Density and Innervation

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

¹Howard Florey Institute, University of Melbourne, Parkville, Victoria, Australia

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-axon 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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Unit Introduction
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 3: Stereological Fractionator Design for Estimating Terminal Tree Size of Neurons

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 Basic 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

Figure 1.13.1

(A) Photomicrograph of a neutral red–labeled section through the mid-to-rostral portion of the normal mouse substantia nigra pars compacta (SNpc). The delineated boundaries highlight the densely packed SNpc cells that can be distinguished from less densely populated neighboring regions including the substantia nigra pars reticulata (SNpr) ventrally and the ventral tegmental area (VTA) dorsally. (B) An example of the fractionator sampling scheme for estimating the number of neurons in the SNpc. For each section containing the relevant nucleus, an unbiased counting frame was then superimposed over the tissue. Counts were then made at regular predetermined intervals (140 × 140 µm in mice) so that all areas of the nucleus had an equal chance of being sampled. This micrograph shows the systematic placement of the counting frames superimposed over a SNpc section. Note that the upper right-hand corner of the counting frame must lie within the delineated boundaries for a counting frame to be included in sampling. Hence the uppermost counting frame on the right would be excluded because the upper right-hand corner of the counting frame is not within the designated boundary of the SNpc section, whereas both counting frames in the bottom row would be included in this example, despite the location of the majority of these particular counting frames. (C) Schematic of a counting frame. Here, two varicosities were counted in total and included those cells where the nucleolus lay either within the counting frame (cell 2) or on the inclusion (upper and right solid green) lines (cell 1). Cell 3 was not counted as it lay on one of the exclusion (lower and left dashed red) lines.

View Image
Figure 1.13.2

(A) In mice, an area bounded by the most dorsal point on the surface of the striatum and a point 400 µm (1.5 mm in rats) ventral was sampled for determination of terminal density. Abbreviations: ac, anterior commissure; cc, corpus callosum; Lv, lateral ventricle. (B) Photomicrograph of dopamine transporter–immunoreactive (DAT-ir) varicosities in the dorsal caudate putamen (CPu) of mice. Scale bar, 25 µm. (C) Schematic of the counting frame seen in B. Here, five varicosities were counted in total, including the gray varicosities within the frame as well as the black varicosities that lie on the inclusion (upper and right solid green) lines. The white varicosity was not counted as it lay on an exclusion (lower and left dashed red) line.

View Image

Literature Cited

Literature Cited
	Adams, J.C. 1981. Heavy metal intensification of DAB-based HRP reaction product. J. Histochem. Cytochem. 29:775.
	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.
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.