Chromogenic Optical Fractionator
The total number of stained cells (e.g. tyrosine hydroxylase positive, TH+, neurons) and
unstained cells (e.g. TH- neurons) in the region(s) of interest (e.g. substantia nigra pars
compacta and/or ventral tegmental area) were counted using the optical fractionator and
our previously described counting criteria (see references below. Briefly, neurons were
counted as TH+ if they showed: (a) TH immunoreactivity within the cell body, (b) part of
the nucleus of that cell was inside the counting frame without touching the avoidance
lines, and (c) a portion of the nucleolus within that nucleus was in focus between the top
and bottom boundaries of the counting frame or not in the guard zone. TH- neurons were
counted if (a) a neuronal nucleus was visualized without cytoplasmic staining, (b) the
nucleus must have a size equal to or greater than the diameter of the average TH+
nucleus, (c) it has a macronucleolus and more than one small nucleoli, (d) the shape of
the nucleus is round to oval, (e) the nuclear membrane typically smooth, (f) the nucleus
was partially or entirely inside the counting frame without touching the avoidance lines,
and (f) a portion of the nucleolus within that nucleus was in focus within the top and
bottom boundaries of the sampling frame, i.e. not in the guard zone.
The sections were ordered from rostral to caudal by visual inspection at low
power. The regions of interest were outlined at low magnification (4x objective) and
sampled at high magnification (100x oil immersion objective) using StereoInvestigator
(Microbrightfield, VT). We performed IHC using every 4th section from the midbrain, but
performed stereology on every 8th section using a random first section to start. This
provided two sets of sections should one set be uncountable due to a damaged or folded
section. We counted and report data from one side of the substantia nigra and identified
the right side using a hole as a marker. We determined our sampling criteria empirically
based on initial testing and confirmation with different experimental manipulations as
previously reported. We count enough TH+ neurons to yield a Nest having a coefficient of
error (C.E.)  10%. We accept a larger C.E. for TH-/SYN- neurons as they are more
difficult to identify, fewer in number, and not the primary object of our research. To
achieve these we sampled every 8th section of the SNpc and we space our sampling
frames every 200 µm (along the x-axis) and 100 µm (along the y axis) from each other
based on the shape of the region. We use a sampling frame size of 70 µm (x-axis) x 50
µm (y-axis) which allows for a reasonable border using our microscope with a 100x
objective and our camera.
We used an Olympus Provis AX-70 microscope equipped with 4x and 100x oil
immersion objectives, a Heidenhain linear encoder mounted to a LEP motorized XYZ
controlled stage connected a MAC 5000 driver. An Optronics DEI-750 CE camera was
used for real time collection of video images and was connected to a Dell computer using
a frame grabber board.
References
Thiruchelvam, M., Richfield, E. K., Baggs, R. B., Tank, A. W., and Cory-Slechta, D. A.
(2000) J. Neuroscience 20, 9207-9214
Barlow, B. K. , Thiruchelvam, M., Richfield, E. K., and Cory-Slechta, D. A. (2004) Dev.
Neurobiology 26, 11-23
Thiruchelvam, M., Powers, J., Cory-Slechta, D. A., and and Richfield, E. K. (2004) Eur.
J. of Neuroscience 19, 845-854