The Neurotoxicity of DOPAL: Behavioral and Stereological Evidence for Its Role in Parkinson Disease Pathogenesis
Galvin JE (2010) The Neurotoxicity of DOPAL: Behavioral and Stereological Evidence for Its Role in Parkinson
Disease Pathogenesis. PLoS ONE 5(12): e15251. doi:10.1371/journal.pone.0015251
The Neurotoxicity of DOPAL: Behavioral and Stereological Evidence for Its Role in Parkinson Disease Pathogenesis
W. Michael Panneton 0
V. B. Kumar 0
Qi Gan 0
William J. Burke 0
James E. Galvin 0
Stephen D. Ginsberg, Nathan Kline Institute/New York University, United States of America
0 1 Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine , St. Louis , Missouri, United States of America, 2 Department of Geriatrics, Saint Louis University School of Medicine, St Louis, Missouri, United States of America, 3 Department of Neurology, Saint Louis University School of Medicine , St. Louis , Missouri, United States of America, 4 Department of Neurology and Department of Psychiatry, Center of Excellence on Brain Aging, New York University Langone Medical Center , New York, New York , United States of America
Background: The etiology of Parkinson disease (PD) has yet to be fully elucidated. We examined the consequences of injections of 3,4-dihydroxyphenylacetaldehyde (DOPAL), a toxic metabolite of dopamine, into the substantia nigra of rats on motor behavior and neuronal survival. Methods/Principal Findings: A total of 800 nl/rat of DOPAL (1 mg/200 nl) was injected stereotaxically into the substantia nigra over three sites while control animals received similar injections of phosphate buffered saline. Rotational behavior of these rats was analyzed, optical density of striatal tyrosine hydroxylase was calculated, and unbiased stereological counts of the substantia nigra were made. The rats showed significant rotational asymmetry ipsilateral to the lesion, supporting disruption of dopaminergic nigrostriatal projections. Such disruption was verified since the density of striatal tyrosine hydroxylase decreased significantly (p,0.001) on the side ipsilateral to the DOPAL injections when compared to the noninjected side. Stereological counts of neurons stained for Nissl in pars compacta of the substantia nigra significantly decreased (p,0.001) from control values, while counts of those in pars reticulata were unchanged after DOPAL injections. Counts of neurons immunostained for tyrosine hydroxylase also showed a significant (p = 0.032) loss of dopaminergic neurons. In spite of significant loss of dopaminergic neurons, DOPAL injections did not induce significant glial reaction in the substantia nigra. Conclusions: The present study provides the first in vivo quantification of substantia nigra pars compacta neuronal loss after injection of the endogenous toxin DOPAL. The results demonstrate that injections of DOPAL selectively kills SN DA neurons, suggests loss of striatal DA terminals, spares non-dopaminergic neurons of the pars reticulata, and triggers a behavioral phenotype (rotational asymmetry) consistent with other PD animal models. This study supports the ''catecholaldehyde hypothesis'' as an important link for the etiology of sporadic PD.
Funding: This work was supported by Saint Louis University School of Medicine. The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Parkinson disease (PD) is the most common neurodegenerative
movement disorder, affecting 2% of individuals over age 65 and
45% over 85 years . PD is characterized phenotypically by
bradykinesia, tremor at rest, rigidity and postural rigidity, and
pathologically by the loss of dopaminergic neurons in the
substantia nigra (SN), severe dopamine (DA) loss in the striatum
and the accumulation of alpha-synuclein (a-syn). Although the
exact causes of PD remain unknown, it is likely a combination of
Many interrelated hypotheses have been postulated about the
death of dopaminergic neurons including genetic defects [2,3],
environmental toxins , inflammation , deficiencies in
the mitochondrial respiratory chain [10,11], and reduced capacity
of transmitters, including monoamine storage vesicles [12,13] and
glutamate metabolism [14,15]. However, no experimental animal
models testing these hypotheses show all the features
characterizing PD. Moreover, most animal models of PD use exogenous
toxins to kill dopaminergic neurons in the SN, which may not
relate to cases of idiopathic PD in humans.
The involvement of DA or one of its metabolites also may be
important in the death of DA SN neurons [16,17]. The
catecholaldehyde hypothesis of PD proposes that an
accumulation of a toxic intermediate of dopamine metabolism,
3,4dihydroxyphenylacetaldehyde (DOPAL), is toxic to nigral neurons
and leads to PD. DOPAL is the catabolic product of dopamine via
oxidative deamination by monoamine oxidase (MAO), and is
quickly cleaved by aldehyde dehydrogenase (ALDH1A1) into
3,4dihydroxyphenylacetic acid (DOPAC). DOPAL is an endogenous
toxin found in dopaminergic cells in human SN [18,19] and could
contribute to the development of PD. Here we examine whether
DOPAL selectively kills dopaminergic neurons in the SN.
Our laboratories have shown that DA itself is not sufficiently
toxic at physiological levels to induce either neuronal death
[19,20] or aggregation of a-synuclein , thus implicating a
metabolite of DA. Investigations in several laboratories have
implicated a metabolite of DA as an endogenous toxin which
triggers DA neuron loss [19,20,2229]. DOPAL levels of 23 mM
are normally present in SN from neurologically intact human
patients at autopsy . However, DOPAL levels increase in the
SN and striatum in PD  while ALDH1A1 mRNA, protein and
activity decrease in the SN and striatum [31,32,3234],
implicating DOPAL as a potential endogenous toxin. Moreover, we have
shown that DOPAL is toxic to neurons at physiological
concentrations in vitro [19,21] and also triggers aggregation of
asynuclein . Earlier experiments provided
immunohistochemical evidence of DOPAL toxicity in vivo by showing loss of tyrosine
hydroxylase immunoreactivity (THir) after DOPAL injections into
rat SN [20,21]. However these studies did not exclude the
possibility that DOPAL injections may have decreased tyrosine
hydroxylase (TH) synthesis and protein levels resulting in
decreased THir as was shown for DA . Here we determined
that DOPAL induces loss of striatal DA in vivo using tyrosine
hydroxylase immunohistochemistry and show that DOPAL is
toxic to DA neurons in vivo with definitive neuronal counts using
unbiased stereology . In addition we show that DOPAL
injections into SN produce a behavioral model of PD. The
experiments provided herein strongly reinforce the notion that
DOPAL is an endogenous neurotoxin, and implicate it as the
trigger which kills dopaminergic neurons in the SN and leads to
Rotational asymmetry was assessed to quantify the effect of
unilateral depletions of striatal dopamine from disruptions of
nigrostriatal circuitry. We show that rats significantly (p,0.05)
prefer rotating to the side ipsilateral to the unilateral DOPAL
injections versus control rats (Fig. 1) after injections of apomorphine.
Neuropathological Evaluation: Immunohistochemistry
In all cases there was a decrease in immunoreactivity of TH in
the SN ipsilateral to the injections of DOPAL (Fig. 2B, yellow
arrowhead) compared to the contralateral, non-injected side
(Fig. 2A). There also was significantly (p,0.001) less TH
immunoreactivity in the striatum on the side ipsilateral to the
DOPAL injections (Fig. 2D, arrows; Fig. 2E) compared to the
noninjected contralateral side (Fig. 2C, arrows; Fig. 2E). After
background densities were subtracted, we calculated a 28%
reduction in immunoreactivity in the striatum on the side
ipsilateral to the DOPAL injections, suggesting a loss of DA
terminals on the injected side. We noted that the ventrolateral
striatum through levels of the globus pallidus were especially
denervated (Fig. 2D, red circles). Spot density measurements
contralateral (17.864.5 units) versus ipsilateral to the DOPAL
injections (3.565.9 units) here were reduced 80%.
Neuropathological Evaluation: Stereology
The SN was included in 810 sections of all cases counted, and
its total length was approximately 1.25 mm. Mean volume of the
SNpc of control rats was 268,639,250 m3, while that of the SNpr
was 777,696,500 m3. Mean volume of the SNpc in the
DOPALinjected rats was 264,674,833 m3 while that of the SNpr was
760,212,500 m3. There was no significant difference in mean
volumes of SNpc or SNpr between controls and DOPAL injected
We first counted TH immunoreactive neurons in the SNpc on
the side of the DOPAL injection and compared them to those on
the non-injected side. When only TH immunoreactive neurons
were counted, the mean number of TH immunoreactive neurons
ipsilateral to the DOPAL injections side was 50% less than that of
the contralateral non-injected side, significantly different
(p = 0.032) using the paired samples T-test by difference method
(Table 1). However, we noted that numerous SNpc neurons
sometimes were not stained for TH despite robust labeling of
others (Fig. 2F). Thus, we compared the number of Nissl stained
profiles in sections immunostained with a-syn rather than TH in
the SNpcs ipsilateral to the DOPAL injections to those of control
rats which had received injections of a buffered saline solution into
their SNs (Table 1). The number of Nissl-stained neurons in the
SNpc (compare Figs. 3A, B) of the DOPAL injected rats was 43%
less than that of the saline-injected rats (Fig. 3C) which was
significantly different (p#0.001). We then determined whether
DOPAL was toxic to neurons in the subjacent pars reticulata of the
SN. The number of neurons in the SNpr of the DOPAL-injected
rats was not different from the saline-injected rats (Table 1;
Fig. 3C). This suggests that DOPAL is selectively lethal to
dopaminergic neurons in the SNpc, further supporting the
Neuropathological Evaluation: Activation of Glia
We immunostained a series of sections against antibodies to glial
fibrillary protein (GFAP), a marker for astrocytes, and to OX-42,
which stains microglia, to determine the relationship between
neuronal loss following DOPAL injections and the activation of
glial cells. Reactive astrocytes were defined as cells with
upregulation of GFAP having pronounced hypertrophy of cell body
and processes with considerable extension of these processes
beyond the normal domains of individual astrocytes . Reactive
astrocytes and their processes were localized to and surrounded
antibodies against tyrosine hydroxylase (F, yellow arrowheads) were sometimes seen in the SNpc of control brains surrounded by numerous neurons
stained only for Nissl (F, black arrows), suggesting that counting only TH-immunostained neurons may be problematic. Abbreviations: ac, anterior
commissure; SNpc, pars compacta of substantia nigra; SNpr, pars reticulata of substantia nigra. *** p,0.001.
the injection site in some of our cases (Figs. 4A, B). There were no
reactive processes streaming throughout the SNpc or SNpr
though, suggesting that the major losses of neurons in the SNpc
[compare neuronal density of the SNpc ipsilateral to injections
(Figs. 3B, 4A, 4C) to that contralateral (Figs. 3A, 4D, 4F)] was not
due to astrogliosis. Mild astrogliosis was noted in the ventromedial
parts of the SNpr both ipsilateral and contralateral to DOPAL
Activated microglia also were relatively few in absolute number
after DOPAL injections (Fig. 4C, arrow) with only few activated
cells (Fig. 4E, arrow). We counted 60 engorged microglia
immunoreactive to OX-42 in the ipsilateral SNpc of the rats
receiving DOPAL injections (n = 6), and 15 in the adjacent SNpr.
However, most activated cells were detected in three DOPAL
cases, with few or no activated microglia in other injected or
Our results show that DOPAL induces a behavioral phenotype
(asymmetrical rotation), significantly decreased
TH-immunoreactivity of nigrostriatal projections, and is lethal to neurons in the pars
compacta of the SN but not those in the adjacent pars reticulata in
rats. This study is the first to quantify in vivo the death of neurons in
the SN due to an endogenous toxin naturally produced in
dopaminergic neurons. It augments our in vitro data implicating
DOPAL as a toxin and supports our contention that intracellular
accumulations of DOPAL trigger death in dopaminergic neurons
in the SNpc and may be relevant to the pathogenesis of PD in
Measurable levels of DOPAL are found in post-mortem human
brains  and increased amounts of DOPAL are in autopsy
brains of PD [30,32]. We have shown previously that DOPAL is
toxic to PC12 cells in vitro at dosages as low as 6.6 mM  and
triggers aggregation of a-synuclein in vitro at dosages as low as
1.5 mM . Previous studies with intracerebral injections of
DOPAL used doses ranging between 0.050.75 mg/200 nl, with
doses above 0.1 mg/200 nl destroying at least some of the DA
neurons in the substantia nigra/ventral tegmental area of the
midbrain [20,21]. However, these studies did not exclude that loss
of THir after DOPAL injections into SN was due to decreased TH
synthesis . In the present study we made three injections of
200 nl-400 nl-200 nl along the rostrocaudal extent of the SN,
hoping to include all the DA neurons in the presumed ellipsoid
shape of the injection. The DOPAL injections (1 mg/200 nl)
caused neuronal loss only in the SNpc, while sparing neurons in
the juxtaposed SNpr. This is of interest since between 7180% of
neurons in SNpc are dopaminergic [39,40] while those in SNpr
are approximately 7080% GABAergic , suggesting that
DOPAL may be selectively lethal to DA neurons.
We elected unilateral injections since bilateral disruption often
results in aphagia, adipsia and high mortality rates [41,42]. Most
studies on rats inducing hemi-Parkinson symptoms use rather large
(i.e., $4 ml) injections of 6-OHDA into their median forebrain
bundles . Numerous dopamine neurons in the ipsilateral
SNpc are killed after such injections, resulting in loss of dopamine
in the ipsilateral striatum but also in the prefrontal cortex, nucleus
accumbens, septum and olfactory tubercles. Injecting DOPAL
unilaterally into the SN also resulted in the ipsilateral loss of
striatal TH immunoreactivity, and perhaps DA in nerve terminals
of the striatum. Unilateral depletion of striatal DA also allows for
tests comparing the dopamine innervation on either side of the
Tests such as rotational asymmetry determine imbalances in
dopaminergic innervation and are easily evaluated .
Rotational behavior after unilateral nigral lesions is hypothesized to be
dependent on the balance between striatal dopamine release and
hypersensitivity of striatal dopamine receptors on the two sides
. We evaluated rotational asymmetry in rats injected with the
endogenous toxin DOPAL into their SN, and show significant
asymmetry with rats turning ipsilateral to the injection. Our rats
turned to the same side as the lesion after apomorphine injection,
similar to other studies after intranigral injections of toxins .
This finding is consistent with the typical asymmetrical onset of
PD in humans.
Rotational asymmetry also is dependent on which neurons of
the basal ganglia circuitry are involved. For example, turning
behavior can be manipulated with lesions/stimulation of the
prefrontal cortex , the centromedian-parafasicular-thalamic
complex , the subthalamic nucleus [50,51] or the SNpr
[52,53]. Indeed, killing neurons in both SNpc and SNpr induce
different behaviors in rats than killing neurons in SNpc alone
[52,54]. Although our experiments targeted SNpc neurons,
quantitative measurements show that SNpr neurons were spared.
Thus the DOPAL model recapitulates many features of nigral
degeneration in sporadic PD. We cannot however fully discount
potential toxicity to non-dopaminergic neurons.
The importance of correct counts of neurons in the SN in the
various animal models of PD has been emphasized , especially
if comparisons of degeneration of nigral dopaminergic neurons
Means (SD); n.s. = not significant.
The effect of DOPAL injections into the substantia nigra on neurons in either the pars compacta (SNpc) or the pars reticularis (SNpr) are shown. Control animals were
injected with buffered saline while experimental animals were injected with DOPAL (4 mg/800 nl). Unbiased stereology was used to assess the number of neurons (see
Figure 3. Photomicrographs of sections through the SN stained for Nissl with neutral red. Red lines mark the boundaries enclosing the
substantia nigra, pars compacta, while green lines encompass the substantia nigra, pars reticulata. Unbiased stereological counts using optical
fractionator probes were made of neurons in both SNpc and SNpr in sections from animals injected with buffer (A; case R2546) and those injected
with DOPAL (B, case R2505). Note the significant (p#0.001) loss of SNpc neurons in rats (C) after the DOPAL injection when compared to control rats,
while no loss of neurons was seen in the adjacent SNpr.
among different studies are to be accurate. The volumes of either
the SNpc or SNpr nuclei were similar in both our control and
DOPAL-injected animals, suggesting uniformity in our
interpretation of the nuclear outlines. Since shrinkage of tissue can be
problematic when comparing counts from different processing
methods, all tissues were processed similarly using free-floating
sections. Moreover, since neurons in both the SNpc and SNpr
subnuclei are neither homogeneous in size or distribution, the
whole rostrocaudal extent of these subnuclei was included in
analysis. The estimated number of neurons determined by these
methods in the SNpc of our control rats (1192661084) compares
favorably with those using similar methodologies in both rats
[39,56] and mice (reviewed by Baquet et al., 2009), with numerous
studies indicating between 8,00012,000 TH-positive neurons in
We used Nissl stained sections for neuronal cell counts in the SN
since there was large variability in counts of TH immunostained
neurons, similar to observations of others [57,58]. Our
densitometry measurements of the whole striatum were also variable
between cases, but collectively showed a 28% decrease on the side
ipsilateral to the DOPAL injections. This number reflects the total
TH immunostaining of the striatum, however, which includes
catecholaminergic innervation from numerous sources such as the
SNpc, the ventral tegmental and retrorubral areas, as well as the
locus coeruleus . For example, an area just lateral to the
anterior commissure (Fig. 2D, yellow arrowhead), always was
densely labeled ipsilateral to the DOPAL injections, and this area
receives projections from a subdivision of the ventral tegmental
nucleus . The ventrolateral portion of the striatum through
levels of the globus pallidus was especially devoid of
immunoreactivity after DOPAL injections; spot density of this portion
showed an 80% reduction over the control side. Nevertheless,
studies have shown that neurons utilizing monoamines as
transmitters/messengers vary their metabolism throughout the
day [61,62] and are asymmetrically lateralized ; this is
especially true for neurons utilizing dopamine. These results
should provide caution to those quantifying TH immunopositive
neurons as their sole data to determine the extent of lesions of SN
Although reactive astrocytes and microglia have been
implicated in the etiology of Parkinson disease [7,8,37,64], we saw few
reactive astrocytes or activated microglia in DOPAL-injected
animals. Inflammatory cells are proposed to induce or mediate
death to dopamine neurons in the SNpc [79,64], however our
results do not support a primary role of glial activation in SNpc
degeneration, but instead may be a later event in the pathogenesis
The present study provides the first in vivo quantification of
neuronal loss after injection of an endogenous toxin. The results
demonstrate that injections of DOPAL kill SN DA neurons with
loss of striatal DA terminals and also induce rotational asymmetry
in rats. These results add to an increasing body of evidence
obtained in our laboratories that the endogenous metabolite of
dopamine, DOPAL, is toxic to dopaminergic neurons. We already
have provided evidence that DOPAL, but not other dopamine
metabolites, induces cell death in vitro  and in vivo , induces
aggregation of a-synuclein , and disrupts mitochondrial
function and creates reactive oxygen species [19,25,65]. DOPAL,
like its analogue 3,4-dihydroxyphenylglycolaldehyde, also activates
the mitochondrial permeability pore which can lead to apoptotic
neuronal death [19,6567]. Moreover, this data is supported by
work from others showing that DOPAL is increased in both the
SN and striatum in PD brains [30,32]. Here we provide definitive
evidence that DOPAL is toxic in vivo, triggering a behavioral
phenotype consistent with other PD animal models. These data
thus support the catecholaldehyde hypothesis on the etiology of
Materials and Methods
Ten adult male Sprague Dawley rats (275299 g) were
purchased commercially (Harlan Laboratories, Indianapolis, IN)
and a housed in the Department of Comparative Medicine at
Saint Louis University. All protocols were approved by the Animal
Care Committee of Saint Louis University and followed the
guidelines of the National Institutes of Health Guide for Care and
Handling of Laboratory Animals.
A commonly used measurement of unilateral dopaminergic
denervation of the rodent striatum is rotational asymmetry
[47,68]. Rats were introduced to the test one week prior
to surgery to establish baseline control data. Rats were injected
subcutaneously with the dopamine agonist apomorphine (0.4 mg/
kg) dissolved in 0.1% ascorbate saline solution . After waiting
5 min, the rats were placed in a hemispheric rotation bowl 40 cm
wide and 20 cm deep and the number of complete turns to the
right or the left quantified by observation. This test was performed
both prior to DOPAL injection and again one day prior to
The rats were anesthetized with injections (IP; 0.1 ml/kg) of a
cocktail of ketamine (60 mg/ml) and xylazine (40 mg/ml) and
mounted in a stereotaxic frame. DOPAL was synthesized as
previously described  and dissolved immediately prior to
injection in 1% benzyl alcohol then diluted to the final
concentration (1 mg/200 nl) with phosphate buffered saline
(PBS; pH 7.4) and red Fluorospheres (Molecular Probes, Eugene,
OR). Three injections (200400 nl-200 nl) of DOPAL were made
through the rostral-caudal extent of the SN of six rats, using a glass
micropipette (tip diameter 2030 mm) attached to a 1 ml Hamilton
syringe at coordinates AP +3.0, 3.6, 4.2; ML 2.0, 2.2, 2.1; DV+2.3,
2.2, 2.0. Control rats (n = 4) were injected similarly with the same
volume of PBS into their SN. The micropipette remained in place
for 5 min to help prevent spread of the injection. The wound was
irrigated with saline and closed with wound clips. After survival
(Control 32-33d; Experimental 40-61d), the animals were deeply
anesthetized with a Euthanasia solution (IP; 40 mg/kg) and
perfused through the heart using a peristaltic pump first with PBS
with 0.25% procaine, and then with a fixative of 4%
paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.3). The brains were
removed, soaked in the fixative containing 20% sucrose, and then
subsequently cut (40 mm) on a freezing microtome and sections
collected in PB. Injections in all cases were verified in the SN.
A 1:4 series of sections from injected brains were rinsed three
times in stock serum (PB containing 0.3% triton and serum from
the secondary antibody), and then soaked in the stock serum
solution containing either mouse anti-tyrosine hydroxylase (TH;
1:7500; ImmunoStar, Inc.) or a-synuclein (1:20,000) overnight on
a shaker table at room temperature. A 1:8 series was processed
similarly for rabbit anti-glial fibrillary acidic protein (GFAP;
1:3500; Abcam, Inc.) or mouse monoclonal anti-CD11b/c
(OX42; 1:8000; Abcam, Inc.). The next morning the tissues were
washed three times in PB, and again in stock serum, and then
soaked for one hour in stock serum to which the secondary
antibody had been added. Secondary antibodies were
ratadsorbed biotinylated donkey anti-mouse (1:500; Sigma) or
biotinylated goat anti-rabbit (1:500; Sigma). After washing 3 more
times, the sections were incubated in Vectastain ABC Elite
solution (1:200; Vector Laboratories) for 1 hour, washed in three
rinses of PB, and reacted with diaminobenzidine dihydrochloride
(DAB) intensified with nickel ammonium sulfate for 410 min.
The sections then were counterstained with Neutral Red,
dehydrated in an ascending series of alcohols, defatted in xylenes,
and cover slipped with Permount. Photomicrographs were taken
with a digital camera and saved with Northern Eclipse software
(Empix Imaging, Inc.). Images were processed in Adobe
Photoshop software (version 7.0), adjusting them with levels, brightness
The density of the striatum was calculated (Northern Eclipse)
both ipsilateral and contralateral to the DOPAL injections at five
different rostrocaudal levels, creating an N = 30 for either side.
Sections surveyed were separated by approximately 200 microns;
care was made to avoid the bundles of fibers in the globus pallidus,
which was not sampled. Since the striatum sampled included both
the black immunohistochemical precipitate as well as the neurons
stained with Neutral Red, densitometry measurements also were
obtained from 20 sections of the striatum which were processed
similarly (with antibodies against CGRP and vasopressin) but had
no immunohistochemical precipitate in their striata. All sections
showed a narrow range of scores, with an average density of 60.4.
This number was considered baseline background, and was
subtracted from the densities with immunopreciptate ipsilateral
and contralateral to the DOPAL injections so that the loss of
immunoreactivity could be calculated.
Unbiased stereological methods [36,40,55] were used to analyze
total volume of the SN as well as the neuronal loss in its pars
compacta (SNpc) and pars reticulata (SNpr) subnuclei after the
DOPAL injections. SNpc and SNpr subnuclei were determined by
anatomical landmarks and regional variations in cell density,
orientation and morphology as outlined by others [40,55]. The
boundaries of the SNpc and SNpr were outlined in the sections
with an E800 Nikon microscope through a 106 objective
equipped with a motorized stage and a stereological imaging
system (StereoInvestigator; MicroBrightField, Inc.). The SN
subnuclei were reconstructed serially and their volume calculated
with StereoInvestigator software.
Unbiased stereology was performed with the 1006 objective of
the microscope. The optical fractionator stereological probe was
used to determine neuronal loss in the SNpc and SNpr in sections
stained for both Nissl and a-synuclein and dopaminergic neuron
loss in another series of sections immunostained for TH after
DOPAL injections. Sampling grid sizes were 140 mm 6140 mm
(area, 19600 mm2) with an unbiased counting frame (25625 m,
625 mm2). A guard height of 2 mm was used on sections between
10.513.0 mm thick. Only cells coming into focus through the
sampling brick were counted. Eight to 10 sections were analyzed/
case; 102125 sampling sites for control cases and 85126
sampling sites after DOPAL injections were analyzed in the SNpc
while 252320 sampling sites were analyzed for control cases and
212314 sampling sites in the SNpr after DOPAL injections (the
larger SNpr required more sampling sites).
Means and standard errors (M 6 S.E.) were determined for
experimental and control groups. Group differences for rotational
asymmetry, neuronal counts and densitometry were determined
using T-tests (SPSS software; v. 13). The number of neurons
stained with Nissl in the SNpc and SNpr of injected brains were
compared to control brains while TH-stained neurons were
compared between injected and non-injected sides. The changes
in behavior of the rats after injections of DOPAL were compared
to the percentage loss of DA neurons.
The Paired Samples T-test was used to calculate significance for
densitometry measurements, a similar test by difference method
for counts of TH neurons, while the Independent Samples T-test
was used for rotational asymmetry and counts of Nissl-stained
neurons. Data are presented as M 6 S.E. and p-values considered
less than p,0.05 were considered significant. Graphs were drawn
with GraphPad Prism software. Box plots present a vertical view of
the data and show the shape of its distribution, its central value,
and its spread. The box itself represents 50% of the data, 75th
percentile marks the top of the box, the 25th percentile marks the
bottom, while the median (50th percentile) is shown as a line
through the box. Whiskers show the most extreme (maximum and
minimum) values in the data set and extend a maximum of 1.5
times the range in the box. Data outside these parameters are
considered outliers. Outliers for the present study (not illustrated)
were associated only with a single neuronal count (4817) of
Nisslstained neurons ipsilateral to the DOPAL injections.
We acknowledge the assistance of Philip Clerc, Jason Le and Rob
Livergood for analysis of the behavior of these rats.
Conceived and designed the experiments: WMP WJB. Performed the
experiments: WMP QG. Analyzed the data: WMP QG WJB. Contributed
reagents/materials/analysis tools: WMP VBK JEG. Wrote the paper:
WMP WJB JEG.
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