Neurobiology of Disease
Unregulated Cytosolic Dopamine Causes Neurodegeneration
Associated with Oxidative Stress in Mice
Amber D. Van Laar,
Teresa G. Hastings,
Un Jung Kang,
and Xiaoxi Zhuang
Committee on Neurobiology, The University of Chicago, Chicago, Illinois 60637, and
Neurology, The University of Pittsburgh, Pittsburgh, Pennsylvania 15261
The role of dopamine as a vulnerability factor and a toxic agent in Parkinson’s disease (PD) is still controversial, yet the presumed
dopamine toxicity is partly responsible for the “DOPA-sparing” clinical practice that avoids using L-3,4-dihydroxyphenylalanine (L-
DOPA), a dopamine precursor, in early PD. There is a lack of studies on animal models that directly isolate dopamine as one determining
factor in causing neurodegeneration. To address this, we have generated a novel transgenic mouse model in which striatal neurons are
engineered to take up extracellular dopamine without acquiring regulatory mechanisms found in dopamine neurons. These mice devel-
oped motor dysfunctions and progressive neurodegeneration in the striatum within weeks. The neurodegeneration was accompanied by
oxidative stress, evidenced by substantial oxidative protein modifications and decrease in glutathione. Ultrastructural morphologies of
degenerative cells suggest necrotic neurodegeneration. Moreover, L-DOPA accelerated neurodegeneration and worsened motor dysfunc-
tion. In contrast, reducing dopamine input to striatum by lesioning the medial forebrain bundle attenuated motor dysfunction. These
data suggest that pathology in genetically modified striatal neurons depends on their dopamine supply. These neurons were also super-
sensitive to neurotoxin. A very low dose of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (5 mg/kg) caused profound neurodegeneration
of striatal neurons, but not midbrain dopamine neurons. Our results provide the first in vivo evidence that chronic exposure to unregu-
lated cytosolic dopamine alone is sufficient to cause neurodegeneration. The present study has significant clinical implications, because
dopamine replacement therapy is the mainstay of PD treatment. In addition, our model provides an efficient in vivo approach to test
therapeutic agents for PD.
Key words: cytosolic dopamine toxicity; neurodegeneration; L-DOPA; oxidative stress; glutathione; dopamine autoxidation
The mechanism for the relatively selective loss of dopamine neu-
rons in Parkinson’s disease (PD) remains unknown. Dopamine
has long been suspected as a toxic agent and a vulnerability factor
for neurodegeneration in PD (Hastings et al., 1996; Barzilai et al.,
2003). This presumed dopamine toxicity is responsible for the
“DOPA-sparing” clinical practice that avoids using
L-DOPA), a dopamine precursor, in
early PD. However, there is a lack of in vivo studies on animal
models that directly isolate dopamine as the determining factor.
Dopamine is a very reactive molecule compared with other
neurotransmitters, and dopamine degradation naturally pro-
duces oxidative species (Graham, 1978). More than 90% of do-
pamine in dopamine neurons is stored in abundant terminal
vesicles and is protected from degradation (Eisenhofer et al.,
2004). However, a small fraction of dopamine is cytosolic, and it
is the major source of dopamine metabolism and presumed tox-
icity. Cytosolic dopamine undergoes degradation to form 3,4-
dihydroxyphenylacetic acid (DOPAC) and homovanillic acid
(HVA) as well as hydrogen peroxide via the monoamine oxidase
pathway (Graham, 1978; Stokes et al., 1999). Alternatively, dopa-
mine undergoes oxidation to form superoxide, hydrogen perox-
ide, and o-quinone or reacts with cysteine residues on glutathione
or proteins to form cysteinyl-dopamine and cysteinyl-DOPAC
conjugates (Hastings and Zigmond, 1994; Sulzer and Zecca,
2000; Barzilai et al., 2003).
The empirical evidence for dopamine as a toxic agent respon-
sible for neurodegeneration in PD remains controversial (Agid et
al., 1999; Fahn, 2005). Cell culture studies have shown that extra-
cellular dopamine or
L-DOPA is either toxic or protective against
cell death (Walkinshaw and Waters, 1995; Simantov et al., 1996;
Koshimura et al., 2000). In vivo, one of us demonstrated that a
high dose of dopamine injected into the striatum caused selective
dopamine terminal loss (Hastings et al., 1996). Sustained eleva-
tion of extracellular dopamine causes striatal neuron degenera-
tion in the dopamine transporter (DAT) knock-out mice (Cyr et
Received Aug. 8, 2007; revised Nov. 8, 2007; accepted Nov. 29, 2007.
This work was supported in part by the Parkinson’s Disease Foundation (X.Z.); the American Parkinson Disease
Ministry of Science and Technology, Republic of Korea (U.J.K.); National Institute of Neurological Disorders and
Stroke (NINDS) Grant NS043286 (U.J.K.); and NINDS Grant NS044076 (T.G.H.). We thank Manuel Utset and Robert
Burke for their help with neuropathology; William Dauer for critical reading of this manuscript; and Nancy Tian,
Sandra Rokosik, Khalid Fakhro, Hyun Ah Yoon, Ali Hussain, and Sarah Manning for technical assistance.
Correspondence should be addressed to either of the following: Linan Chen, Department of Neurobiology, Uni-
versity of Chicago, 924 East 57th Street, Knapp R222, Chicago, IL 60637, E-mail: email@example.com; or Un
Jung Kang, Department of Neurology, University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637,
Copyright © 2008 Society for Neuroscience 0270-6474/08/280425-09$15.00/0
The Journal of Neuroscience, January 9, 2008 • 28(2):425– 433 • 425
al., 2003). Both studies suggest that extracellular dopamine is
toxic while leaving the role of cytosolic dopamine on neurode-
generation unanswered. In dealing with cytosolic dopamine, two
recent studies examined mice with severely reduced vesicular
monoamine transporter 2 (VMAT2) expression [5% of wild-type
(WT) level]. Colebrooke et al. (2006) found age-related decline of
striatal dopamine and motor performance in the absence of ni-
gral cell loss. Using the same transgenic line, Caudle et al. (2007)
reported mild substantia nigra dopamine neuron loss in aged
mice, suggesting that cytosolic dopamine-mediated toxicity
could be the potential cause. However, direct in vivo evidence that
isolates cytosolic dopamine as the determining factor responsible
for neurodegeneration is lacking.
In the present study, we have generated transgenic mice in
which striatal neurons are engineered to take up extracellular
dopamine released from dopaminergic terminals without acquir-
ing the regulatory mechanisms typically found in dopamine neu-
rons. These mice develop motor dysfunctions and progressive
neurodegeneration in the striatum within weeks. Therefore, we
were able to isolate cytosolic dopamine as the determining factor
responsible for the neurodegeneration and provide definitive ev-
idence that chronic exposure to unregulated cytosolic dopamine
alone is sufficient to produce neurodegeneration in vivo.
Materials and Methods
Mice. For most experiments, mice were kept on dietary doxycycline
(DOX) rodent chow (200 mg/kg; Bio-Serv, Frenchtown, NJ) from gesta-
tion. Two- to four-month-old CaMKII
-tTA; tetO-DAT double trans-
genic mice (CamDAT mice) were used after DOX withdrawal. When
CamDAT mice maintained on DOX treatment were used as controls,
sex- and age-matched littermates were used. When tetO-DAT single
transgenic mice (TetDAT mice) were used as controls, hemizygous
-tTA mice were bred with homozygous tetO-DAT mice to gen-
erate CamDAT experimental group and TetDAT control group. All mice
were kept on a 6:00 A.M. to 6:00 P.M. light cycle with ad libitum food and
water. Behavioral tests were performed during the light period. All ani-
mal procedures were approved by the Institutional Animal Care and
Usage Committee of The University of Chicago.
In situ hybridization and immunohistochemistry. Mice were perfused
with 4% paraformaldehyde. For DAT protein expression pattern, 40
sections were incubated with rat anti-mouse DAT antibody (1:5000, Mil-
lipore, Billerica, MA). For GFAP and
-synuclein immunostaining, sec-
tions from the same brains were incubated with rabbit anti-GFAP pri-
mary antibody (1:2500, DAKO Cytomation, Fort Collins, CO) or mouse
-synuclein primary antibody (1:2000, BD Transduction, Lexing-
ton, KY). Sections were then incubated with secondary antibody (Jack-
son ImmunoResearch, West Grove, PA) and ABC (Vector Labs, Burlin-
game, CA). DAB was used for visualization.
Frozen sections (20
m) were used for in situ hybridization protocol.
In situ probe for mouse DAT corresponds to the sequence from 1053 to
1801 nt (GenBank accession number NM_010020). In situ probes for
mouse preprodynorphin (Pdyn) and mouse preproenkephalin (PPE)
correspond to the sequence from 120 to 614 nt (GenBank accession
number U64968) and the sequence from 342 to 1025 nt (GenBank ac-
cession number M13227), respectively. Digoxigenin nonradioactive in
situ hybridization was used to detect DAT expression (Chen et al., 2005),
S radioactive in situ hybridization was used to detect Pdyn and PPE
expression (Ding et al., 2007).
Western blot. For quantification of DAT protein expression levels,
brains were lysed in NaCl-Tris-EDTA buffer, and 50
g of protein was
separated on SDS-polyacrylamide gel. The membrane was then blocked
and incubated with rat anti-mouse DAT antibody (1:2000, Millipore)
and mouse anti-mouse glyceraldehyde-3-phosphate dehydrogenase (1:
200,000, Abcam, Cambridge, MA) sequentially. Signals were then de-
tected by horseradish peroxidase-conjugated secondary antibodies
(Jackson ImmunoResearch) and enhanced chemiluminescence (Fisher,
Hanover Park, IL).
Unbiased stereological cell counting and brain volume estimation. Mice
were perfused with 4% paraformaldehyde. Brains were sectioned into 40
m serial sections. One of six sections were stained with mouse anti-
mouse NeuN antibody (1:1000, Millipore) and visualized with DAB. The
estimation of the total number of NeuN
neurons in the dorsal striatum
was determined using the optical fractionator probe (Stereo Investigator
6 from MicroBrightField, Williston, VT) under 100⫻ oil lens. The 300 ⫻
m grid and 18 ⫻ 18
m counting frame were used. The Gunder-
sun’s coefficient of error was 0.05 (m ⫽ 1) in this study. To quantify
dopamine neurons in substantia nigra pars compacta (SNc), one of three
sections was used. The detailed method was described in our previous
publication (Chen et al., 2005).
Slides were scanned, and the areas of cerebral cortex, striatum, and
olfactory bulb were measured using ImageJ (NIH). Volumes of these
structures were estimated by Cavalier principles.
Transmission electron microscopy. Mice (n ⫽ 3 for DOX group and n ⫽
3 for no DOX group) were perfused with 4% paraformaldehyde and 2%
glutaraldehyde. Striatum was dissected and postfixed in the same fixative.
Then blocks were processed by osmium tetroxide, dehydrated, and em-
bedded in epoxy resin. Ninety nanometer sections were cut and exam-
ined under Tecnai F30 scanning transmission electron microscope.
HPLC. Tissue dopamine, metabolites, and protein cysteinyl-catechols
were measured according to previously published methods (LaVoie and
Hastings, 1999). Briefly, tissues were homogenized in 1 ml of cold 0.1N
perchloric acid and centrifuged (30,000 ⫻ g, 20 min), and the resulting
supernatant was separated from the protein pellet. An aliquot of the
supernatant was filtered before analysis for dopamine, DOPAC, and
HVA levels on HPLC with electrochemical detection. The precipitated
protein pellet was hydrolyzed in 6N HCl (100°C, under vacuum, 24 h),
dried, and alumina extracted before analysis by HPLC with electrochem-
ical detection for cysteinyl-catechol conjugates. All catechols were iden-
tified and quantified by comparison to standards.
Open field locomotor activity and food consumption. Each mouse was
placed in an open field chamber (40 cm long ⫻ 40 cm wide ⫻ 37 cm high,
Med Associates, Georgia, VT). Illumination of open filed was set to 20
lux. Locomotor activity was monitored by infrared beams that record the
animal’s location and path. Data were collected in 20 min sessions with a
preceding 10 min habituation period.
For home cage food consumption, mice were individually housed.
Preweighed rodent chow was given, and the amount of consumed rodent
chow was recorded after 24 h. For small pellet consumption, mice were
tested weekly in transparent cylinder with small pellets (20 mg of each)
for 5 min. The number of pellets consumed was recorded.
Unilateral lesion of dopamine neurons and forepaw adjust stepping test.
Mice were anesthetized and put into stereotaxics. Desipramine (25 mg/
kg, Sigma-Aldrich, St. Louis, MO) was injected intraperitoneally to pro-
tect the norepinephrine neurons. Thirty minutes later, 6-OHDA (1
3 mg/ml dissolved in 0.01% ascorbate in saline, Sigma-Aldrich) was in-
jected into left medial forebrain bundle (coordinates: anteroposterior,
⫺1.3 mm from bregma; lateral, 1.3 mm from bregma; dorsoventral,
⫺4.9 to ⫺5.2 mm from the skull surface) via a 28-gauge stainless-steel
cannula that stayed in the brain for 5 min after the injection before being
The hindpaws of mice were held up and the front paws put on a
treadmill as the belt moved forward for a complete cycle. Each mouse was
tested in three nonconsecutive cycles. Stepping was videotaped and
scored by a blind observer afterward. The numbers of left and right paw
steps were counted.
MPTP treatment and silver staining. Two weeks after DOX withdrawal,
mice received saline or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP) (Sigma-Aldrich) at dosages of 4 ⫻ 2 mg/kg or 4 ⫻ 5 mg/kg
intraperitoneally at 2 h intervals (n ⫽ 5). One week later, mice were
perfused with 4% paraformaldehyde and cryoprotected with 30% su-
crose. Brains were sliced on a cryostat. Silver staining was done with FD
NeuroSilver Kit (FD NeuroTechnologies, Ellicott City, MD).
L-DOPA treatment. Four weeks after DOX withdrawal, mice were
divided into two groups to receive daily intraperitoneal injections of
L-DOPA (300 mg/kg, Sigma-Aldrich) plus benserazide (100 mg/kg,
Sigma-Aldrich) or saline for 4 weeks. Benserazide is an inhibitor of pe-
426 • J. Neurosci., January 9, 2008 • 28(2):425–433 Chen et al. • Cytosolic DA Causes Neurodegeneration in Mice
ripheral DOPA decarboxylase to prevent the conversion of L-DOPA in
periphery. Body weight was measured daily during treatment. By the end
of fourth week of
L-DOPA treatment, mice were perfused, and unbiased
stereological cell counting was performed to assess neuron number in
Glutathione measurement. Four weeks after DOX withdrawal, mice
were killed, and striatum, cerebral cortex, and cerebellum were dissected
on ice and then stored in a ⫺80°C freezer. Reduced glutathione (GSH)
and oxidized glutathione (GSSG) content were measured using a gluta-
thione assay kit (Cayman Chemical, Ann Arbor, MI) (n ⫽ 5). GSH and
GSSG contents were normalized to protein content and presented as
nmol/mg protein. Protein was quantified with BCA protein quantifica-
tion kit (Fisher).
Statistical analysis. Data were analyzed using StatView 5.0.1. Unpaired
two-tailed Student’s t tests were used when genotype or DOX treatment
was the only grouping variable. ANOVA were used when additional
factors were considered. Repeated-measures ANOVA were used when
data were collected in multiple trials. In figures, * indicates p ⬍ 0.05, **
indicates p ⬍ 0.01, and *** indicates p ⬍ 0.001. The exact p value (if it is
not ⬍0.001) is indicated in figure legends.
Generation of transgenic mice with ectopic DAT
overexpression in the forebrain
The transgenic mice with inducible forebrain DAT overexpres-
sion were engineered using the tetracycline inducible system
(Gossen and Bujard, 1992) (Fig. 1A). A tetO-DAT knock-in line
was generated by inserting the tetO promoter into the 5⬘ untrans-
lated region of DAT, placing DAT under the transcriptional con-
trol of the tetO promoter. In homozygous tetO-DAT mice, DAT
expression is reduced to ⬃5% of WT expression levels (data not
shown). The tetO-DAT mice were then crossed with the well
characterized forebrain-specific CaMKII
-tTA mice (Mayford et
al., 1996) (from The Jackson Laboratory, Bar Harbor, ME) to
generate CamDAT mice. In these mice, ectopic DAT expression
is driven by tTA-dependent tetO activity in a forebrain-specific
manner. In addition, DAT is also expressed in dopamine neurons
from the remaining wild-type allele.
To characterize DAT expression, homozygous tetO-DAT
mice were mated with hemizygous CaMKII
-tTA mice to gener-
ate CamDAT mice and TetDAT mice. All mice were kept on
dietary DOX (200 mg/kg rodent chow, from Bio-Serv) from ges-
tation until the experiments started, inhibiting ectopic DAT ex-
pression. DOX was then removed to induce ectopic DAT expres-
sion in adult mice. After 1 week of DOX withdrawal, ectopic DAT
mRNA expression was detected in the dor-
sal striatum, nucleus accumbens, olfactory
tubercle, olfactory bulb, cortex, and hip-
pocampus (for lower-power photomicro-
graph, see Fig. 1B; for a high-
magnification image at the cellular level,
see supplemental Fig. 1, available at www.
jneurosci.org as supplemental material).
Ectopically expressed DAT protein was
highly expressed in the striatum and mod-
erately expressed in the cortex and hip-
pocampus. As expected for a protein en-
riched in axon terminals, DAT was most
abundantly present in two main striatum
efferent nuclei, globus pallidus and sub-
stantia nigra pars reticulata, that receive
inputs from striatal D
-positive and D
positive medium spiny neurons, respec-
tively (Fig. 1C). DAT protein was also ex-
pressed on cell bodies of neurons in
striatum of CaMDAT mice (supplemental
Fig. 1, available at www.jneurosci.org as supplemental material).
We used Western blot analysis to quantify DAT protein levels. In
the forebrain, DAT protein levels in the CamDAT mice after 3
weeks of DOX withdrawal were approximately three times the
levels of DOX-treated mice (n ⫽ 4 per group, unpaired t test, p ⬍
Ectopic DAT overexpression in the forebrain significantly
altered dopamine turnover in the striatum
Figure 2 A illustrates a schematic of the predicted dopamine turn-
over in the CamDAT mice and WT mice, which is supported by
our HPLC data (Fig. 2B). In CamDAT mice after 3 weeks of DOX
withdraw, striatal tissue dopamine content decreased by 76%,
whereas DOPAC and HVA increased by 68 and 368%, respec-
tively. Similarly, in cerebral cortex, tissue dopamine decreased by
66%, whereas DOPAC and HVA increased by 38 and 192%, re-
spectively. In contrast, in the control tissue cerebellum, where
dopamine levels were 0.2% of the striatal level because of the lack
of dopamine input or DAT expression, dopamine levels were not
changed by DOX withdrawal, whereas DOPAC and HVA levels
increased by 31 and 47%, respectively. To exclude the possibility
that decreased dopamine level results from loss of dopamine neu-
rons in midbrain, we quantified dopamine neurons by unbiased
stereological cell counting. The dopamine neuron numbers in
SNc were comparable between the DOX group and no DOX
group of CamDAT mice (5938 ⫾ 535 and 6300 ⫾ 364, respec-
tively; data are given as mean ⫾ SEM).
The sharp decrease of dopamine and increases of DOPAC and
HVA in CamDAT mice suggest that postsynaptic neurons act as a
“sink” that quickly sequesters presynaptic dopamine (Fig. 2 A)
and that dopamine is turned over at a high rate, a phenomenon
that has been described in the DAT knock-out mice (Jones et al.,
1998). The increased dopamine metabolites in the cerebellum
suggest diffusions from dopamine terminals. This is consistent
with the observed increases of HVA and DOPAC in the cerebel-
L-DOPA-treated animals (el Gemayel et al., 1986).
Ectopic DAT overexpression in the forebrain led to motor
dysfunction, body weight loss, and lethality
Ectopic DAT expression in the forebrain had severe behavioral
consequences (Fig. 3A). If not treated with dietary DOX, Cam-
DAT pups were smaller, and most of them died ⬃1 week after
Figure 1. Generation of transgenic mice with forebrain expression of DAT (CamDAT mice). A, Inducible forebrain DAT expres-
sion mice were generated by breeding forebrain-specific CaMKII
-tTA mice and tetO-DAT mice. B, DAT mRNA expression was
detected in the striatum (STR), cerebral cortex (CTX), hippocampus (HIP), olfactory bulb (not shown), and olfactory tubercle (not
shown). Endogenous DAT mRNA was detected in SNc. In control mice (CTL), no DAT mRNA expression was detected in the
forebrain. C, DAT proteins were detected in regions with mRNA expression as well as in globus pallidus (arrow) and substantia
nigra pars reticulata (arrowhead) that are main striatum efferent nuclei. Top, Forebrain region shown in more rostral sections.
Bottom, Midbrain region shown in more caudal sections.
Chen et al. • Cytosolic DA Causes Neurodegeneration in Mice J. Neurosci., January 9, 2008 • 28(2):425– 433 • 427
weaning. If CamDAT mice were treated
with DOX from gestation stage on, they
were indistinguishable from TetDAT
mice. However, if DOX treatment was
withdrawn at any time point, most mice
died 4 – 8 weeks afterward (Fig. 3A).
We noted that lethality in CamDAT
mice after DOX withdrawal was preceded
by severe hypolocomotion and lack of
feeding. CamDAT mice developed se-
verely impaired locomotor activity after 2
weeks of DOX withdrawal (Fig. 3B). We
monitored animals’ body weight and food
intake throughout DOX withdrawal pe-
riod. Significant body weight loss was ob-
vious in CamDAT mice but not in TetDAT
(Fig. 3C) or CamDAT mice on DOX (data
not shown). Body weight loss was appar-
ently caused by lack of feeding (Fig. 3D).
However, when food intake was assessed
using easily accessible food (20 mg pellets
on the floor), CamDAT mice after 2 weeks
of DOX withdrawal (already underweight)
consumed more than the control group
(Fig. 3E). Therefore, the lack of feeding is
most likely a result of motor dysfunction
rather than the lack of appetite.
Neuropathology in forebrain DAT
To explore whether neuropathological
changes might underlie the behavioral ab-
normalities of CamDAT mice, mice were
perfused, and brain sections were immuno-
stained with a neuron-specific marker
(NeuN) after 4 weeks of DOX withdrawal.
We first measured the volume of striatum
and cortex as well as olfactory bulb. We
found significant striatal and cortical atro-
phy in CamDAT mice after DOX with-
drawal compared with those kept on DOX
(Fig. 4A,C). These areas have both ectopic
DAT expression and dopaminergic input. In
contrast, there was no change in the olfactory
bulb, which also has ectopic DAT expression
but has no significant dopamine afferents
(Fig. 4C). The dopamine neurons in the glo-
merular layer do not project to the granule
cell layer, where ectopic DAT expressing
neurons are localized.
We analyzed the striatal neuropathol-
ogy further by unbiased stereology counting of NeuN-positive
cells. We found 10% neuron loss in the striatum of CamDAT
mice after DOX withdrawal compared with those kept on DOX
(Fig. 4D). Although the neuronal loss strongly suggested dopa-
mine toxicity, the degree of the cell loss was not sufficient to account
for the behavioral changes. Therefore, we sought further evidence of
the neuronal dysfunction that precedes actual cell loss.
We investigated ultrastructural evidence for neurodegenera-
tion by transmission electron microscopy. Striatal neurons
showed various degrees of abnormal morphologies. Some af-
fected neurons only displayed dilated organelles, such as Golgi
apparatus and mitochondria. Other affected neurons displayed
prominent cytoplasmic organelle disintegration, although the
morphology of their nucleus remained relatively normal. The
more severely affected neurons displayed vacuolization and dis-
integration of the nucleus (Fig. 4 F). The latter two types of
pathology account for ⬃50% of striatal neurons. The typical
apoptotic morphology, such as cell shrinkage, chromatin con-
densation, membrane blebbing, and phagocytosis, was lacking.
We also used apoptotic markers to examine potential apoptosis.
Likewise, we found no increase of active caspase-3 or terminal
deoxynucleotidyl transferase-mediated biotinylated UTP nick
end labeling-positive neurons in CamDAT mice with ectopic
DAT expression (data not shown).
Figure 2. Alternation of dopamine turnover in CamDAT mice. A, A schematic illustration of genetically engineered synapse in
the striatum. Most dopamine released by presynaptic dopamine terminal is taken up by postsynaptic neurons expressing ectopic
DAT. There is no VMAT2 expression in postsynaptic neurons. Uptaken dopamine is degraded in cytoplasm and produces quinine,
, and other reactive species in postsynaptic neurons. B, After 3 weeks of DOX withdrawal, striatum and cortex dopamine
content decreased and its metabolites, DOPAC and HVA, increased. In cerebellum, dopamine level was unchanged, whereas HVA
and DOPAC levels increased. Note the differences in y-axis scales (n ⫽ 8 per group, unpaired t test, p ⬍ 0.001 for dopamine in
striatum, p ⬍ 0.001 for DOPACin striatum, p ⫽ 0.01 for HVA in striatum, p ⬍ 0.001 for dopamine in cortex, p ⫽ 0.018 for DOPAC
in cortex, p ⬍ 0.001 for HVA in cortex, p ⫽ 0.015 for DOPAC in cerebellum, p ⫽ 0.013 for HVA in cerebellum).
428 • J. Neurosci., January 9, 2008 • 28(2):425–433 Chen et al. • Cytosolic DA Causes Neurodegeneration in Mice
To explore further potential functional changes in striatal
neurons, we measured dynorphin and enkephalin mRNA expres-
sion, which are neuropeptides specifically found in D
-positive neurons, respectively. Semiquantitative radioac
tive in situ hybridization for dynorphin and enkephalin mRNA in
striatum did not reveal differences in expression levels 4 weeks
after DOX removal in CamDAT mice compared with control
group (supplemental Fig. 2, available at www.jneurosci.org as
supplemental material), suggesting that these peptidergic pheno-
types in D
-positive and D
-positive neurons were preserved,
although there were profound changes in morphology.
In addition to neurodegeneration, immunohistochemical
staining with an antibody against GFAP revealed obvious astro-
gliosis in striatum and cortex of CamDAT mice after 4 weeks of
DOX withdrawal but not in control mice (Fig. 4B). Although
-synuclein accumulation was observed in several PD models, we
did not find
-synuclein accumulation or upregulation in striatal
neurons by immunostaining and Western blot (supplemental
Fig. 3, available at www.jneurosci.org as supplemental material),
suggesting that neurodegeneration in CamDAT mice is likely to
Neurodegeneration was accompanied by oxidative protein
modifications and decreased tissue glutathione content
To further investigate the mechanism of neurodegeneration, we
examined evidence for oxidative stress. Dopamine undergoes ox-
idation to dopamine-quinone, which reacts with cysteine resi-
dues on proteins to form cysteinyl-dopamine and cysteinyl-
DOPAC conjugates (Hastings and Zigmond, 1994; Sulzer and
Zecca, 2000; Barzilai et al., 2003). To assess the abundance of
quinone-modified proteins, HPLC analyses of 5-cysteinyl-
dopamine and 5-cysteinyl-DOPAC levels were used to assess for
quinone modified protein (Hastings et al., 1996; LaVoie and
Hastings, 1999). We found significant increases of dopamine-
quinone by 527% and DOPAC-quinone by 149% evidenced by
modified cysteine residues in CamDAT mice after 3 weeks of
DOX withdrawal compared with animals kept on DOX. DOPA-
quinone modified cysteine residues were unchanged (Fig. 5A).
Similarly, in cerebral cortex, we found increases of dopamine-
quinone by 138% and DOPAC-quinone by 88% noted by mod-
ified cysteine residues. DOPA-quinone modified cysteine resi-
dues were unchanged (Fig. 5A). In contrast, in the cerebellum, no
change was found for dopamine, DOPA, or DOPAC-quinone
modified cysteine residues (Fig. 5 A).
In addition to reactive quinones, dopamine metabolism also
and superoxide, which may contribute to oxida
tive stress. Therefore, glutathione content was examined. Com-
pared with control mice, total glutathione (GSH⫹GSSG), GSH,
and GSSG content in striatum decreased by 26% ( p ⬍ 0.01), 29%
( p ⬍ 0.01), and 27% ( p ⬍ 0.05), respectively, in CamDAT mice
4 weeks after DOX withdrawal (n ⫽ 5), which alone could be suffi-
cient to cause mitochondria damage in brain (Jain et al., 1991). In
cerebral cortex and cerebellum, total glutathione (GSH⫹GSSG),
GSH, and GSSG content appeared normal (Fig. 5B).
L-DOPA accelerated neurodegeneration and body weight loss
If cytosolic dopamine is responsible for the aforementioned neu-
L-DOPA would be predicted to accelerate such a
process by boosting dopamine supply. Four weeks after DOX
withdrawal, we treated CamDAT mice with another 4 weeks of
daily intraperitoneal injections of 300 mg/kg
L-DOPA plus 100
mg/kg benserazide, and body weight was monitored.
treated CamDAT mice lost more body weight than saline control
(41.5 vs 25% by the end of fourth week, respectively, n ⫽ 5, p ⬍
0.001) (Fig. 6A). Unbiased stereological cell counting in striatum
revealed significant NeuN
cell loss in L-DOPA-treated Cam
DAT mice compared with saline-treated CamDAT mice (n ⫽ 4,
12% loss, p ⬍ 0.05) (Fig. 6 B).
Removing dopamine supply stopped progression of
Striatal neurons in CamDAT mice express ectopic DAT without
the capacity to synthesize dopamine, and therefore the accumu-
lation of cytoplasmic dopamine in these mice depends on supply
of extracellular dopamine. To assess whether the behavioral im-
pairment is dopamine dependent (therefore a putative dopamine
toxicity effect), we performed unilateral lesions of the medial
forebrain bundle by 6-hydroxydopamine before DOX with-
drawal to remove the major source of dopamine supply for stri-
One month after the lesion, CamDAT mice’s usage of individ-
ual forelimbs was assessed weekly by stepping behavior on a mo-
torized treadmill. Stepping of the contralateral forelimb con-
trolled by the lesion side was significantly impaired (16% of that
of intact side), consistent with our previous observation of motor
deficit resulting from dopaminergic deafferentation (Chang et
al., 1999). The 6-OHDA-lesion paradigm used in current study
damages ⬎90% dopamine neurons, which is confirmed by im-
munostaining with tyrosine hydroxylase antibody (data not
Figure 3. Behavioral phenotypes of CamDAT mice. A, DOX treatment was required for the
survival of CamDAT mice. B, In the open field, locomotor activity of CamDAT mice was normal 1
week after DOX withdrawal. However, their locomotor activity sharply declined in the second
weekof DOX withdrawal(n ⫽ 4 per group,repeated-measures ANOVA, p ⬍ 0.001). C, Progres-
sive body weight loss in CamDAT mice after DOX withdrawal (n ⫽ 6, repeated-measures
ANOVA, p ⬍ 0.001 for group ⫻ week interaction). D, Four weeks after DOX withdrawal, Cam-
DAT mice in their home cages consumed very little regular rodent chow (n ⫽ 5 per group,
individually housed, unpaired t test, p ⬍ 0.001). E, Two weeks after DOX withdrawal, when
provided with easily accessible chocolate flavored pellets (20 mg each) on the cage floor, Cam-
DAT mice ate more than TetDAT mice (n ⫽ 6 per group, repeated-measures ANOVA, p ⬍ 0.001
for group ⫻ week interaction).
Chen et al. • Cytosolic DA Causes Neurodegeneration in Mice J. Neurosci., January 9, 2008 • 28(2):425– 433 • 429
shown). After DOX withdrawal, stepping
of the ipsilateral forelimb controlled by the
intact side declined drastically (Fig. 7)
(stepping of the ipsilateral forelimb con-
trolled by the intact side before DOX with-
drawal was used as baseline). In contrast,
there was no further deterioration of the
stepping of the contralateral limb. In fact, a
gradual partial improvement of stepping
of the contralateral forelimb controlled by
the lesion side was noted (Fig. 7). These
data suggest that the stepping dysfunction
of the intact side after DOX withdrawal
was caused by uptake of released dopa-
mine from presynaptic terminals, and
such toxicity was spared in the lesion side,
where the source of dopamine was re-
moved. The partial improvement of the
contralateral limb is intriguing. There are
at least two possibilities. One is gradual be-
havioral adaptation after acute damage.
The second possibility is that forced use of
this particular limb after drastic deteriora-
tion of the other limb induced functional re-
covery from the lesion. Functional and ana-
tomical recovery after 6-OHDA lesion
through exercise or forced use in rodents has
been reported by other groups (Tillerson et
al., 2001; Dobrossy and Dunnett, 2003). To-
gether, the above data strongly suggest that
the behavioral abnormality in CamDAT
mice is dependent on dopamine supply.
Low-dose MPTP causes
neurodegeneration in the striatum
MPTP-treated mice usually develop dopamine neuron degener-
ation and are commonly used as PD models (Przedborski et al.,
2004). The selective loss of dopamine neurons in the MPTP
model depends on DAT expression in dopamine neurons (Gai-
netdinov et al., 1997; Donovan et al., 1999). Our data suggest that
CamDAT mice are especially vulnerable to dopamine-induced
neurodegeneration. To examine whether they are also especially
vulnerable to MPTP and to confirm that their ectopically ex-
pressed DAT is functional, MPTP was administrated to mice, and
neurodegeneration in striatum, cortex, and hippocampus was ex-
amined. Two weeks after DOX withdrawal, a single dose of MPTP
(30 mg/kg, i.p.) killed all seven CamDAT mice within 24 h, whereas
CamDAT mice kept on DOX remained active. Therefore, we low-
ered the dose of MPTP to 2 mg/kg and 5 mg/kg. All mice were alive
1 week after MPTP treatment. Using silver staining, degenerative
neurons in striatum were found in CamDAT mice treated with both
dosages of MPTP (Fig. 8). This type of degenerating neuron was not
found in saline-treated mice or in low-dose MPTP-treated Cam-
DAT mice kept on DOX. At this low dose of MPTP, no neurodegen-
eration was observed in the substantia nigra. These data suggest that
ectopically expressed DAT in striatal neurons are functional, and
these neurons are more susceptible to MPTP treatment than dopa-
mine neurons in substantia nigra, which have protective mecha-
nisms including the presence of VMAT2.
One important feature of the present in vivo model is that we were
able to isolate dopamine as the determining factor in causing
neurodegeneration. In our model, dopamine supply determines
the progression of neurodegeneration, which is accelerated by
L-DOPA treatment and attenuated by removing dopaminergic
afferents. Therefore, we provide definitive evidence that chronic
exposure to unregulated cytosolic dopamine alone is sufficient to
produce neurodegeneration in vivo. Moreover, severe neuropa-
thology and motor dysfunctions develop within weeks, making it
a practical model for studying mechanisms and testing therapeu-
In the transgenic mice, striatal neurons were engineered to
take up extracellular dopamine released from dopaminergic ter-
minals without acquiring intrinsic regulatory mechanisms typi-
cally found in dopamine neurons. Therefore, we were able to
isolate the contribution of dopamine without introducing other
confounding factors. In normal dopamine neurons, there are
major regulatory mechanisms that include the VMAT2 and reg-
ulated dopamine synthesis and activity. Those mechanisms
maintain cytosolic dopamine homeostasis and keep cytosolic do-
pamine within relatively nontoxic levels (Zigmond et al., 1989;
Kumer and Vrana, 1996; Eisenhofer et al., 2004). Therefore, it
may require decades of exposure to low levels of cytosolic dopa-
mine or may require impaired protective mechanisms for neuro-
pathology to take place.
How does cytosolic dopamine cause toxicity? Our data suggest
that the detrimental effects of dopamine in the present animal
model are mediated via dopamine metabolism and oxidation.
and other reactive oxidative species produced naturally in
dopamine degradation pathways may oxidize proteins, lipids,
and DNA, which ultimately cause cell dysfunction and neurode-
Figure 4. Neuropathology in the forebrain of CamDAT mice. A, C, Four weeks after DOX withdrawal, the volumes of cerebral
cortex and striatum decreased by 15 and 10% respectively, whereas volume of olfactory bulb was unchanged (n ⫽ 4 per group,
unpaired t test, p ⬍ 0.001 for both striatum and cortex). B, Astrogliosis was detected by GFAP staining in the cerebral cortex and
striatum. n ⫽ 6 per group. D, NeuN-positive cells in the striatum decreased by 10% (n ⫽ 4, unpaired t test, p ⫽ 0.04). E,
Transmission electron microscopic image of a normal striatal neuron from a CamDAT mouse under DOX treatment. F, Abnormal
morphologies were found in striatum of CamDAT mice after 4 weeks of DOX withdrawal. A severely affected neuron displayed
vacuolization (V) and disintegration of the nucleus (N). Arrowheads indicate the nuclear envelope. Scale bar, 2
430 • J. Neurosci., January 9, 2008 • 28(2):425–433 Chen et al. • Cytosolic DA Causes Neurodegeneration in Mice
generation (Stokes et al., 1999). Of particular importance is the
modification of cysteinyl residues by dopamine- and DOPAC-
quinones. Cysteinyl residues, the prime target of electrophilic
quinones, often reside at the active sites of proteins, and thus,
their covalent modifications by quinones could inactivate func-
tion, alter conformation, and promote aggregation of proteins
(Graham, 1978). One of the main defense mechanisms for such
detrimental effects is glutathione. Glutathione is ready to bind
dopamine- and DOPAC-quinones by their thiol group so that
other proteins are spared of their detrimental modifications. In
agreement with cytosolic dopamine toxicity in our transgenic
model, there are dramatic increases of 5-cysteinyl-dopamine and
5-cysteinyl-DOPAC and decrease of total glutathione, similar to
what have been found in PD patients (Schulz et al., 2000).
Dopamine toxicity-induced neurodegeneration may involve
-synuclein, including its oxidative modification. It has been
found that dopamine and other catechols inhibit
fibrillization and lead to accumulation of
brils. These include studies in both cell culture (Montine et al.,
1997; Conway et al., 2001; Cappai et al., 2005; Norris et al., 2005)
and in animal models (Li et al., 2004; Mazzulli et al., 2006). It has
been further shown that dopamine-autoxidized products but not
dopamine itself are responsible for modifying
mainly by noncovalent modification on
dopaminochrome, DA-quinone, DOPA-quinone, and DOPAC-
quinone. Therefore, we investigated the possibility that
-synuclein is a major mediator of dopamine toxicity, but did not
-synuclein expression or accumulation, suggesting
that neurodegeneration in CamDAT mice is likely to be
Our data show severe motor impairment after DOX removal
in CamDAT mice. Because striatal neuron loss is modest, striatal
neuron dysfunction is more likely to be the cause of their motor
impairment. Our EM ultrastructural data indicate that ⬃50%
Figure 5. Oxidative stress in transgenic mice. A, Oxidation-induced protein modification by dopamine. Using HPLC, we found significant increases of cys-dopamine and cys-DOPAC in CamDAT
mice after 3 weeks of DOX withdrawal. Similar increases were found in cerebral cortex. In contrast, no change was found in the cerebellum (n ⫽ 8 per group, unpaired t test, ***p ⬍ 0.001). B,
Decreased glutathione content in brain tissues. Total glutathione (GSH⫹GSSG), GSH, and GSSG were 26% ( p ⬍ 0.01), 29% ( p ⬍ 0.01), and 27% ( p ⬍ 0.05) lower, respectively, in striatum of
CamDATmice after 4 weeks of DOX withdrawal compared with controlmice (n ⫽ 5 per group,unpaired t test,*p ⬍ 0.05, **p ⬍ 0.01). No change was foundeither in thecortex or cerebellum( p ⬎
0.05, n ⫽ 5 per group).
Figure 6. L-DOPA accelerates neurodegeneration and body weight loss. Four weeks after
DOX withdrawal, CamDAT mice were injected daily with 300 mg/kg
L-DOPA plus 100 mg/kg
benserazide,andbody weightwasmonitored. A,
weight than saline control (41.5% vs 25%, respectively, n ⫽ 5 per group, unpaired t test, p ⬍
0.001). Day 0 indicates the first day of
L-DOPA treatment. Blank data points indicate the days
without treatment. B,
L-DOPA accelerated neurodegeneration in striatum assayed by unbiased
stereological cell counting (12% more loss of NeuN
neurons in L-DOPA-treated mice, n ⫽ 4
per group, unpaired t test, p ⫽ 0.03). *p ⬍ 0.05, **p ⬍ 0.01, ***p ⬍ 0.001.
Figure 7. Motor dysfunction in CamDAT mice is dopamine supply dependent. DOX treat-
ment was removed 1 month after unilateral 6-OHDA lesion, and stepping of the forelimb con-
trolled by the intact side worsened progressively, dropping from 100 to 15% of baseline level
during 4 weeks DOX withdrawal, whereas stepping of the forelimb controlled by the lesion side
improved from 16 to 50% of baseline level (n ⫽ 5 per group, repeated-measures ANOVA, p ⬍
0.001 for side ⫻ week interaction).
Chen et al. • Cytosolic DA Causes Neurodegeneration in Mice J. Neurosci., January 9, 2008 • 28(2):425– 433 • 431
striatal neurons have degenerative mor-
phologies. Low striatal dopamine content
might be another contributor. However,
our unilateral lesion data clearly indicate
that dopamine toxicity is a much more sig-
nificant contributor than dopamine defi-
ciency to the motor impairment pheno-
type in CamDAT mice.
The major pathological phenotype of
our mice bears some similarities to that of
models of Huntington’s disease, which is
characterized by neuropathology in stria-
tal medium spiny neurons. Interestingly,
extracellular dopamine has been shown to
worsen neuronal loss caused by mutant
huntingtin in both cultured cells and mice
models (Hastings et al., 1996; Jakel and
Maragos, 2000; Cyr et al., 2003, 2006;
Charvin et al., 2005; Tang et al., 2007). In
comparison, the CamDAT mouse is a cy-
tosolic dopamine toxicity model. There-
fore dopamine is likely to be a toxic agent,
whether elevated inside or outside of
Our data highlight the importance of cellular regulatory
mechanisms for cytosolic dopamine homeostasis in preventing
dopamine induced neurotoxicity and the importance of protec-
tive biochemical pathways such as glutathione. Under normal
conditions, those protective mechanisms along with others such
as the ubiquitin-proteasome system might work in concert to
prevent dopamine neurons from degeneration. However, the
detrimental effects of dopamine could be exaggerated under con-
ditions of genetic defects or environmental challenges. For exam-
ple, the disruption of dopamine vesicular storage by
has been suggested (Conway et al., 2001; Lotharius and Brundin,
2002; Cooper et al., 2006). DJ-1-deficient mice have been shown
to exhibit elevated DAT function and total tissue dopamine con-
tent (Chen et al., 2005). Parkin-deficient mice have been reported
to have increased extracellular dopamine (Bonifati et al., 2003;
Goldberg et al., 2003). Conversely, parkin has been reported to be
one of the targets modified by dopamine quinone (LaVoie et al.,
2005). Synuclein protofibrils can also be modified and stabilized
by catechols related to dopamine but not other compounds
(Conway et al., 2001).
Our results have significant clinical implications for the po-
tential detrimental effects of dopamine replacement therapies for
L-DOPA and some of the gene therapy approaches.
Results from the recent large-scale clinical trials are mixed.
L-DOPA improved the signs and symptoms of the disease, sug-
gesting neuroprotection. However, the same trial showed re-
duced uptake of DAT substrates, suggesting more rapid dopa-
mine terminal loss in
L-DOPA-treated patients (Rascol et al.,
2000; Parkinson Study Group, 2002; Whone et al., 2003; Hollo-
way et al., 2004; Fahn, 2005). Our
L-DOPA treatment data indi-
cate that under conditions of compromised defense mechanisms,
dopamine replacement therapies will accelerate neurodegenera-
tion. This point is also relevant for many of the PD gene therapy
approaches that involve the expression of tyrosine hydroxylase
L-amino acid decarboxylase either via direct in vivo
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(Kang et al., 2001; Liste et al., 2004). Virus-infected neurons
(mostly striatal neurons) and most engineered cells (e.g., fibro-
blasts) do not have the complete cellular machinery to protect
them from cytosolic dopamine toxicity. Therefore, reducing cy-
tosolic dopamine toxicity might be necessary to prevent any po-
tential damages associated with such therapies.
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Chen et al. • Cytosolic DA Causes Neurodegeneration in Mice J. Neurosci., January 9, 2008 • 28(2):425– 433 • 433
[Show abstract] [Hide abstract] ABSTRACT: The dyskinesia of Parkinson's Disease is most likely due to excess levels of dopamine in the striatum. The mechanism may be due to aberrant synthesis but also, a deficiency or absence of the Dopamine Transporter. In this study we have examined the proposition that reinstating Dopamine Transporter expression in the striatum would reduce dyskinesia. We transplanted c17.2 cells that stably expressed the Dopamine Transporter into dyskinetic rats. There was a reduction in dyskinesia in rats that received grafts expressing the Dopamine Transporter. Strategies designed to increase Dopamine Transporter in the striatum may be useful in treating the dyskinesia associated with human Parkinson's Disease.
- "This potential risk of overexpression of DAT would be best tested in non human primates where the bradykinesia can be more readily assessed. There is also a risk in using cell that do not normal transport dopamine into the cell that the dopamine will cause toxicity . While the possibility that a proportion of grafted DAT expressing cells degenerated because of dopamine toxicity cannot be discounted, it was not significant enough to obscure the observed improvement in dyskinesia. "
[Show abstract] [Hide abstract] ABSTRACT: The regulation of the dopamine transporter (DAT) impacts extracellular dopamine levels after release from dopaminergic neurons. Furthermore, a variety of protein partners have been identified that can interact with and modulate DAT function. In this study we show that DJ-1 can potentially modulate DAT function. Co-expression of DAT and DJ-1 in HEK-293T cells leads to an increase in [3H] dopamine uptake that does not appear to be mediated by increased total DAT expression but rather through an increase in DAT cell surface localization. In addition, through a series of GST affinity purifications and co-immunoprecipitations, we provide evidence that the DAT can be found in a complex with DJ-1, which involve distinct regions within both DAT and DJ-1. Using in vitro binding experiments we also show that this complex can be formed in part by a direct interaction between DAT and DJ-1. Co-expression of a mini-gene that can disrupt the DAT/DJ-1 complex appears to block the increase in [3H] dopamine uptake by DJ-1. Mutations in DJ-1 have been linked to familial forms of Parkinson's disease, yet the normal physiological function of DJ-1 remains unclear. Our study suggests that DJ-1 may also play a role in regulating dopamine levels by modifying DAT activity.
- "Therefore, a dysregulation of cytoplasmic DA levels could potentially generate pathological oxidative stress and may serve as a common pathway in both sporadic and familial forms of PD leading to nigral degeneration. Transgenic mice that either overexpress DAT  or exhibit low expression of VMAT2 , which would contribute to increased cytosolic DA levels, exhibit neurodegeneration in affected neurons. Thus, it is conceivable that altered DAT activity could facilitate pathological intracellular accumulation of DA or DA-like molecules, which would subsequently lead to the production of reactive free radical metabolites that are neurotoxic to dopaminergic cells. "
[Show abstract] [Hide abstract] ABSTRACT: The aim of this study was to evaluate chitosan (CS)-, glycolchitosan (GCS)- and corresponding thiomers-based nanoparticles (NPs) for delivering dopamine (DA) to the brain by nasal route. Thus, the polyanions tripolyphospate and sulfobutylether-β-cyclodextrin (SBE-β-CD), respectively, were used as polycation crosslinking agents and SBE-β-CD also in order to enhance the DA stability. The most interesting formulation, containing GCS and SBE-β-CD, was denoted as DA GCS/DA-CD NPs. NMR spectroscopy demonstrated an inclusion complex formation between SBE-β-CD and DA. X-ray photoelectron spectroscopy analysis revealed the presence of DA on the external surface of NPs. DA GCS/DA-CD NPs showed cytotoxic effect towards Olfactory Ensheathing Cells only at higher dosage. Acute administration of DA GCS/DA-CD NPs into the right nostril of rats did not modify the levels of the neurotransmitter in both right and left striatum. Conversely, repeated intranasal administration of DA GCS/DA-CD NPs into the right nostril significantly increased DA in the ipsilateral striatum. Fluorescent microscopy of olfactory bulb after acute administration of DA fluorescent-labeled GCS/DA-CD NPs into the right nostril showed the presence of NPs only in the right olfactory bulb and no morphological tissue damage occurred. Thus, these GCS based NPs could be potentially used as carriers for nose-to-brain DA delivery for the Parkinson's disease treatment. Copyright © 2015. Published by Elsevier B.V.
- "This may be in agreement with previous studies reporting the generation of toxic metabolites by DA autoxidation or through the action of monoamine oxidase, a mitochondrial enzyme. DA autoxidation leads to the production of toxic oxygen radicals and quinines causing neurodegeneration associated with oxidative stress585960. However, a correlation between the in vitro results to the in vivo studies is not simple, principally because no gross tissue damage was observed in the olfactory bulb of rats administered with DA GCS/DA CD (Fig. 8). "