Korean J Physiol Pharmacol
Vol 17: 89－97, February, 2013
ABBREVIATIONS: MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyri-
dine; BH4, (6R)-5,6,7,8-tetrahydrobiopterin; PD, Parkinson’s disease.
Received December 10, 2012, Revised December 28, 2012,
Accepted January 8, 2013
Corresponding to: Hak Rim Kim, Department of Pharmacology,
College of Medicine, Dankook University, 119, Dandaero, Cheonan
330-714, Korea. (Tel) 82-41-550-3935, (Fax) 82-41-551-3866, (E-mail)
This is an Open Access article distributed under the terms of the
Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial
use, distribution, and reproduction in any medium, provided the original work
is properly cited.
Differential Effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
on Motor Behavior and Dopamine Levels at Brain Regions in Three
Different Mouse Strains
Keun-Sung Lee, Jin-Koo Lee, Hyung-Gun Kim, and Hak Rim Kim
Department of Pharmacology, College of Medicine, Dankook University, Cheonan 330-714, Korea
Developing an animal model for a specific disease is very important in the understanding of the
underlying mechanism of the disease and allows testing of newly developed new drugs before human
application. However, which of the plethora of experimental animal species to use in model develop-
ment can be perplexing. Administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a
very well known method to induce the symptoms of Parkinson’s disease in mice. But, there is very
limited information about the different sensitivities to MPTP among mouse strains. Here, we tested
three different mouse strains (C57BL/6, Balb-C, and ICR) as a Parkinsonian model by repeated MPTP
injections. In addition to behavioral analysis, endogenous levels of dopamine and tetrahydrobiopterin
in mice brain regions, such as striatum, substantia nigra, and hippocampus were directly quantified
by liquid chromatography-tandem mass spectrometry. Repeated administrations of MPTP significantly
affected the moving distances and rearing frequencies in all three mouse strains. The endogenous
dopamine concentrations and expression levels of tyrosine hydroxylase were significantly decreased
after the repeated injections, but tetrahydrobiopterin did not change in analyzed brain regions.
However, susceptibilities of the mice to MPTP were differed based on the degree of behavioral change,
dopamine concentration in brain regions, and expression levels of tyrosine hydroxylase, with C57BL/6
and Balb-C mice being more sensitive to the dopaminergic neuronal toxicity of MPTP than ICR mice.
Key Words: Dopamine, Mass spectrometry, Mouse strains, MPTP, Parkinson’s disease
A good experimental animal model is vital to understand
the underlying mechanisms of the particular disease [1,2].
A suitable experimental animal model is also an ideal plat-
form for the testing of newly developed drugs prior to hu-
man use for the possible cure of the target disease [2,3].
The neurotoxic effect of 1-methyl-4-phenyl-1,2,3,6-tetra
hydropyridine (MPTP) on dopaminergic neurons at sub-
stantia nigra has been established in various animals [3,4].
MPTP has been used to induce symptoms of Parkinson’s
disease (PD) in a number of animals. Repeated MPTP ad-
ministration over several days produces a Parkinsonian
syndrome, such as decreased excise abilities, neurochemical
changes, and histopathological differences .
The active accumulation of 1-methyl-4-phenylpyridinium
(MPP+) in dopaminergic terminals via the dopamine trans-
porter occurs because of the specific neurotoxic action of
MPTP on dopaminergic neurons [6,7]. The oxidation of
MPTP to MPP+ by monoamie oxidase B (MAO-B) is essen-
tial to the neurotoxic effect, especially at the substantia
nigra. It has been suggested that the toxic effects of MPP+
are related to its accumulation within mitochondria , its
inhibition of mitochondrial respiration at complex I [9,10],
and the depletion of ATP in the affected cells . The en-
dogenous levels of dopamine, as a biogenic amine, are in-
volved in emotion, reward systems, and motor control in
human [12,13]. Tetrahydrobiopterin is an essential cofactor
of tyrosine hydroxylase, a rate limiting enzyme for syn-
thesis for dopamine synthesis [14,15].
Differences in susceptibility to MPTP by different animal
species have been recognized [6,7,16]. Mice are sensitive to
the neurotoxic effects of MPTP, with primates being more
sensitive [17,18]. Although several mouse strains have been
used for the development of a Parkinsonian animal model
by the repeated injection of MPTP [6,7,16,19], the differ-
ential susceptibilities between mouse strains following
MPTP injection as measured by behavior changes and al-
teration in the content of the endogenous dopamine, tetra-
KS Lee, et al
hydrobiopterin, and tyrosine hydroxylase have not been
In this study, we evaluated three mouse strains in
MPTP-induced PD by determining the endogenous concen-
tration of tetrahydrobiopterin and dopamine, and the ex-
pression levels of tyrosine hydroxylase in brain regions. We
directly quantified the endogenous levels of dopamine and
tetrahydrobiopterin at the striatum three commonly-used
mice strains − C57BL/6, Balb-C, and ICR − using liquid
chromatography-tandem mass spectrometry (LC-MS/MS)
after MPTP administration . We also measured the
striatum expression levels of tyrosine hydroxylase in the
mouse strains and evaluated behavioral changes after sub-
cutaneous (s.c.) MPTP administration using the Rota-rod
test and an open field behavior recording system. The re-
sults revealed differences in behavior and dopamine level
in the brains of the mouse strains.
(6R)-5,6,7,8-Tetrahydrobiopterin dihydrochloride (BH4
2HCl), dopamine hydrochloride (dopamine HCl), MPTP,
monoclonal anti-tyrosine hydroxylase antibody, and mono-
clonal anti-β-actin antibody were purchased from Sigma-
Aldrich (St. Louis, MO, USA). Epilson-acetamidocaproic
acid (AACA) was donated by Kuhnil Pharmaceuticals
(Seoul, Korea). T-PERⓇ tissue protein extraction reagent
and protease inhibitor cocktail were purchased from
Thermo Scientific (St. Louis, MO, USA). IRDyeⓇ 800CW
conjugated goat (polyclonal) anti-mouse IgG was purchased
from LI-CORⓇ Biosciences (Lincoln, NB, USA).
Animal treatment and sample preparation
All procedures in this study were performed according to
protocol approved by the Institutional Animal Care and Use
Committee at Dankook University. Six-week-old C57BL/6,
Balb-C, and ICR mice were purchased from Daehan-Bio
Link (Seoul, Korea). They were housed in cages in groups
of 10 or 11 animals under controlled environmental con-
ditions (23±2oC; relative humidity 50±10%; 12:12-h light-
dark cycle until the initiation of the experiment; food and
water ad libitum) before use in experiments. Each mouse
was injected subcutaneously (s.c) with MPTP (20∼30
mg/kg). After testing by Rota-rod and open field test sys-
tems as detailed below, mice were killed by cervical dis-
location, and each brain was quickly retrieved. The stria-
tum, substantia nigra, and hippocampus were quickly dis-
sected on ice under a dissecting microscope. The dissected
brain regions were quick-frozen in a deep-freezer (−80oC)
until further analysis.
Preparation of stock solution, calibration standards, quality
control, and sample preparation for LC-MS/MS system
were done as previously described . The liquid chroma-
tography system was an Accela system (Thermo Fisher
Scientific, Waltham, MA, USA) equipped with a nanospace
SI-2 3133 solvent delivery module as an auto-sampler
(Shiseido, Tokyo, Japan), and connected to a Discovery Max
(Thermo Fisher Scientific) quadrupole tandem mass spec-
trometer coupled with electrospray ionization (ESI-MS/MS).
Chromatographic separation was achieved by using hydro-
philic interaction chromatography (HILIC) using a Sepax
Polar-Imidazole (2.1×100 mm internal diameter, 3μm par-
ticle size) high-pressure liquid chromatography (HPLC) col-
umn (Sepax Technologies, Newark, DE, USA) with a 4×2
mm C18 guard column (Phenomenex, Torrance, CA, USA).
The ESI mass spectrometer was operated in the positive
ion mode. Multiple reaction monitoring (MRM) of the pre-
cursor-product ion transitions was m/z 242.1 → m/z 166.0
for BH4, m/z 154.1 → m/z 90.0 for dopamine, and m/z 174.1
→ m/z 114.0 for AACA. The optimized ESI conditions could
sensitively detect BH4, dopamine, and IS with positive ion
detection mode. The most abundant protonated ion peaks
in the Q1 mass spectra of BH4 and dopamine were at 242.1
m/z and 154.1 m/z, respectively. There was no evidence of
fragmentation and adduct formation. The product ions
([M+H]+) in the Q3 mass spectra and proposed fragmenta-
tion patterns were BH4 at m/z 166.0 (2-amino-7,8-dihy-
dropteridin-4(1H)-one) by loss of propane-1,2-diol, and dop-
amine at m/z 90.9 (butane-1,2-diol) by loss of (E)-3-methyl-
pent-3-en-1-amine. The interpretation and analysis of data
followed previously published methods .
Mice were trained for two consecutive days to stay on
a rota-rod (Ugo Basile, Comerio VA, Italy) at least for 600
seconds (16 rpm, fixed speed) before the start of testing.
On the test day, baseline data was collected and then mice
were evaluated for their latency to fall after s.c admin-
istration of 20∼30 mg/kg of MPTP. Rota-rod testing was
performed twice per mouse with an intervening interval of
at least 10 minutes. The times in seconds from the two tests
Open field behavior test
Each mouse was put in a white or black plastic rec-
tangular box (40×27×27 cm) after s.c administration of 20∼
30 mg/kg MPTP. The mice injected with the same volume
of saline were used as the control group. The mice were
observed with a CCD camera connected to a recording
system. The recorded behavior was analyzed by EthoVision
Version 2.3 (Noldus information Technology, Wageningen,
The Netherlands). An automated video tracking system
was used for calculation of moving distances and rearing
frequencies. The mice were observed for 6 hours in normal
lighting. The white or black plastic rectangular box was
cleaned with 70% alcohol and water between trials.
Quick-frozen tissue samples were homogenized and ana-
lyzed via immunoblotting as previously described .
Briefly, the homogenized samples were boiled in Laemmli
sample buffer and the proteins separated by 10% sodium
dodecyl sulfate-polyacrylamide gel electrophoresis. The re-
solved proteins were transferred to a polyvinylidene di-
fluoride membrane (Millipore, Billerica, MA, USA). Protein
transfer was confirmed by staining membranes with Pon-
ceau Red. Each membrane was incubated with an appro-
priate primary antibody for overnight at 4oC. After ex-
tensive washing with Tris-buffered saline containing Tween
20, each membrane was incubated with the appropriate sec-
Mouse Model of Parkinson’s Disease
Fig. 1. Effects of MPTP (30 mg/kg, s.c)
on Rota-rod performance. Animals were
trained to stay on an operating Rota-rod
for 600 seconds. On the test day, animals
were evaluated on the Rota-rod 6 and 24
hours after MPTP administration. (A)
MPTP (C57BL/6, 30 mg/kg, s.c.), (B)
MPTP (ICR, 30 mg/kg, s.c.), (C) MPTP
(C57BL/6, ICR, Balb C, 20 mg/kg, s.c.),
(D) body weight after MPTP admini-
stration. Data are expressed as mean
latency to fall off the Rota-rod (vertical
bars represent standard error of the
mean). *p＜0.05, **p＜0.01 vs 0 hour
ween 6 hours and 24 hours groups by
Student's t-test, n=6∼10.
#p＜0.05 compared bet-
Fig. 2. Summary of experimental design. Each groups received
MPTP treatment for 3 days followed by one MPTP-free day. At the
end of behavioral test of day 4, animals were euthanized for brain
ondary antibody. Densitometry of protein bands was per-
formed with the software for Odyssey Infrared Imaging
System (LI-COR Biosciences).
All values given in the text are mean±SEM. Differences
between means were evaluated using a Student’s t-test. For
comparison between strains, Two-way ANOVA with the fac-
tors “strain” (ICR, Balb C, and C57BL/6) and “day after
treatment” (1, 2, and 3 days) were computed using Prism
(version 5.0) (GraphPad Software, San Diego, CA, USA).
Post-hoc analysis of significant differences was performed
using Bonferroni post-tests for multiple comparision. Stati-
stical significant differences were taken at the p＜0.05
level. The n values given represent numbers of tissues or
animals used in each experiment.
Rota-rod performance is decreased but recovers after
C57BL/6 mice were trained to stay on the Rota-rod for
600 seconds when the system was operating at a fixed rate
of 16 rpm. The trained animals were injected with MPTP
(30 mg/kg s.c.) and then evaluated their latency to fall off
the Rota-rod. Fall latency 6 hours after injection was sig-
nificantly decreased (Fig. 1A). The decreased latency per-
sisted until 24 hours post-injections, although the decrease
was significantly greater at 6 hours than at 18 hours (Fig.
1A). Similar findings were obtained with ICR mice (Fig.
1B). However, the latency to fall of ICR mice was greater
than for C57BL/6 mice 24 hours after injection.
KS Lee, et al
Fig. 3. Effect of MPTP (20 mg/kg, s.c) on moving distance for 1
hour following MPTP administration. (A) C57BL/6 mice, (B) Balb
C mice, (C) ICR mice. Data are expressed as accumulated total
moving distances in cages (vertical bars represent standard error
of the mean). *p＜0.05, **p＜0.01, ##p＜0.01 vs day 0 (n=6∼10).
Fig. 4. Effect of MPTP (20 mg/kg, s.c) on total rearing frequencies
until 1 hour after MPTP administration. (A) C57BL/6 mice, (B) Balb
C mice, (C) ICR mice. Data are expressed as accumulated number of
rearing frequencies in cages (vertical bars represent standard error
of the mean). *p＜0.05, **p＜0.01 vs day 0 (n=6∼10).
Multiple injections of MPTP (30 mg/kg, s.c.) were given
at 24 hour intervals and mice were tested their behavioral
changes (Fig. 2). This treatment produced extensive mortal-
ity and the experiment was terminated day 2, so the MPTP
dosing regimens have to be changed. To limit mortality, the
three different mouse strains were treated with a lower
dose of MPTP (20 mg/kg, s.c), and with multiple injections
of this lower dose, and were evaluated for their latency to
fall on the Rota-rod. However, the injection of lower dose
of MPTP (20 mg/kg, s.c.) didn’t decrease the latency on ro-
ta-rod at 24 hours after MPTP injection (Fig. 1C). Moreover,
multiple injection of MPTP (20 mg/kg, s.c.) at 24 hours in-
tervals for 3 days didn’t significantly decrease latency to
fall on a rota-rod (data not shown). There were no sig-
nificant changes in the body weights of the examined mice
during the 3-day period of MPTP injection and 24 hours
after MPTP injection in all three mouse strains (Fig. 1D).
MPTP injections decrease moving distances and rear-
We further evaluated the mice after MPTP injections
with alternative methods to see whether the lower dose of
MPTP was insufficient to be toxic to dopaminergic neurons
or whether the Rota-rod test was not sensitive enough to
detect behavioral changes. To assess the behavioral effects,
the movements of C57BL/6, Balb C, and ICR mice after in-
jecting MPTP (20 mg/kg, s.c.) were evaluated by the open
field test for 6 hours. The total moving distances recorded
and analyzed until 1 hour after MPTP injection were sig-
Mouse Model of Parkinson’s Disease
Table 1. Total moving distances after MPTP injection
C57BL/6Balb C ICR
3 hrs (m)6 hrs (m)3 hrs (m)6 hrs (m)3 hrs (m)6 hrs (m)
The values are mean of 6 individual animals (mean±standard error of the mean). m, meter; *p＜0.05, **p＜0.01, ##p＜0.01 vs day
Table 2. Rearing frequencies after MPTP injection
C57BL/6Balb C ICR
3 hrs (n)6 hrs (n) 3 hrs (n)6 hrs (n)3 hrs (n) 6 hrs (n)
The values are mean of 6 individual animals (mean±standard error of the mean). n, frequency number; *p＜0.05, **p＜0.01, ##p＜0.01
vs day 0.
Fig. 5. Comparison of moving distances
and total rearing frequencies among
three mouse strains Effect of MPTP (20
mg/kg, s.c) on moving distances (A) and
total rearing frequencies (B) until 1 hour
after MPTP administration over three
days. In each case, the mean results
were normalized by the mean of day 0.
Vertical bars represent standard error of
the mean and negative SE bars have
been deleted for clarity. Two-way
ANOVA, *p＜0.05, **p＜0.01 vs ICR
nificantly decreased at day 2 and day 3 in C57BL/6 mice
(Fig. 3A). However, the moving distances were still recov-
ered after 24 hours (day 4) similarly as day 0 if the mice
were not treated with MPTP (Fig. 3A).
In parallel, Balb C mice were similarly tested. The total
moving distances were significantly decreased from day 1
and further decreased following the repeated injection of
MPTP (20 mg/kg, s.c.). Moreover, the total moving dis-
tances at day 4 (without MPTP injection) were markedly
increased by two-fold compared with day 0 (Fig. 3B). The
observations were consistent with the hyperactivity of the
mice at day 4. Of interest, ICR mice did not respond well
at the lower dose of MPTP (20 mg/kg, s.c.) in the same ex-
periments and showed no specific changes at day 4 (Fig. 3C).
Concerning rearing frequencies, all three strains of mice
responded well to the MPTP injection (Fig. 4). The rearing
frequencies until 1 hour after MPTP injection were sig-
nificantly decreased from day 1 and its effects lasted until
day 3. But, this decrease observed with multiple MPTP in-
jections recovered after 24 hours (day 4) with a frequency
similar to that noted on day 0 if the mice were not injected
with MPTP (Fig. 4).
We further analyzed the data until 3 hours and 6 hours
in the same experiments. The results showed a very similar
pattern at both times in the total moving distances (Table
1) and rearing frequencies (Table 2). The total moving dis-
tances were significantly decreased following the repeated
injection of MPTP (20 mg/kg, s.c.) in both C57BL/6 and Balb
C mice. At the same time, the total moving distances at
day 4 (without MPTP injection) were recovered to similar
distances . Similarly, ICR mice did not display sig-
nificant differences at 3 hours or 6 hours evaluation when
the lower dose of MPTP was used. Interestingly, Balb C
mice at day 4 moved an extended distance (up to 3-fold)
compared to day 0. These data indicated that mice, espe-
cially Balb C mice, are hyperactive during the recovery time
KS Lee, et al
Fig. 6. Effects of MPTP (20 mg/kg, s.c) on
dopamine concentration in mouse brain
section. The endogenous level of dopa-
mine in mice brain scetions ((A) stria-
tum, (B) substantia nigra, (C) hippo-
campus) were measured by LC-MS/MS
in three different mice species. (D) in-
ternal calibration for dopamine. Data is
expressed as mean±s.e.m. *p＜0.05, **p
at day 4 (Table 1).
All three strains of mice displayed significantly decreased
rearing frequencies from day 1 to day 3, but still showed
robust recovery of activities at day 4 (Table 2). Especially,
the rearing frequencies at day 4 were markedly increased
up to 5-fold in ICR mice. But, there were no increased rear-
ing activities at day 4 either in C57BL/6 or Balb C mice.
These data suggest that MPTP can differentially affect the
rearing behavior of different mouse strains.
Although, repeated administrations of MPTP signifi-
cantly affected the moving distances and rearing frequen-
cies in all three mouse strains, we further analyzed the da-
ta to see mice strains are differently susceptible (Fig. 5).
In both parameters, ICR strain is statistically less sensitive
to MPTP than C57BL/6 or Balb C strains.
MPTP injections decrease endogenous dopamine levels,
but not tetrahydrobiopterin, in mouse brain
MPTP destroys dopaminergic neurons in the substantia
nigra, which causes Parkinsonism [11,16]. Recently, we re-
ported a direct detection method for tetrahydrobiopterin
(BH4) and dopamine in rat brain using LC-MS/MS .
Dopamine and BH4 were presently measured in brain re-
gions of the three strains of mice after administration of
MPTP (Fig. 6 and 7). Three injections of MPTP (20 mg/kg
s.c.) for a 24 hours interval resulted in an approximately
60% decrement of dopamine in the striatum in all three
strains (Fig. 6A). In parallel, endogenous levels of dopamine
in the three strains of mice were measured in the sub-
stantia nigra (Fig. 6B) and in the hippocampus (Fig. 6C).
The three injections of MPTP did not statistically affect the
substantia nigra in any of the strains (Fig. 6B). But, the
dopamine concentration in the hippocampus of C57BL/6
mice was slightly decreased by the multiple MPTP in-
jections (Fig. 6C).
BH4 is an essential cofactor for dopamine synthesis
through its action on dopamine hydroxylase. BH4 has been
intensively studied concerning the development and pro-
gression of PD [23-26]. The endogenous levels of BH4 in
brain section were measured after three doses of MPTP (20
mg/kg s.c.) delivered at 24 hours intervals. No statistically
significant differences were evident in the striatum, sub-
stantia nigra, and hippocampus among the three strains
of mice (Fig. 7). These data suggested that the admin-
istration of MPTP can decrease the endogenous level of dop-
amine in mouse brain by destroying dopaminergic neurons,
mainly at striatum, with no effect on BH4 levels in mouse
MPTP injections decrease expression of tyrosine hydro-
xylase in mouse striatum
Based on the assay of dopamine and tetrahydrobiopterin
at striatum, substantia nigra, and hippocampus by
LC-MS/MS, the dopamine concentration was greatest in the
striatum. To ascertain the activity of tyrosine hydroxylase
following MPTP administration, we quantified the total ex-
pression levels of tyrosine hydroxylase at striatum in the
three strains of mice (Fig. 8). The expression levels of ty-
Mouse Model of Parkinson’s Disease
Fig. 7. Effects of MPTP (20 mg/kg, s.c)
on tetrahydrobiopterin (BH4) concentra-
tion in brain regions. The endogenous
level of BH4 in mice brain regions ((A)
striatum, (B) substantia nigra, (C) hip-
pocampus) were measured by LC-MS/MS
in three different mice strains. (D) Inter-
nal calibration for BH4. Data is ex-
pressed as mean±standard error of the
Fig. 8. Effects of MPTP (20 mg/kg, s.c) on the expression of tyrosine
hydroxylase in the striatum in the three strains of mice. (A)
Representative immunoblots of tyrosine hydroxylase (TH) and β-
actin expression. (B) Densitometry plot of TH expression immu-
noblots normalized to the control levels, with and without MPTP
treatment (n=4). *p＜0.05 and **p＜0.01 vs. black bar (control
rosine hydroxylase in the striatum were significantly de-
creased by the systemic administration of MPTP (20 mg/kg,
s.c.) in all three strains. However, the tyrosine hydroxylase
decrease was least for ICR mice.
Development of an animal model for the specific disease
under investigation is very important to further the under-
standing of the underlying mechanism of disease, and pro-
vides the chance to test newly developed new drugs before
their human application [1,2]. However, the plethora of ex-
isting animal models can make the decision of the most
appropriate model challenging. It is true for investigating
the interactions in brain on the pharmacological and/or tox-
icological effects of certain agents that link to delineate
neural mechanisms underlying in a specific disease .
PD is a progressive neurodegenerative disease caused by
the destruction of dopaminergic neurons in the substantia
nigra . The underlying mechanisms are still not fully
understood . Moreover, the current approach to the treat-
ment of PD involves suppressing disease progression rather
than achieving a cure . This approach is unsatisfactory,
given the high prevalence of PD worldwide, with its con-
comitant morbidity and mortality; improved understanding
of the underlying mechanisms and novel therapeutics are
Neuromelanin-containing dopamine neurons in the sub-
stantia nigra projecting to the striatum in the brain are
KS Lee, et al
selectively degenerated in this disease . In PD animal
model, several agents effective against dopaminergic neu-
rons are available . Of these, the administration of
MPTP is an established and valid method to induce PD
symptoms in mice [3,4]. The effect of the parkinsonism-in-
ducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropy-
ridine on central catecholamine neuron in C57BL/6 mice
revealed that MPTP caused a severe reduction of endoge-
nous DA in substantia nigra and striatum which was fol-
lowing by an increase in the 3,4-dihydroxyphenylacetic acid
(DOPAC)/ dopamine ratio . But, little is known about
MPTP susceptibilities between different mice strains.
C57BL/6, Balb-C, and ICR strains of mice, among others,
have been used to develop disease models. However, there
is limited information about the different responses among
those mice strains after injection of MPTP, which hinders
research concerning the underlying mechanisms of PD.
In this study, we evaluated behavioral changes after sub-
cutaneous injection of MPTP using the Rota-rod test and
open field behavior recording. In parallel, we evaluate the
MPTP-induced mouse model of PD by determining the lev-
els of BH4 and dopamine in brain regions of three selected
mouse strains. BH4 is an essential cofactor for hydrox-
ylation of cyclic amino acids including dopamine catalyzed
in a rate limiting fashion by tyrosine hydroxylase [14,15].
Recently, we reported a novel method for direct detection
of BH4 and dopamine in rat brain using LC-ESI-MS .
Here, the method was applied to the MPTP-induced mouse
models of PD to monitor the changes of dopamine concen-
tration and the levels of endogenous BH4. We directly
quantified the endogenous levels of dopamine in ICR,
C57BL/6, and Balb-C mouse strains using LC-MS/MS. The
changes of neurotransmitter concentration in several brain
regions are influential in the development of a variety of
psychiatric and neurodegenerative diseases [25,31]. There-
fore, it is clearly necessary to precisely determine the level
of dopamine as a means of diagnostically evaluating PD
and for the screening of potential therapeutic products to
modulate dopamine levels .
Presently, repeated administration of MPTP affected ex-
ercise abilities of mice, including moving distances and
rearing frequencies. However, the generally used methods
for evaluation of motor-activities appeared to be too in-
sensitive to detect the fine behavioral changes in the
Parkinsonian mice models. The endogenous dopamine con-
centrations were significantly decreased after repeated
MPTP injection, but BH4 was unchanged in mouse brain
regions. In addition, the expression levels of tyrosine hy-
droxylase in the striatum were significantly decreased by
MPTP injection. These results suggest that the decreased
levels of dopamine in the striatum are mainly due to the
extensive damage of dopaminergic neurons in this region.
Consistent with previous reports [3,6,7,33], marked differ-
ences were evident in the sensitivity of the three strains
of mice to MPTP, although the reasons for the strain-re-
lated differences in response to MPTP remain unclear. The
fundamental difference in the sensitivity to toxic MPTP
may be one possibility.
In conclusion, we tested the effects of repeated MPTP in-
jedctions, an animal model of Parkinson’s disease, in three
strains of mice on rota-rod performance, locomotor activity,
as well as striatal, hippocampal, and substantia nigra lev-
els of dopamine and tetrahydrobiopterin and striatal levels
of tyrosine hydroxylase. Our results showed that the ICR
strain was generally less sensitive to MPTP on rota-rod per-
formance, locomotor suppression, and striatal tyrosine hy-
droxylase suppression. We found that C57BL/6 and Balb-C
mice were more sensitive to the dopaminergic neuronal tox-
icity of MPTP than was ICR mice based on open field test,
tyrosine hydroylase expression.
The present research was conducted by the research fund
of Dankook University in 2010.
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