Zonisamide ameliorates levodopa-induced dyskinesia and reduces expression of striatal genes in Parkinson model rats

Abstract

To investigate the difference in results according to the mode of levodopa administration and the effect of zonisamide (ZNS), we analyzed the mRNA expression of dopaminergic and non-dopaminergic receptors in the striatum of Parkinson model rats in relation to the development of levodopa-induced dyskinesia (LID). Unilateral Parkinson model rats were subdivided into 4 groups and treated as follows: no medication (group N), continuous levodopa infusion (group C), intermittent levodopa injection (group I), and intermittent levodopa and ZNS injection (group Z). Two weeks after the treatment, LID was observed in group I and Z, but less severe in group Z. The level of both D1 and D2 receptor mRNAs was elevated in groups I and Z, but only D2 receptor mRNA expression was elevated in group C. Adenosine A2A receptor mRNA showed increased expression only in group I. The level of endocannabinoid CB1 receptor mRNA was elevated in groups N, C, and I, but not in group Z. Intermittent injection of levodopa caused LID, in association with elevated expression of D1 and A2A receptors. ZNS ameliorated the development of LID and inhibited up-regulation of A2A and CB1 receptors. Modulation of these receptors may lead to therapeutic approaches for dyskinesia.

Full text

Please
cite
this
article
in
press
as:
Oki,
M.,
et
al.,
Zonisamide
ameliorates
levodopa-induced
dyskinesia
and
reduces
expression
of
striatal
genes
in
Parkinson
model
rats.
Neurosci.
Res.
(2017),
http://dx.doi.org/10.1016/j.neures.2017.04.003
ARTICLE IN PRESS
G Model
NSR-4033;
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of
Pages
6
Neuroscience
Research
xxx
(2017)
xxx–xxx
Contents
lists
available
at
ScienceDirect
Neuroscience
Research
jo
ur
nal
homepage:
www.elsevier.com/locate/neures
Zonisamide
ameliorates
levodopa-induced
dyskinesia
and
reduces
expression
of
striatal
genes
in
Parkinson
model
rats
Mitsuaki
Okia,
Satoshi
Kanekoa,∗,
Satoshi
Morisea,
Norihiro
Takenouchib,
Takanori
Hashizumec,
Ayako
Tsugea,
Masataka
Nakamuraa,
Reika
Watea,
Hirofumi
Kusakaa
aDepartment
of
Neurology,
Kansai
Medical
University,
Osaka,
Japan
bDepartment
of
Microbiology,
Kansai
Medical
University,
Osaka,
Japan
cLaboratory
of
Drug
Metabolism
&
Pharmacokinetics,
Faculty
of
Pharmacy,
Osaka
Ohtani
University,
Osaka,
Japan
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
1
February
2017
Received
in
revised
form
29
March
2017
Accepted
12
April
2017
Available
online
xxx
Keywords:
Parkinson’s
disease
Levodopa-induced
dyskinesia
Zonisamide
Adenosine
Endocannabinoid
a
b
s
t
r
a
c
t
To
investigate
the
difference
in
results
according
to
the
mode
of
levodopa
administration
and
the
effect
of
zonisamide
(ZNS),
we
analyzed
the
mRNA
expression
of
dopaminergic
and
non-dopaminergic
receptors
in
the
striatum
of
Parkinson
model
rats
in
relation
to
the
development
of
levodopa-induced
dyskinesia
(LID).
Unilateral
Parkinson
model
rats
were
subdivided
into
4
groups
and
treated
as
follows:
no
medi-
cation
(group
N),
continuous
levodopa
infusion
(group
C),
intermittent
levodopa
injection
(group
I),
and
intermittent
levodopa
and
ZNS
injection
(group
Z).
Two
weeks
after
the
treatment,
LID
was
observed
in
group
I
and
Z,
but
less
severe
in
group
Z.
The
level
of
both
D1
and
D2
receptor
mRNAs
was
elevated
in
groups
I
and
Z,
but
only
D2
receptor
mRNA
expression
was
elevated
in
group
C.
Adenosine
A2A
receptor
mRNA
showed
increased
expression
only
in
group
I.
The
level
of
endocannabinoid
CB1
receptor
mRNA
was
elevated
in
groups
N,
C,
and
I,
but
not
in
group
Z.
Intermittent
injection
of
levodopa
caused
LID,
in
association
with
elevated
expression
of
D1
and
A2A
receptors.
ZNS
ameliorated
the
development
of
LID
and
inhibited
up-regulation
of
A2A
and
CB1
receptors.
Modulation
of
these
receptors
may
lead
to
therapeutic
approaches
for
dyskinesia.
©
2017
Elsevier
Ireland
Ltd
and
Japan
Neuroscience
Society.
All
rights
reserved.
1.
Introduction
Dopaminergic
replacement
therapy
is
a
basic
strategy
for
the
treatment
of
Parkinson’s
disease
(PD).
The
development
of
motor
complications
such
as
wearing-off
and
dyskinesia
along
with
the
disease
progression
has
a
negative
impact
on
the
quality
of
life
of
PD
patients
(Chapuis
et
al.,
2005),
and
it
also
interferes
with
the
treatment.
It
has
been
clinically
noticed
that
the
mode
of
levodopa
administration
should
be
continuous
rather
than
intermittent
to
delay
or
reduce
the
development
of
dyskinesia
(Olanow
et
al.,
2006;
Stocchi
et
al.,
2008).
Intermittent
administration
of
high-
dose
levodopa
to
animal
models
induces
dyskinesia
more
strongly
than
continuous
or
low-dose
administration
(Tsironis
et
al.,
2008).
Many
non-dopaminergic
neurotransmitters
have
been
implicated
∗Corresponding
author.
E-mail
address:
kanekosa@takii.kmu.ac.jp
(S.
Kaneko).
in
modulation
of
the
basal
ganglia
function,
and
they
have
emerged
as
promising
targets
for
novel
treatments.
To
investigate
the
difference
in
results
according
to
the
effect
of
the
mode
of
levodopa
administration
on
the
mechanism
of
levodopa-induced
dyskinesia
(LID)
development,
we
analyzed
the
mRNA
expression
of
dopaminergic
and
non-dopaminergic
recep-
tors
in
the
striatum
of
PD
model
rats
in
relation
to
LID.
The
target
mRNAs
chosen
were
those
of
dopamine
D1
(Drd1)
and
D2
(Drd2)
receptors,
neuropeptides
prodynorphin
(Pdyn)
and
pre-
proenkephalin
(Penk),
the
adenosine
A2A
(A2A)
receptor,
and
the
endocannabinoid
CB1
(CB1)
receptor.
We
also
investigated
the
molecular
mechanism
for
the
effect
of
zonisamide
(ZNS),
which
is
known
not
to
worsen
dyskinesia
clinically
in
PD
patients
(Murata
et
al.,
2007),
on
LID
development
and
expression
of
these
target
mRNAs
in
response
to
intermittent
levodopa
administration.
http://dx.doi.org/10.1016/j.neures.2017.04.003
0168-0102/©
2017
Elsevier
Ireland
Ltd
and
Japan
Neuroscience
Society.
All
rights
reserved.

Please
cite
this
article
in
press
as:
Oki,
M.,
et
al.,
Zonisamide
ameliorates
levodopa-induced
dyskinesia
and
reduces
expression
of
striatal
genes
in
Parkinson
model
rats.
Neurosci.
Res.
(2017),
http://dx.doi.org/10.1016/j.neures.2017.04.003
ARTICLE IN PRESS
G Model
NSR-4033;
No.
of
Pages
6
2
M.
Oki
et
al.
/
Neuroscience
Research
xxx
(2017)
xxx–xxx
2.
Materials
and
methods
2.1.
Preparation
of
unilateral
Parkinson’s
model
rats
and
drug
administration
Adult
female
Sprague-Dawley
rats
(Japan
SCL,
Inc.
Shizuoka,
Japan)
of
220–250
g
body
weight
were
anesthetized
and
placed
in
a
stereotaxic
instrument
(SR-5R-HT,
Narishige,
Tokyo,
Japan).
All
animal
experiments,
approved
by
the
institutional
experimental
animal
usage
committee,
were
in
accordance
with
Guidelines
for
Proper
Conduct
of
Animal
Experiments,
Science
Council
of
Japan.
6-
hydroxydopamine
(6-OHDA)
was
stereotaxically
injected
into
the
left
medial
forebrain
bundle
(stereotaxic
coordinates
from
bregma:
anterior
3.7
mm,
lateral
1.7
mm,
depth
from
brain
surface
7.8
mm:
(Paxinos
and
Watson,
2007)
via
a
26-gauge
needle
(Microsyringe
MS-N10AOC,
Ito
Corporation,
Shizuoka,
Japan).
Six
␮l
of
6-OHDA-
HBr
(Sigma-Aldrich,
MO,
USA)
(3
␮g/␮l
of
6-OHDA-HBr
with
0.02%
ascorbic
acid
in
saline)
was
slowly
injected
over
a
6-min
period.
Two
weeks
after
the
injection
of
6-OHDA,
the
apomorphine-
induced
behavioral
test
was
performed
to
evaluate
the
success
of
the
operation.
Namely,
10
min
after
the
intraperitoneal
injection
(0.5
mg/kg)
of
apomorphine-HCl
(Tocris
Bioscience,
MO,
USA),
the
rats
were
placed
in
a
hemispheric
bowl.
More
than
20
rotations
towards
the
contralateral
side
from
the
operated
side
over
5
min
was
judged
as
a
positive
result.
Successfully
operated
rats
were
subdivided
into
4
groups
and
treated
for
2
weeks
as
follows:
1)
no
medication
(group
N,
n
=
13),
2)
continuous
levodopa
infusion
(group
C,
n
=
7),
3)
intermittent
levodopa
injection
(group
I,
n
=
7),
and
4)
intermittent
levodopa
and
ZNS
injection
(group
Z,
n
=
7)
(Fig.
1).
Levodopa
methyl
ester
hydrochloride
(Santa
Cruz
Biotech-
nology,
Inc.
TX,
USA)
was
always
combined
with
benserazide
hydrochloride
at
a
4:1
ratio
(Sigma-Aldrich)
and
dissolved
in
phys-
iological
saline
with
L-ascorbic
acid
(Sigma-Aldrich,
0.2
mg/ml)
as
an
antioxidant.
ZNS
was
dissolved
in
physiological
saline
contain-
ing
10%
DMSO.
ZNS
was
provided
by
Sumitomo
Dainippon
Pharma
Co.
Ltd.,
Osaka,
Japan.
For
continuous
levodopa
infusion,
ALZET
osmotic
mini-
pumps
(Model
2ML4,
DURECT
Corp.
CA,
USA)
containing
levodopa/benserazide
(24/6
mg/kg/day)
were
implanted
subcu-
taneously
under
anesthesia
and
infusion
was
continued
for
2
weeks
(Lebel
et
al.,
2010).
For
intermittent
levodopa
injection,
levodopa/benserazide
(12/3
mg/kg)
was
injected
subcutaneously
twice
daily
(at
0900
and
1700)
for
2
weeks.
For
intermittent
levodopa
and
ZNS
injection,
levodopa/benserazide
(12/3
mg/kg)
and
zonisamide
(26
mg/kg
solution)
were
injected
subcutaneously
twice
daily
(at
0900
and
1700)
for
2
weeks
(Wallingford
et
al.,
2008).
Plasma
concentrations
of
zonisamide
in
blood
from
a
tail
vein
were
measured
by
HPLC
after
1
and
2
weeks
of
treatment.
2.2.
Evaluation
of
LID
LID
of
each
rat
in
group
C,
I,
and
Z
was
evaluated
after
the
last
administration
of
levodopa
after
2
weeks
of
treatment.
Amplitude
and
severity
of
axial
and
forelimb
abnormal
involuntary
move-
ments
were
scored
at
every
20
min
after
levodopa
administration
for
180
min
(Winkler
et
al.,
2002).
2.3.
Tissue
preparation
After
evaluation
of
LID,
animals
were
decapitated
under
deep
anesthesia.
Brains
were
removed
and
quickly
frozen
on
pow-
dered
dry
ice.
Frozen
brains
were
coronally
sectioned
by
cryostat
at
1
mm
thickness
(coordinates,
0.24-1.2
mm
anterior
to
bregma;
(Paxinos
and
Watson,
2007)).
Approximately
0.7
mg
of
brain
tis-
sues
at
lateral
striatum
on
both
sides
were
punched
out
by
use
of
an
18-G
non-bevel-tipped
needle
(Terumo,
Tokyo,
Japan).
The
tis-
sues
were
digested
with
RNase-free
proteinase
K,
and
total
RNAs
were
extracted
by
using
a
NucleoSpin
RNA
kit
(Macherey-Nagel,
Düren,
Germany).
Random
hexamer
was
used
to
generate
cDNA
fragments.
The
remaining
tissue
was
sectioned
and
stained
with
rabbit
anti-
tyrosine
hydroxylase
(TH)
antibody
(AB152,
Chemicon,
MA,
USA)
to
confirm
homogeneity
of
dopaminergic
denervation.
2.4.
Gene
expression
analyses
Primers
and
real-time
RT-PCR
settings
for
neuropeptides
pro-
dynorphin
and
preproenkephalin,
dopamine
D1
and
D2
receptors,
adenosine
A2A
receptor,
and
endocannabinoid
CB1
receptors
are
listed
in
the
Table
1.
Expression
of
mRNAs
on
the
operated
and
unoperated
sides
was
quantitated
in
duplicate
by
performing
real-
time
PCR
using
SYBR
Green
(Takara
Bio
Inc.
Shiga,
Japan).
The
hypoxanthine
phosphoribosyltransferase
1
gene
was
used
as
an
internal
control.
2.5.
Statistical
analyses
The
Wilcoxon
signed-rank
test
was
used
to
compare
the
expres-
sion
difference
of
mRNAs
between
operated
and
unoperated
sides.
The
Mann-Whitney
U
test
was
used
for
the
evaluation
of
LID.
3.
Results
The
mean
and
standard
deviation
of
apomorphine-induced
rotation
test
for
group
N,
C,
I
and
Z
were
49
±
23.4,
56.3
±
12.5,
49.9
±
19.4,
and
60.9
±
34.9,
respectively,
and
there
was
no
statis-
tically
significant
difference
by
Mann-Whitney
U
test
(Fig.
2A).
TH
immunoreactivity
in
the
lesioned
side
of
the
stratum
was
uniformly
decreased
in
all
animals
which
were
positive
for
apomorphine-induced
rotation
test
(Fig.
2B).
LID
was
not
observed
in
group
C.
On
the
other
hand,
LID
appeared
20
min
after
levodopa
administration,
peaked
at
80
min
and
lasted
up
to
140
min
in
group
I.
LID
was
also
developed
in
group
Z,
but
it
was
less
severe
and
lasted
for
a
shorter
period
of
time
than
those
in
group
I.
Especially,
the
mean
forelimb
AIMs
score
of
group
Z
at
140
min
was
significantly
lower
than
that
of
group
I
(Fig.
2C).
The
trough
plasma
concentration
of
ZNS
reached
to
15–20
␮g/ml
after
1
week,
and
remained
in
the
same
range
after
2
weeks.
The
expression
of
Pdyn
mRNA
was
decreased,
whereas
that
of
Penk
mRNA
was
increased
on
the
operated
side
in
group
N.
Pdyn
mRNA
expression
was
not
changed,
but
Penk
mRNA
expression
was
increased
in
group
C.
Pdyn
mRNA
was
increased,
but
Penk
mRNA
was
not
changed,
in
group
I
or
Z
(Fig.
3A).
The
expression
of
dopamine
receptors
was
not
changed
in
group
N.
The
expression
of
D1
receptor
was
not
changed,
but
that
of
D2
receptor
was
increased
in
group
C.
The
expression
of
both
D1
and
D2
receptors
was
increased
in
groups
I
and
Z
(Fig.
3B).
Adenosine
A2A
receptor
expression
was
increased
only
in
group
I,
and
it
was
not
changed
in
group
N,
C
or
Z
(Fig.
3C).
The
expression
of
endocannabinoid
CB1
receptor
was
increased
in
groups
N,
C
and
I,
but
it
was
not
changed
in
group
Z
(Fig.
3D).
4.
Discussion
All
animals
in
groups
I
and
Z
developed
LID,
whereas
LID
did
not
appear
in
group
C
as
well
as
in
group
N.
Considering
that
Pdyn
mRNA
expression
was
decreased
in
group
N
but
not
in
group
C
(Fig.
3A),
dopaminergic
stimulation
by
continuous
levodopa
infusion
in
this
study
was
not
insufficient.
Therefore,
pul-
satile
dopaminergic
stimulation
by
intermittent
levodopa
injection

Please
cite
this
article
in
press
as:
Oki,
M.,
et
al.,
Zonisamide
ameliorates
levodopa-induced
dyskinesia
and
reduces
expression
of
striatal
genes
in
Parkinson
model
rats.
Neurosci.
Res.
(2017),
http://dx.doi.org/10.1016/j.neures.2017.04.003
ARTICLE IN PRESS
G Model
NSR-4033;
No.
of
Pages
6
M.
Oki
et
al.
/
Neuroscience
Research
xxx
(2017)
xxx–xxx
3
Fig.
1.
Time
schedule
of
the
experiments.
Two
weeks
after
stereotaxic
injection
of
6-OHDA,
the
apomorphine-induced
behavioral
test
was
performed
to
evaluate
success
of
the
operation.
Positive
rats
were
subdivided
into
4
groups
and
treated
as
shown.
After
2
weeks
of
treatment,
LID
was
evaluated:
and
then
the
rats
were
sacrificed.
appears
to
have
induced
dyskinesia
in
groups
I
and
Z.
This
result
is
consistent
with
our
clinical
experience
on
dyskinesia
induction
in
patients
with
PD
and
suggests
that
the
present
experimental
sys-
tem
is
a
valuable
model
to
study
LID.
However,
LID
was
less
severe
in
group
Z
compared
with
group
I,
in
agreement
with
the
clinical
effect
of
ZNS
on
PD
patients
(Murata
et
al.,
2007),
(Murata
et
al.,
2015).
The
expression
of
Pdyn
and
Penk
mRNAs
is
differentially
regulated
by
dopamine.
Increased
dopaminergic
input
results
in
increased
Pdyn
and
decreased
Penk
expression.
Conversely,
decreased
dopaminergic
input
results
in
decreased
Pdyn
and
increased
Penk
levels.
Expression
of
Pdyn
and
Penk
mRNAs
indi-
cates
the
activity
of
medium
spiny
neurons
(MSNs)
of
direct
and
indirect
pathways,
respectively
(Gerfen
et
al.,
1990).
Accord-
ing
to
the
changes
in
expression
levels
of
these
neuropeptides,
the
indirect
pathway
was
predominant
in
the
operated
side
of
rats
in
groups
N
and
C.
On
the
other
hand,
the
direct
pathway
predominated
on
the
operated
side
of
intermittently
injected,
LID-
expressing
rats
in
groups
I
and
Z.
In
the
striatum,
the
dopamine
D1
receptors
are
expressed
selec-
tively
by
direct
pathway
MSNs,
whereas
the
dopamine
D2
receptors
are
expressed
by
those
in
the
indirect
pathway.
The
expression
level
of
dopamine
D2
receptor
mRNA
was
not
changed
in
group
N,
but
it
was
elevated
in
groups
C
and
I
regardless
of
the
mode
of
levodopa
administration.
It
was
also
elevated
in
group
Z.
On
the
other
hand,
the
mRNA
expression
of
the
dopamine
D1
receptor
was
not
increased
in
group
N
or
C,
but
was
elevated
in
groups
I
and
Z.
The
D1
receptor
is
primarily
in
a
low-affinity
state,
whereas
the
D2
one
shows
high
affinity
for
dopamine
(Richfield
et
al.,
1989).
Continuous
levodopa
infusion
could
have
been
sufficient
to
stim-
ulate
the
high-affinity
D2
receptors
but
not
sufficient
to
stimulate
the
low-affinity
D1
receptors.
Intermittent
levodopa
injection
pro-
vokes
pulsatile
elevation
of
dopamine
in
the
operated
striatum
(Miller
and
Abercrombie,
1999),
which
might
be
enough
to
stimu-
late
low-affinity
dopamine
D1
receptors
and
increase
D1
receptor
mRNA
expression.
Moreover,
increased
D1
receptor
expression
might
be
related
to
predominant
direct
pathway
and
LID
expression
in
intermittently
injected
rats.
Table
1
Primers
and
real-time
RT-PCR
settings
for
each
gene.
Gene
Sequence
(5→
3)
Primer
GC%
Primer
TM
(◦C)
Amplicon
(bp)
Drd1
dopamine
receptor
D1
F-
CGTGATCAGCGTGGACAGGTA
57.1
64.5
86
R-
TCAGGATGAAGGCTGCTTTGG
52.4
64.8
Pdyn
prodynorphin
F-
GATCGGCCATCCTATCACCTG
57.1
63.9
146
R-
GGACCACGCCATTCTGTATCAC
54.5
63.8
Drd2
dopamine
receptor
D2
F-
AGCTCCAAGCGCCGAGTTA
57.9
63.4
128
R-
AAGGCAGGGTTGGCAATGATA
47.6
63.8
Penk
proenkephalin
F-
TGGCTACAGTGCAGGCAGA
57.9
61.8
199
R-
TTGTACATGTCGATGTTATCCCAAG
40
62.1
A2A
adenosine
A2a
receptor
F-
TCCTCACGCAGAGTTCCATCTTTA
45.8
64.4
100
R-
ACACCTGTCACCAAGCCATTGTA
47.8
63.8
CB1
endocannabinoid
receptor
1
F-
TGTCTCCCATTTCAAGCAAGGAG
47.8
64.8
142
R-
CATTCGAGCCCACGTAGAGGA
57.1
64.7
Hprt1
hypoxanthine
phophoribosyltransferase
1
F-
TCCTCATGGACTGATTATGGACA
43.5
61.6
132
R-
TAATCCAGCAGGTCAGCAAAGA
45.5
62.1
Component
Volume(␮l)
PCR
cycle
Temp
Time
cDNA
9.6
SYBR
Premix
Ex
Taq
GC
10
1
cycle
95 ◦C
2
min
(Perfect
Real
Time) ®50
cycle
95 ◦C
Primer
−
forward
(20
␮M)
0.2
60 ◦C
60
s,
real
time
Primer
−
reverse
(20
␮M)
0.2
Melt
curve
65 ◦C
10
s,
0.5 ◦C
each
→
95 ◦C
Total
20

Please
cite
this
article
in
press
as:
Oki,
M.,
et
al.,
Zonisamide
ameliorates
levodopa-induced
dyskinesia
and
reduces
expression
of
striatal
genes
in
Parkinson
model
rats.
Neurosci.
Res.
(2017),
http://dx.doi.org/10.1016/j.neures.2017.04.003
ARTICLE IN PRESS
G Model
NSR-4033;
No.
of
Pages
6
4
M.
Oki
et
al.
/
Neuroscience
Research
xxx
(2017)
xxx–xxx
Fig.
2.
(A)
The
mean
and
standard
deviation
of
apomorphine-induced
rotation
test
for
each
groups.
(B)
TH
immunoreactivity
in
the
lesioned
side
of
the
stratum
was
uniformly
decreased.
(C)
Evaluation
of
LID
in
groups
C,
I,
and
Z.
Amplitude
and
severity
of
axial
and
forelimb
abnormal
involuntary
movements
(AIMs)
were
scored
at
every
20
min
after
levodopa
administration
for
180
min.
Adenosine
A2A
receptors
are
exclusively
expressed
in
indirect
pathway
MSNs.
A2A
receptors
enhance
GABA
release
from
the
nerve
terminals
and
work
antagonistically
to
dopamine
D2
recep-
tors
in
indirect
pathway
MSNs
(Mori,
2014).
In
contrast
with
the
mRNA
expression
of
dopamine
D1
and
D2
receptors,
which
was
increased
in
both
groups
I
and
Z,
adenosine
A2A
receptor
was
increased
only
in
group
I,
which
developed
LID
strongly.
Expres-
sion
levels
of
the
A2A
receptor
were
stable
not
only
in
group
N
or
LID-free
group
C,
but
also
in
group
Z,
which
presented
mild
LID.
According
to
the
results
of
histopathological
(Calon
et
al.,
2004)
and
PET
(Mishina
et
al.,
2011;
Ramlackhansingh
et
al.,
2011)
stud-
ies,
adenosine
A2A
receptor
expression
is
increased
in
PD
patients
with
LID.
Considering
that
A2A
receptors
activate
indirect
pathway
MSNs,
the
elevated
expression
of
A2A
mRNA
receptor
might
be
a
compensatory
mechanism
against
the
predominant
direct
path-
way
and
LID
development,
but
not
a
cause
of
LID.
ZNS
ameliorated
LID
and
A2A
receptor
mRNA
up-regulation,
indicating
that
ZNS
inhibited
LID
development
and
a
compensatory
up-regulation
of
A2A
receptors.
Co-administration
of
ZNS
with
levodopa
may
have
a
prophylactic
effect
on
LID.
Endocannabinoid
CB1
receptors,
presynaptically
localized
in
glutamatergic
neurons
projecting
from
the
cerebral
cortex
to
MSNs
in
the
striatum,
regulate
presynaptic
glutamate
release.
When
the
glutamatergic
input
is
in
excess,
glutamate
will
spillover
and
stim-

Please
cite
this
article
in
press
as:
Oki,
M.,
et
al.,
Zonisamide
ameliorates
levodopa-induced
dyskinesia
and
reduces
expression
of
striatal
genes
in
Parkinson
model
rats.
Neurosci.
Res.
(2017),
http://dx.doi.org/10.1016/j.neures.2017.04.003
ARTICLE IN PRESS
G Model
NSR-4033;
No.
of
Pages
6
M.
Oki
et
al.
/
Neuroscience
Research
xxx
(2017)
xxx–xxx
5
Fig.
3.
Expression
levels
of
mRNA
for
each
gene.
(A)
prodynorphin
(Pdyn)
and
preproenkephalin
(Penk).
(B)
dopamine
D1
receptor
(Drd1)
and
D2
receptor
(Drd2).
(C)
adenosine
A2A
receptor
(A2A).
(D)
endocannabinoid
CB1
receptor
(CB1).
Expression
levels
of
mRNA
for
each
gene
on
the
control
side
(Co)
and
the
lesioned
side
(Le)
were
compared
for
each
group.
P
values
of
a
significant
increase
(*)
or
decrease
(**)
in
the
lesioned
side
are
indicated.
ulate
postsynaptically
localized
metabotropic
glutamate
receptors.
Then
endocannabinoids
are
synthetized
by
MSNs
and
act
as
a
retro-
grade
messenger
to
stimulate
presynaptic
CB1
receptors,
providing
protection
as
a
circuit
breaker
against
excess
presynaptic
activity
(Katona
and
Freund,
2008).
In
our
present
study,
the
expression
of
CB1
receptors
was
elevated
in
groups
N,
C,
and
I,
regardless
of

Please
cite
this
article
in
press
as:
Oki,
M.,
et
al.,
Zonisamide
ameliorates
levodopa-induced
dyskinesia
and
reduces
expression
of
striatal
genes
in
Parkinson
model
rats.
Neurosci.
Res.
(2017),
http://dx.doi.org/10.1016/j.neures.2017.04.003
ARTICLE IN PRESS
G Model
NSR-4033;
No.
of
Pages
6
6
M.
Oki
et
al.
/
Neuroscience
Research
xxx
(2017)
xxx–xxx
levodopa
administration
or
LID.
Therefore,
the
elevation
of
CB1
receptors
might
be
an
early
compensatory
mechanism
against
dopaminergic
denervation
in
operated
striatum
to
maintain
stabil-
ity
of
the
basal
ganglia
circuit.
Elevation
of
CB1
receptors
was
not
observed
in
group
Z,
suggesting
that
ZNS
acted
on
a
different
non-
dopaminergic
mechanism
to
inhibit
a
compensatory
up-regulation
of
CB1
receptors.
In
summary,
intermittent
injection
of
levodopa
caused
LID,
in
association
with
elevated
expression
of
D1
and
A2A
receptors.
ZNS
ameliorated
LID
development
and
inhibited
the
up-regulation
of
A2A
and
CB1
receptors.
Further
studies
are
needed
to
explore
the
precise
pathomechanisms
of
these
receptors
with
respect
to
LID
development,
but
modulation
of
expression
of
these
receptors
may
lead
to
therapeutic
approaches
for
the
prevention
or
amelioration
of
motor
complications
in
PD.
Conflict
of
interest
statement
The
authors
have
no
conflict
of
interest.
Acknowledgments
We
thank
Ms.
Yoshiko
Shimada
for
technical
assistance.
This
work
was
performed
as
a
collaborative
study
with,
and
under
finan-
cial
support
from
Sumitomo
Dainippon
Pharma
Co.
Ltd.
This
work
was
also
supported
by
a
research
grant
(D2)
from
Kansai
Medical
University.
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