Neuroscience & Medicine, 2013, 4, 299-318
Published Online December 2013 (http://www.scirp.org/journal/nm)
http://dx.doi.org/10.4236/nm.2013.44044
Open Access NM
299
Involvement of CRH Receptors in the Neuroprotective
Action of R-Apomorphine in the Striatal 6-OHDA Rat
Model
Mustafa Varçin1, Eduard Bentea1, Steven Roosens1, Yvette Michotte1, Sophie Sarre1,2*
1Department of Pharmaceutical Chemistry and Drug Analysis, Center for Neurosciences, Vrije Universiteit Brussel, Faculty of
Medicine and Pharmacy, Brussels, Belgium; 2Belgian Pharmacists Association, Medicines Control Laboratory, Brussels, Belgium.
Received October 21st, 2013; revised November 20th, 2013; accepted December 10th, 2013
Copyright © 2013 Mustafa Varçin et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
The dopamine D1-D2 receptor agonist, R-apomorphine, has been shown to be neuroprotective in different models of
Parkinson’s disease. Different mechanisms of action for this effect have been proposed, but not verified in the striatal
6-hydroxydopamine rat model. In this study, the expression of a set of genes involved in 1) signaling, 2) growth and
differentiation, 3) neuronal regeneration and survival, 4) apoptosis and 5) inflammation in the striatum was measured
after a subchronic R-apomorphine treatment (10 mg/kg/day, subcutaneously, during 11 days) in the striatal 6-hydroxy-
dopamine rat model. The expression of 84 genes was analysed by using the rat neurotrophins and receptors RT2 Pro-
filer™ PCR array. The neuroprotective effects of R-apomorphine in the striatal 6-hydroxydopamine model were con-
firmed by neurochemical and behavioural analysis. The expression data suggest the observed neuroprotection involved
the alteration of the gene and the protein expression levels of the anti-inflammatory corticotropin releasing hormone
receptor (CRHR) 1 and the pro-inflammatory CRHR2 receptor confirming its potential anti-inflammatory action.
Keywords: Apomorphine; Gene Expression; Inflammation; Neuroprotection; Parkinson’s Disease; Striatal
6-Hydroxydopamine Rat Model
1. Introduction
Until today, the available treatments for Parkinson’s dis-
ease do not stop or slow down the progressive nature of
the disease and are based on dopamine (DA) replacement
strategies, such as the use of DA agonists and/or the DA
precursor, levodopa (L-DOPA) [1]. Due to the long pre-
symptomatic phase of the disease, therapeutic intervene-
tions that result in the protection, restoration and/or res-
cuing of the dopaminergic neurons are of extreme im-
portance to improve the quality of life of the patients
[1,2].
Although R-apomorphine has been introduced as a
drug a certain time ago, it is still available as treatment
for patients with Parkinson’s disease and with advanced
Parkinson’s disease and for the treatment of persistent
and disabling motor fluctuations which do not respond to
L-DOPA [3-7]. It activates D1-like (D1, D5) and D2-like
(D2, D3, D4) receptors, serotonin receptors (5HT1A,
5HT2A, 5HT2B and 5HT2C) and α-adrenergic receptors
(α1B, α1D, α2A, α2B, α2C) [8].
R-apomorphine has been shown to be neuroprotective
both in vitro and in vivo, including the 1-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine (MPTP) [9-12], 6-hydroxy-
dopamine (6-OHDA) [13-15] and methamphetamine
(METH) [12,16] rodent models of Parkinson’s disease
and more recently, a mouse model of Alzheimer’s dis-
ease [17]. The in vivo neuroprotective properties of R-
apomorphine have been shown to be dose dependent, and
mainly linked to the administration of high doses of R-
apomorphine. These findings also suggest that the neu-
roprotective action may involve other pathways rather
than activation of DA receptors. Among the proposed
mechanisms of action are its radical scavenging activity
[13,15,18,19] and iron chelating properties [20], inhibi-
*Corresponding author.

Involvement of CRH Receptors in the Neuroprotective Action of R-Apomorphine
in the Striatal 6-OHDA Rat Model
300
tion of mitochondrial iron-induced lipid peroxidation and
protein oxidation [13,18,19], activation of nuclear tran-
scription factor NF-E2-related factor 2 (Nrf2) [21], inhi-
bition of monoamine oxidase (MAO)-A and MAO-B
[22], its enhancement of glutathione peroxidase activity
[23], anti-apoptotic [7,24], anti-inflammatory [9], mito-
genic [25] and trophic effects [26-29]. We previously
showed that a short-term treatment with R-apomorphine
(10 mg/kg/day, s.c., during 11 days), started before or 24
hours after lesions, has neuroprotective actions in the
striatal 6-OHDA rat model, as demonstrated by an im-
proved motor behavior and a significant protection (20%
- 35%) of the integrity of the nigrostriatal dopaminergic
system at the level of the substantia nigra pars compacta
and striatum [14]. As hydroxyl radical formation is con-
sidered as an important event in the neurotoxicity of
6-OHDA [30], several in vivo studies investigated the
interference of R-apomorphine with the hydroxyl radical
formation in the rat striatum [13-15]. However, these
findings are not consistent. Furthermore, Battaglia et al.
[11] have shown that R-apomorphine is still neuropro-
tective after 40 h following MPTP injections, and to-
gether with our previous findings that R-apomorphine is
still neuroprotective 24 h after 6-OHDA administration
[14], it can be suggested that the mechanism of action of
R-apomorphine is not linked to the interference with the
initial effects of the neurotoxins.
Although different mechanisms of action of R-apo-
morphine, as summarised above, have been proposed for
its neuroprotective effect, these have not been exten-
sively explored in the striatal 6-OHDA rat model. In this
study, we screened the expression of a set of genes in-
volved in 1) signaling, 2) growth and differentiation, 3)
neuronal regeneration and survival, 4) apoptosis and 5)
inflammation in the central nervous system after a sub-
chronic R-apomorphine treatment (10 mg/kg/day, s.c.,
during 11 days) in the unilateral striatal 6-OHDA parkin-
sonian rat model.
Our data confirm the neuroprotective effects of R-apo
morphine in the unilateral striatal 6-OHDA parkinsonian
rat model and suggest that they may involve the altera-
tion of the striatal gene and the protein expression levels
of the anti-inflammatory corticotropin releasing hormone
receptor (CRHR) 1 and the pro-inflammatory CRHR2 re-
ceptor.
2. Experimental Procedure
All the chemical compounds where no supplier is men-
tioned, are supplied by Sigma-Aldrich, Brussels, Belgium.
2.1. Animals
In all experiments, male albino Wistar rats (Charles
River, Sulzfeld, Germany) weighing 175 - 200 g were
used. Animals were kept under standardised conditions
(25˚C, 12 h light-dark cycle) with free access to food and
tap water. At the end of the experiments, rats were sacri-
ficed with an overdose of pentobarbital (Nembutal®,
Ceva Sante Animale, Brussels, Belgium). Animal ex-
periments were carried out according to the national
guidelines on animal experimentation and were approved
by the Ethical Committee for Animal Experiments of the
Faculty of Medicine and Pharmacy of the Vrije Univer-
siteit Brussel. All efforts were made to minimise animal
suffering and the minimal number of animals necessary
to produce reliable scientific data was used.
2.2. Effects of R-Apomorphine on the Progress of
Neurodegeneration in Rats Unilaterally
Lesioned with 6-OHDA
2.2.1. Experimental Design
The experimental design is described in Figure 1. The
rats were divided into 3 groups. Rats in group I were
used as non-lesioned controls, receiving subcutaneous
injections of saline. Rats in groups II were injected uni-
laterally with 3 µl of 6-OHDA solution (6.7 µg/µl in
0.1% ascorbic acid) in the left striatum and subcutane-
ously with saline. Rats in group III were lesioned with
Figure 1. Experimental design. The rats were divided into 3 groups. Rats in group I were used as non-lesioned controls, rats
in groups II were injected intrastriatally with 6-OHDA and subcutaneously with saline, and rats in group III were lesioned
with 6-OHDA similar to rats of group II, but also received daily subcutaneous injections of R-apomorphine (10 mg/kg/day).
Further details are described in Materials and Methods. Abbreviations: R-APO: R-apomorphine, s.c. subcutaneously.
Open Access NM

Involvement of CRH Receptors in the Neuroprotective Action of R-Apomorphine
in the Striatal 6-OHDA Rat Model
301
6-OHDA similar to rats of group II, but also received
daily subcutaneous injections of R-apomorphine (10 mg/
kg/day). The treatment with R-apomorphine started 15
min before the intrastriatal injection of 6-OHDA and
continued for another 10 days. The dose of the R-apo-
morphine was based on previous findings [10,11,14].
Briefly, Grünblatt et al had already demonstrated that a
low dose (0.5, 1, 2, and 2.5 mg/kg) of R-apomorphine
does not protect against MPTP induced DA loss in mice,
whereas the higher dose (10 mg/kg) restored the values
to those of control mice [10]. Similar findings were re-
ported in 2001 [22]. Furthermore, Fornai et al demon-
strated a dose dependent neuroprotective effect of R-
apomorphine in methamphetamine induced DA depletion.
Based on these findings, we decided to use 10 mg/kg R-
apomorphine in the 6-OHDA striatal rat model. Animals
were visually checked at regular intervals and weighed
daily before any manipulation until the end of the ex-
periment (day 14). For all the groups there was a wash-
out period of 3 days. Different sets of rats were used for
the initial screening of the relative striatal gene expres-
sion levels on the one hand, and behavioural testing, as-
sessment of DA and DOPAC content, and protein ex-
pression analysis on the other. mRNA of high quality
was used during the gene expression studies, and there-
fore the purity and integrity of all mRNA samples were
checked with advanced methods, such as UV spectro-
photometry and a microfluidics based electrophoresis
system. The specificity of the PCR reaction has been
evaluated by analysing the PCR product (the amplicon)
by conducting at the end of each RTqPCR experiment a
melt curve analysis for each amplicon. The web based
software of the company (SA Biosciences, MD) also
automatically performed a quality control for each PCR
run. The quality of the reference genes has been evalu-
ated by the software program that was provided by the
supplier (SA Biosciences, MD) of the PCR arrays. The
selection of the potential candidate genes for the protein
expression analysis was based on experimental and lit-
erature findings. To mitigate biological and technical
variabilities during a gene expression study, the recom-
mended number of biological replicates is minimum
three, and for technical replicates minimum two [31]. We
managed to have the minimum number of biological rep-
licates for the gene expression study, but unfortunately
technical replicates were not possible due to the limited
sample volume. Despite a lower number of animals for
the gene expression studies, our slightly higher number
of animals for the protein expression favours our obser-
vations at the gene level.
2.2.2. Local Administration of 6-OHDA by Striatal
Stereotaxic Microinjection
Rats were anaesthesized with a mixture of ketamine (50
mg/kg i.p.; Ketamine 1000 Ceva®, Ceva Sante Animale,
Brussels, Belgium) and diazepam (5 mg/kg i.p.; Valium®,
Roche Brussels, Belgium) and placed on a Kopf stereo-
taxic frame (David Kopf Instruments, Tujunga, Califor-
nia, USA). The skull was exposed and a burr hole was
drilled to introduce a syringe for a single injection of the
6-OHDA solution [containing 6.7 µg 6-OHDA per µl in
0.1% ascorbic acid, pH 5.0]. To minimize variability due
to degradation of the toxin, the 6-OHDA solution was
freshly prepared, kept on ice, and protected from expo-
sure to light. The solution was injected in the left stria-
tum at the following coordinates relative to the bregma L:
−3.0, A: +1.0 and V: +5.0, according to the atlas of Pax-
inos and Watson [32]. A total volume of 3 µl 6-OHDA
was injected at a flow rate of 1 µl/min. After injection,
the syringe was left in place for 5 min and then slowly
removed over a 1 - 2 min time period. The skin was su-
tured, the animals received ketoprofen (4 mg/kg i.p.;
Ketofen®, Merial, Brussels, Belgium) as analgesic and
were allowed to recover before returning to the animal
housing facilities [14].
2.3. Behavioral Analysis
Locomotor activity in Open Field
The spontaneous locomotor activity was monitored in a
Plexiglas box (sides, 60 cm; height, 60 cm). At least 1 h
prior to testing, rats were acclimated to the testing room.
The recording started immediately after placing animals
in the open field and continued for 60 min. To neutralise
odor formation, the arena was disinfected and cleaned
with 70% ethanol before each rat was tested. Experi-
ments were performed between 09:00 AM and 06:00 PM.
The rat was placed carefully in the center of the arena,
and allowed to explore the field for 60 min. Each per-
formance was automatically analysed using a video
tracking system and Ethovision 3.0 tracking software
(Noldus, the Netherlands). The following parameters
were recorded: 1) distance moved, 2) movement time, 3)
immobility, 4) velocity, 5) rearing and 6) relative mean-
der. The assessment of spontaneous locomotor behavior
was carried out during the wash-out period of the proto-
col, in order to avoid a possible modulatory effect of the
DA agonist R-apomorphine on motor function, as previ-
ous studies have shown that following DA agonists sig-
nificant alteration of spontaneous motor behavior occurs
[33,34]. We preferred to use the open field test in this
study to evaluate the animals in a drug-free state. Indeed,
amphetamine induced rotation might influence the ex-
pression of genes, and our previous results already indi-
cate that a subchronic R-apomorphine treatment of 6-
OHDA striatally lesioned rats reduced the amphetamine
induced ipsilateral rotations [14].
Open Access NM

Involvement of CRH Receptors in the Neuroprotective Action of R-Apomorphine
in the Striatal 6-OHDA Rat Model
302
2.4. Sacrifice and Brain Prelevation
Seventy-two hours (wash-out period) after the last inject-
tion of R-apomorphine (day 14), the rats were killed with
an overdose of pentobarbital (Nembutal®, Ceva Sante
Animale, Brussels, Belgium) and the brains were quickly
removed without perfusion. From the rostral part (10 mm
from front) of the brain the left and right striatum were
dissected out on an ice cold chilled petri-dish, immedi-
ately snap frozen in dry-ice cooled isopentane and stored
separately at −80˚C until homogenisation and analysis of
1) striatal DA and DOPAC content, 2) striatal gene ex-
pression and 3) protein expression of CRHR1, CRHR2,
and neuropeptide Y (NPY) receptors 1 and 2.
2.5. Neurochemical Determination of the Striatal
DA and DOPAC Content
To establish the extent of DA and DOPAC depletion in
the striatum after 6-OHDA lesioning, the liquid chroma-
tography (LC) method previously described was used
[14], with slight modifications.
The supernatant obtained during protein expression
analysis (4.7) was diluted 5 times in antioxidant (0.05 M
HCl, 0.5% Na2S2O5 and 0.05% Na2EDTA). 20 µl of
samples were injected and analysed directly for DA and
DOPAC content on a narrowbore (C18 column: 15 m,
150 mm × 2.1 mm; Altima; Grace; Lokeren; Belgium)
LC system. The mobile phase consisted of 0.1 M sodium
acetate trihydrate, 20 mM citric acid monohydrate, 1 mM
1-octane sulfonic acid, 0.1 mM Na2EDTA and 1 mM
dibutylamine, adjusted to pH 3.7. Methanol 3% (v/v) was
added as organic modifier. The flow rate was set at 0.2
ml/min. The electrochemical detection (Antec, The
Netherlands) potential was +700 mV versus the reference
electrode (Ag/AgCl). Sensitivity was set at 1 nA full
scale. All samples were injected via a high precision
auto-injector equipped with a cooling system (Kontron,
San Diego, CA, USA). The integration of the chroma-
tograms was done with the Data Apex Clarity software
program (Antec). The tissue DA and DOPAC content
were calculated and expressed as µg/g wet weight of
tissue.
2.6. Gene Expression Analysis
2.6.1. RNA Extraction
Total RNA was extracted from the striatal tissue using
the RNeasy® Lipid Tissue Mini Kit according to the
manufacturer’s protocol (Qiagen, Venlo, The Nether-
lands). The concentration and purity of RNA were de-
termined by measuring the absorbance using the Nano-
drop 1000 (Thermo Scientific). The quality of total RNA
was assessed using the Agilent® Bioanalyzer RNA 6000
Nano Labchip® (Agilent Technologies, Palo Alto, CA).
For each RNA sample the presence of sharp bands/peaks
present for both the 18S and 28S ribosomal RNAs were
verified and only the samples with an RNA Integrity
Number of 7 or higher were used.
One microgram of total RNA was subjected to first
strand cDNA synthesis using the RT2 First Strand Kit
(SA Biosciences, MD). For real-time PCR, a PCR com-
ponents mix consisting of 2× RT2 SYBR Green Fluor
qPCR Mastermix (1350 µl) (SA Biosciences), cDNA
synthesis reaction (102 µl) and RNase-free water (1248
µl) was prepared. The final volume was 2700 µl, provid-
ing an excess volume to perform pipetting steps as pre-
cisely as possible to ensure that each well of the array
receives the required volume. 25 µl of the mixture was
added into each of the wells of the RT2 Profiler PCR Ar-
rays (PARN-031, SA Biosciences, MD)
2.6.2. Quantitative Real-Time PCR
Real-time quantifications were performed using the Bio-
Rad® iCycler® Real-time PCR system (Bio-Rad) using
the recommended cycling conditions for the RT2 Profiler
PCR arrays (SA Biosciences, MD). Each PCR array
contained 84 transcripts, a set of five housekeeping genes
as internal controls and additional controls for efficiency
of reverse transcription, PCR and the absence of con-
taminating genomic DNA.
Relative expression was determined with the ∆∆ CT
method using the PCR Array Data Analysis Web Portal
(www.SABiosciences.com/pcrarraydataanalysis.php) and
the web based software automatically performed quanti-
fication, including the quality control for each PCR run/
array. For normalization of the expression levels of the
genes of interest, the average CT value of the five house-
keeping genes: Ribosomal protein, large, P1 (Rplp1);
hypoxanthine phosphoribosyltransferase 1 (Hprt1); ribo-
somal protein L13A (Rpl13a); lactate dehydrogenase A
(Ldha) and β-actin (Actb) was chosen. If the fold-change
was greater than 1, the result is reported as a fold up-
regulation. If the fold-change was less than 1, the nega-
tive inverse of the results is reported as a fold downregu-
lation.
2.7. Protein Expression Analysis
To measure CRHR1, CRHR2, NPY1R and NPY2R pro-
tein levels, the striatal tissue was homogenised in an ice-
cold PBS-solution (0.02 M, pH 7.0 - 7.2) containing 2%
protease inhibitor cocktail (Sigma, St-Louis, MO, USA)
(25 mg tissue/ml) and subsequently sonicated for 1 min
(Branson Sonifier 250). After centrifugation at 10,000 g
at 4˚C during 10 minutes (Sorvall RC5B refrigated su-
perspeed centrifuge, Dupont Instruments), the super-
natants were collected and stored at −20˚C until use. Af-
ter decantation, the protein content of the supernatant
Open Access NM

Involvement of CRH Receptors in the Neuroprotective Action of R-Apomorphine
in the Striatal 6-OHDA Rat Model
Open Access NM
303
was determined by ELISA and the DA content by LC
(4.5). Tissue levels of CRHR1, CRHR2, NPY1R and
NPY2R were determined in 100 µl aliquots by comer-
cially available ELISA kits (USCN Life Science Inc.,
Wuhan, China) according to the manufacturer’s instruc-
tions. Absorbances were measured using a Bio-Rad 680
microplate (Bio-Rad Laboratories, Belgium) reader set at
450 nm. The protein content was determined using the
Pierce® 660 nm Protein Assay Kit (Thermo Scientific,
USA).
sioned striata of saline treated rats was significantly re-
duced after intrastriatal application of 6-OHDA by 60%,
similar to our previous findings [14]. The DOPAC:DA
ratio in the denervated striatum of the striatally lesioned
rats was significantly increased. Treatment with R-apo-
morphine started 15 min before the intrastriatal injection
of 6-OHDA significantly attenuated the striatal DA de-
pletion and restored the DOPAC:DA ratio. The benefi-
cial neurochemical effects of the treatment with R-apo-
morphine were similar to our previous findings [14].
Recording of the open field activity (Figure 3) re-
vealed that R-apomorphine significantly attenuated the
6-OHDA induced reduction in total distance moved and
increase in relative meander. There was a trend for R-
apomorphine to improve the 6-OHDA lesion induced
reduction in velocity (p = 0.08). R-apomorphine treat-
ment had no effect on movement time, immobility time
and rearing (data not shown).
2.8. Data Analysis
The web-based software of the company SA Biosciences
has been used to analyse the gene expression data. All
data are expressed as mean ± S.E.M. Significant differ-
ences between all the experimental groups were deter-
mined using one-way analysis of variance (ANOVA)
followed by the Bonferroni’s post-hoc test. The signifi-
cance of the change in gene expression between the
groups was evaluated by unpaired Student t-test for each
gene. All statistical analysis was performed with Graph-
Pad Instat 3.0 (GraphPad Prism Software, Inc., San
Diego, USA) at the 5% level of significance.
3.2. Gene Expression Analysis
A list of the genes that have been screened with the cor-
responding fold changes is listed in Table 1.
3.2.1. Gene Expression Profile of the Striatal
6-Hydroxydopamine Rat Model
3. Results
3.1. Neuroprotective Effect of R-Apomorphine in
the Striatal 6-OHDA Rat Model (Figures 2
and 3)
In the striatum, 20 genes out of the 84 genes on the array
were changed in the striatally lesioned rats compared to
non-lesioned rats. Significant differences were detected
between the striatally lesioned rats and the non-lesioned
rats for 14 out of the 20 genes (p < 0.05), with a further 6
genes nearing significance (p < 0.1). The majority of the
genes (13) were upregulated in the striatal 6-OHDA rat
model, whereas 7 were downregulated. Two weeks after
the striatal administration of 6-OHDA, the expression of
CRHR2, GFRA1, GFRA3, GMFG, CD40, CCKAR,
TGFb1, CX3CR1, IL-10rα, IL-6rα, HSPB1, STAT1 and
The neuroprotective effect of R-apomorphine was con-
firmed by determination of the striatal DA content and
DA turnover (Figure 2) and in the open field test (Fig-
ure 3). Two weeks after injection of 6-OHDA, the DA
content of the striata contralateral to the side of the lesion
of rats receiving saline or R-apomorphine were not sig-
nificantly different from those of the control animals
data not shown). However, the DA content of the le- (
Figure 2. Dopamine levels and dopamine turnover. Dopamine (DA) content (left panel) and 3,4-dihydroxyphenylacetic acid
(DOPAC) DOPAC:DA ratio (right panel) of the left striatum of control (n = 6), saline treated 6-OHDA striatal lesioned (n = 6)
and R-apomorphine treated 6-OHDA striatally lesioned rats (n = 6). DA content was determined two weeks after striatal
micro-injection of 6-hydroxydopamine (6-OHDA) and is expressed as µg/g wet tissue (mean ± S.E.M). *Significantly different
(one-way ANOVA followed by a Bonferroni’s Multiple Comparison Test: p < 0.05).

Involvement of CRH Receptors in the Neuroprotective Action of R-Apomorphine
in the Striatal 6-OHDA Rat Model
304
Figure 3. Open field activity. Locomotor activity of control (n = 5), saline treated 6-OHDA striatal lesioned (n = 5) and R-
apomorphine treated 6-OHDA striatally lesioned rats (n = 6). The mean total distance moved (upper left panel), movement
time (upper right panel), velocity (lower left panel) and relative meander (lower right panel) in the open field under un-
habituated conditions are illustrated. The recording started immediately after placing animals in the open field and contin-
ued for 60 min. Each performance was automatically analyzed using a video tracking system and Ethovision 3.0 tracking
software (Noldus, the Netherlands). Data are expressed as mean ± S.E.M. *Significantly different (one-way ANOVA followed
by a Bonferroni’s Multiple Comparison Test: p < 0.05).
Table 1. Striatal gene epression results obtained by quantitative real-time PCR. Gene expression in the control (n = 3), saline
treated 6-OHDA striatal lesioned (n = 4) and R-apomorphine treated 6-OHDA striatal lesioned rats (n = 4) was analyzed us-
ing the Neurotrophins and Receptors RT2 Profiler PCR Arrays (SA Biosciences) as described in Material and Methods. Rela-
tive expression is determined with the ∆∆ CT method. If the fold-change is greater than 1, the result is reported as a fold
upregulation. If the fold-change is less than 1, the negative inverse of the results is reported as a fold downregulation. The
p-values were calculated based on an unpaired Student’s t-test for each gene. All the 84 genes and their functional classes are
shown in the table.
Description Gene symbolGenBank
Fold
regulation
6-OHDA
versus
control
p-value
Fold
regulation
R-APO
versus
control
p-value
Neurotrophins and Receptors
Adenylate cyclase activating polypeptide 1 receptor 1 Adcyap1r1 NM_133511 −1.11 0.6471 1.07 0.9809
Artemin Artn NM_053397 −1.11 0.6000 −1.41 0.1508
Brain−derived neurotrophic factor Bdnf NM_012513 2.22 0.3474 −1.08 0.8819
Ciliary neurotrophic factor Cntf NM_013166 1.10 0.5290 1.06 0.6760
Ciliary neurotrophic factor receptor Cntfr NM_001003929−1.15 0.5044 −1.15 0.5441
Corticotropin releasing hormone Crh NM_031019 −1.80 0.2127 −2.15 0.1367
Corticotropin releasing hormone binding protein Crhbp NM_139183 −1.15 0.5033 −1.16 0.4726
Corticotropin releasing hormone receptor 1 Crhr1 NM_030999 1.04 0.7216 1.39 0.0632
Open Access NM

Involvement of CRH Receptors in the Neuroprotective Action of R-Apomorphine
in the Striatal 6-OHDA Rat Model
305
Continued
Fibroblast growth factor receptor substrate 3 Frs3 NM_0010173821.08 0.5453 1.22 0.1740
Glial cell derived neurotrophic factor Gdnf NM_019139 −1.05 0.7042 −1.17 0.4756
GDNF family receptor alpha 1 Gfra1 NM_012959 1.50 0.0999 1.40 0.1511
GDNF family receptor alpha 2 Gfra2 NM_012750 −1.00 0.9577 −1.14 0.6205
GDNF family receptor alpha 3 Gfra3 NM_053398 1.79 0.0435 1.26 0.0801
Glia maturation factor, beta Gmfb NM_031032 1.10 0.4245 1.26 0.0867
Glia maturation factor, gamma Gmfg NM_181091 1.70 0.0072 1.83 0.0032
Hypocretin (orexin) receptor 1 Hcrtr1 NM_013064 1.19 0.4072 −1.08 0.9414
Hypocretin (orexin) receptor 2 Hcrtr2 NM_013074 −1.05 0.8012 −1.13 0.6321
Metallothionein 3 Mt3 NM_053968 −1.11 0.4487 −1.27 0.2408
Nerve growth factor (beta polypeptide) Ngfb XM_227525 1.20 0.2602 1.36 0.1124
Nerve growth factor receptor (TNFR superfamily,
member 16) Ngfr NM_012610 −1.06 0.8455 1.12 0.6659
Nerve growth factor receptor (TNFRSF16) associated
protein 1 Ngfrap1 NM_053401 −1.48 0.0437 −1.72 0.0339
Nuclear receptor subfamily 1, group I, member 2 Nr1i2 NM_052980 −1.23 0.3919 −1.11 0.7157
Neuregulin 1 Nrg1 NM_031588 −1.20 0.3026 −1.26 0.2108
Neuregulin 2 Nrg2 XM_344662 −1.01 0.9586 1.20 0.3032
Neurotrophin 3 Ntf3 NM_031073 1.19 0.6292 1.78 0.1067
Neurotrophin 5 Ntf5 NM_013184 −1.25 0.3904 −1.96 0.4401
Neurotrophic tyrosine kinase, receptor, type 1 Ntrk1 NM_021589 −1.82 0.0828 −1.42 0.3921
Neurotrophic tyrosine kinase, receptor, type 2 Ntrk2 NM_012731 −1.17 0.3840 −1.44 0.1327
Persephin Pspn NM_013014 −1.03 0.7075 1.04 0.7371
Prostaglandin E receptor 2 (subtype EP2) Ptger2 NM_031088 −1.13 0.6021 1.08 0.9750
Trk−fused gene Tfg NM_0010121441.01 0.9790 1.03 0.8562
CD40 molecule, TNF receptor superfamily member 5 Cd40 NM_134360 2.68 0.0003 3.27 0.0001
Fas (TNF receptor superfamily, member 6) Fas NM_139194 1.29 0.3149 1.64 0.1195
Urocortin Ucn NM_019150 −1.17 0.5582 −1.15 0.4268
VGF nerve growth factor inducible Vgf NM_030997 −1.36 0.3562 −1.42 0.1827
Zinc finger protein 110 Zfp110 NM_001024775−1.43 0.1192 −1.68 0.0479
Zinc finger protein 91 Zfp91 NM_001169120−1.30 0.0316 −1.53 0.0526
Neuropeptides and Receptors
Bombesin Receptors
Gastrin releasing peptide receptor Grpr NM_012706 −1.79 0.0484 −1.77 0.1867
Cholecystokinin Receptors
Cholecystokinin A receptor Cckar NM_012688 2.00 0.0897 2.15 0.1202
Galanin Receptors
Galanin receptor 1 Galr1 NM_012958 −2.10 0.3045 −1.83 0.1559
Open Access NM

Involvement of CRH Receptors in the Neuroprotective Action of R-Apomorphine
in the Striatal 6-OHDA Rat Model
306
Continued
Galanin receptor 2 Galr2 NM_019172 1.03 0.7421 1.01 0.7771
Tachykinin Receptors
Tachykinin receptor 1 Tacr1 NM_012667 −1.24 0.1941 −1.06 0.8856
Other Neuropeptides and Receptors
Neuropeptide FF receptor 2 Npffr2 NM_023980 −1.11 0.9903 −1.32 0.8297
Hypocretin HcRt NM_013179 −1.48 0.2752 −1.23 0.3425
Melanocortin 2 receptor Mc2r NM_0011004911.65 0.3436 1.86 0.1214
Neuropeptide Y Npy NM_012614 −1.10 0.4672 −1.06 0.7078
Neuregulin 1 Nrg1 NM_031588 −1.20 0.3026 −1.26 0.2108
Neurogenesis
Central Nervous System Development
Chemokine (C−X−C motif) receptor 4 Cxcr4 NM_022205 1.19 0.4159 −1.11 0.4334
Fibroblast growth factor receptor 1 Fgfr1 NM_024146 1.25 0.1320 1.26 0.1858
Nerve growth factor receptor (TNFR superfamily,
member 16) Ngfr NM_012610 −1.06 0.8455 1.12 0.6659
Neurotrophin 3 Ntf3 NM_031073 1.19 0.6292 1.78 0.1067
Peripheral Nervous System Development
Artemin Artn NM_053397 −1.11 0.6000 −1.41 0.1508
Glial cell derived neurotrophic factor Gdnf NM_019139 −1.05 0.7042 −1.17 0.4756
GDNF family receptor alpha 3 Gfra3 NM_053398 1.79 0.0435 1.26 0.0801
Nerve growth factor (beta polypeptide) Ngfb XM_227525 1.20 0.2602 1.36 0.1124
Neuregulin 1 Nrg1 NM_031588 −1.20 0.3026 −1.26 0.2108
Neurotrophin 3 Ntf3 NM_031073 1.19 0.6292 1.78 0.1067
Axon Guidance
Artemin Artn NM_053397 −1.11 0.6000 −1.41 0.1508
GDNF family receptor alpha 3 Gfra3 NM_053398 1.79 0.0435 1.26 0.0801
Nerve growth factor receptor (TNFR superfamily,
member 16) Ngfr NM_012610 −1.06 0.8455 1.12 0.6659
Gliogenesis
Fibroblast growth factor 2 Fgf2 NM_019305 1.24 0.3390 1.47 0.0259
Neuregulin 1 Nrg1 NM_031588 −1.20 0.3026 −1.26 0.2108
Neurotrophin 3 Ntf3 NM_031073 1.19 0.6292 1.78 0.1067
Dendrite Morphogenesis
Brain−derived neurotrophic factor Bdnf NM_012513 2.22 0.3474 −1.08 0.8819
Metallothionein 3 Mt3 NM_053968 −1.11 0.4487 −1.27 0.2408
Other Neurogenesis Genes
Bcl2−associated X protein Bax NM_017059 −1.13 0.6256 −1.09 0.5521
FBJ osteosarcoma oncogene Fos NM_022197 1.74 0.1261 3.29 0.0102
Open Access NM

Involvement of CRH Receptors in the Neuroprotective Action of R-Apomorphine
in the Striatal 6-OHDA Rat Model
307
Continued
Galanin receptor 2 Galr2 NM_019172 1.03 0.7421 1.01 0.7771
GDNF family receptor alpha 1 Gfra1 NM_012959 1.50 0.0999 1.40 0.1511
GDNF family receptor alpha 2 Gfra2 NM_012750 −1.00 0.9577 −1.14 0.6205
NEL−like 1 (chicken) Nell1 NM_031069 −1.08 0.5581 1.01 0.9296
Neurotrophin 5 Ntf5 NM_013184 −1.25 0.3904 −1.96 0.4401
Neurotrophic tyrosine kinase, receptor, type 1 Ntrk1 NM_021589 −1.82 0.0828 −1.42 0.3921
Neurotrophic tyrosine kinase, receptor, type 2 Ntrk2 NM_012731 −1.17 0.3840 −1.44 0.1327
Synaptic Transmission
Cerebellin 1 precursor Cbln1 NM_001109127−1.38 0.9382 −1.64 0.6224
Growth and Differentiation
Growth Factors and Receptors
Artemin Artn NM_053397 −1.11 0.6000 −1.41 0.1508
Brain−derived neurotrophic factor Bdnf NM_012513 2.22 0.3474 −1.08 0.8819
Fibroblast growth factor 2 Fgf2 NM_019305 1.24 0.3390 1.47 0.0259
Fibroblast growth factor 9 Fgf9 NM_012952 1.24 0.4508 1.17 0.5779
Fibroblast growth factor receptor 1 Fgfr1 NM_024146 1.25 0.1320 1.26 0.1858
Glial cell derived neurotrophic factor Gdnf NM_019139 −1.05 0.7042 −1.17 0.4756
Glia maturation factor, beta Gmfb NM_031032 1.10 0.4245 1.26 0.0867
Glia maturation factor, gamma Gmfg NM_181091 1.70 0.0072 1.83 0.0032
Interleukin 10 Il10 NM_012854 1.35 0.4768 −1.02 0.7271
Interleukin 1 beta Il1b NM_031512 2.50 0.1685 3.16 0.0035
Interleukin 6 Il6 NM_012589 −1.21 0.3849 1.46 0.6407
Leukemia inhibitory factor Lif NM_022196 1.57 0.3027 2.17 0.0687
Metallothionein 3 Mt3 NM_053968 −1.11 0.4487 −1.27 0.2408
Nerve growth factor (beta polypeptide) Ngfb XM_227525 1.20 0.2602 1.36 0.1124
Neuregulin 2 Nrg2 XM_344662 −1.01 0.9586 1.20 0.3032
Neurotrophin 3 Ntf3 NM_031073 1.19 0.6292 1.78 0.1067
Neurotrophin 5 Ntf5 NM_013184 −1.25 0.3904 −1.96 0.4401
Persephin Pspn NM_013014 −1.03 0.7075 1.04 0.7371
Transforming growth factor alpha Tgfa NM_012671 1.23 0.1619 1.22 0.1567
Transforming growth factor, beta 1 Tgfb1 NM_021578 2.90 0.0029 3.36 0.0001
Transforming growth factor beta 1 induced transcript 1 Tgfb1i1 XM_341934 1.21 0.2067 1.20 0.3595
Tumor protein p53 Tp53 NM_030989 −1.10 0.5051 1.10 0.6728
VGF nerve growth factor inducible Vgf NM_030997 −1.36 0.3562 −1.42 0.1827
Cell cycle
Fibroblast growth factor 2 Fgf2 NM_019305 1.24 0.3390 1.47 0.0259
Fibroblast growth factor 9 Fgf9 NM_012952 1.24 0.4508 1.17 0.5779
Open Access NM

Involvement of CRH Receptors in the Neuroprotective Action of R-Apomorphine
in the Striatal 6-OHDA Rat Model
308
Continued
Interleukin 1 beta Il1b NM_031512 2.50 0.1685 3.16 0.0035
Neurotrophic tyrosine kinase, receptor, type 1 Ntrk1 NM_021589 −1.82 0.0828 −1.42 0.3921
Transforming growth factor alpha Tgfa NM_012671 1.23 0.1619 1.22 0.1567
Transforming growth factor, beta 1 Tgfb1 NM_021578 2.90 0.0029 3.36 0.0001
Tumor protein p53 Tp53 NM_030989 −1.10 0.5051 1.10 0.6728
Cell Proliferation
Bcl2−associated X protein Bax NM_017059 −1.13 0.6256 −1.09 0.5521
Chemokine (C−X−C motif) receptor 4 Cxcr4 NM_022205 1.19 0.4159 −1.11 0.4334
Fibroblast growth factor 2 Fgf2 NM_019305 1.24 0.3390 1.47 0.0259
Fibroblast growth factor 9 Fgf9 NM_012952 1.24 0.4508 1.17 0.5779
Gastrin releasing peptide receptor Grpr NM_012706 −1.79 0.0484 −1.77 0.1867
Interleukin 10 Il10 NM_012854 1.35 0.4768 −1.02 0.7271
Interleukin 1 beta Il1b NM_031512 2.50 0.1685 3.16 0.0035
Myelocytomatosis oncogene Myc NM_012603 1.26 0.2614 1.67 0.0109
Signal transducer and activator of transcription 4 Stat4 NM_0010122261.64 0.2469 1.38 0.4436
Transforming growth factor alpha Tgfa NM_012671 1.23 0.1619 1.22 0.1567
Transforming growth factor, beta 1 Tgfb1 NM_021578 2.90 0.0029 3.36 0.0001
Tumor protein p53 Tp53 NM_030989 −1.10 0.5051 1.10 0.6728
Cell Differentiation
Ciliary neurotrophic factor Cntf NM_013166 1.10 0.5290 1.06 0.6760
Fibroblast growth factor 2 Fgf2 NM_019305 1.24 0.3390 1.47 0.0259
Fibroblast growth factor 9 Fgf9 NM_012952 1.24 0.4508 1.17 0.5779
Neurofibromin 1 Nf1 NM_012609 −1.36 0.0706 −1.34 0.0954
Neuregulin 1 Nrg1 NM_031588 −1.20 0.3026 −1.26 0.2108
Signal transducer and activator of transcription 3 Stat3 NM_012747 1.27 0.3093 1.33 0.2001
Tumor protein p53 Tp53 NM_030989 −1.10 0.5051 1.10 0.6728
Zinc finger protein 91 Zfp91 NM_001169120−1.30 0.0316 −1.53 0.0526
Cytokines and Receptors
Chemokine (C−X3−C motif) receptor 1 Cx3cr1 NM_133534 1.67 0.0315 1.70 0.0635
Chemokine (C−X−C motif) receptor 4 Cxcr4 NM_022205 1.19 0.4159 −1.11 0.4334
Interleukin 10 Il10 NM_012854 1.35 0.4768 −1.02 0.7271
Interleukin 10 receptor, alpha Il10ra NM_057193 1.73 0.0500 1.71 0.0292
Interleukin 1 beta Il1b NM_031512 2.50 0.1685 3.16 0.0035
Interleukin 1 receptor, type I Il1r1 NM_013123 1.22 0.2624 1.20 0.4294
Interleukin 6 Il6 NM_012589 −1.21 0.3849 1.46 0.6407
Interleukin 6 receptor Il6r NM_017020 1.60 0.0157 1.40 0.1988
Interleukin 6 signal transducer Il6st NM_0010087251.08 0.3498 1.10 0.3305
Open Access NM

Involvement of CRH Receptors in the Neuroprotective Action of R-Apomorphine
in the Striatal 6-OHDA Rat Model
309
Continued
Leukemia inhibitory factor Lif NM_022196 1.57 0.3027 2.17 0.0687
Leukemia inhibitory factor receptor alpha Lifr NM_031048 −1.43 0.1643 −1.47 0.1204
Neuregulin 1 Nrg1 NM_031588 −1.20 0.3026 −1.26 0.2108
Signal transducer and activator of transcription 4 Stat4 NM_0010122261.64 0.2469 1.38 0.4436
Apoptosis
Anti−apoptosis
B−cell CLL/lymphoma 2 Bcl2 NM_016993 −1.16 0.4948 −1.34 0.2575
Brain−derived neurotrophic factor Bdnf NM_012513 2.22 0.3474 −1.08 0.8819
Interleukin 10 Il10 NM_012854 1.35 0.4768 −1.02 0.7271
Caspase Activation
Bcl2−associated X protein Bax NM_017059 −1.13 0.6256 −1.09 0.5521
Myelocytomatosis oncogene Myc NM_012603 1.26 0.2614 1.67 0.0109
Tumor protein p53 Tp53 NM_030989 −1.10 0.5051 1.10 0.6728
Induction of Apoptosis
Bcl2−associated X protein Bax NM_017059 −1.13 0.6256 −1.09 0.5521
Myelocytomatosis oncogene Myc NM_012603 1.26 0.2614 1.67 0.0109
Nerve growth factor receptor (TNFR superfamily,
member 16) Ngfr NM_012610 −1.06 0.8455 1.12 0.6659
Nerve growth factor receptor (TNFRSF16) associated
protein 1 Ngfrap1 NM_053401 −1.48 0.0437 −1.72 0.0339
Fas (TNF receptor superfamily, member 6) Fas NM_139194 1.29 0.3149 1.64 0.1195
Tumor protein p53 Tp53 NM_030989 −1.10 0.5051 1.10 0.6728
Other Apoptosis Genes
Heat shock protein 1 Hspb1 NM_031970 3.86 0.0231 4.21 0.0075
Interleukin 6 Il6 NM_012589 −1.21 0.3849 1.46 0.6407
CD40 molecule, TNF receptor superfamily member 5 Cd40 NM_134360 2.68 0.0003 3.27 0.0001
Immune response
Acute−phase response
Interleukin 6 Il6 NM_012589 −1.21 0.3849 1.46 0.6407
Signal transducer and activator of transcription 3 Stat3 NM_012747 1.27 0.3093 1.33 0.2001
Inflammatory response
Interleukin 10 Il10 NM_012854 1.35 0.4768 −1.02 0.7271
Interleukin 1 beta Il1b NM_031512 2.50 0.1685 3.16 0.0035
Transforming growth factor, beta 1 Tgfb1 NM_021578 2.90 0.0029 3.36 0.0001
Lymphocyte activation
Interleukin 10 Il10 NM_012854 1.35 0.4768 −1.02 0.7271
CD40 molecule, TNF receptor superfamily member 5 Cd40 NM_134360 2.68 0.0003 3.27 0.0001
Other immune response genes
Open Access NM

Involvement of CRH Receptors in the Neuroprotective Action of R-Apomorphine
in the Striatal 6-OHDA Rat Model
Open Access NM
310
Continued
Leukemia inhibitory factor Lif NM_022196 1.57 0.3027 2.17 0.0687
Fas (TNF receptor superfamily, member 6) Fas NM_139194 1.29 0.3149 1.64 0.1195
Transcription factors and regulators
Positive regulation of transcription
Fusion (involved in t(12;16) in malignant liposarcoma)
(human) Fus NM_001012137−1.18 0.2780 −1.14 0.3574
Neurotrophin 3 Ntf3 NM_031073 1.19 0.6292 1.78 0.1067
Transforming growth factor beta 1 induced transcript 1 Tgfb1i1 XM_341934 1.21 0.2067 1.20 0.3595
Transcription coactivator activity
Melanoma antigen, family D, 1 Maged1 NM_053409 −1.34 0.0279 −1.36 0.0508
Transforming growth factor beta 1 induced transcript 1 Tgfb1i1 XM_341934 1.21 0.2067 1.20 0.3595
Other transcription factors and regulators
FBJ osteosarcoma oncogene Fos NM_022197 1.74 0.1261 3.29 0.0102
Myelocytomatosis oncogene Myc NM_012603 1.26 0.2614 1.67 0.0109
Nuclear receptor subfamily 1, group I, member 2 Nr1i2 NM_052980 −1.23 0.3919 −1.11 0.7157
Similar to myocyte enhancer factor 2C LOC685671XR_006259 1.08 0.5206 1.20 0.4123
Signal transducer and activator of transcription 1 Stat1 NM_032612 1.82 0.0014 1.61 0.0042
Signal transducer and activator of transcription 2 Stat2 NM_0010119051.23 0.0877 1.22 0.2499
Signal transducer and activator of transcription 3 Stat3 NM_012747 1.27 0.3093 1.33 0.2001
Signal transducer and activator of transcription 4 Stat4 NM_0010122261.64 0.2469 1.38 0.4436
Tumor protein p53 Tp53 NM_030989 −1.10 0.5051 1.10 0.6728
Zinc finger protein 110 Zfp110 NM_001024775−1.43 0.1192 −1.68 0.0479
STAT2 was upregulated, whereas the expression of
NGFRAP1, NTRK1, ZFP91, GRPR, NPY2R, NF1,
MAGED1 was downregulated.
3.2.2. Gene Expression Profile in the R-Apomorphine
Treated Striatally 6-Hydroxydopamine
Lesioned Rats
R-apomorphine treatment of the striatally lesioned 6-
OHDA rats changed the striatal expression of 20 genes
out of the 84 genes on the array compared to the non-
lesioned rats. Significant differences were detected be-
tween the R-apomorphine treated striatally lesioned rats
and the non-lesioned rats for 13 genes out of 20 genes (p
< 0.05), with a further 7 genes nearing significance (p <
0.1). The majority of the genes (15) were upregulated in
the R-apomorphine treated striatally lesioned rats,
whereas 5 were downregulated. An upregulation was
found for the genes CRHR1, GFRA3, GMFB, GMFG,
CD40, FGF2, FOS, IL-1b, LIF, TGFb1, MYC, CX3CR1,
IL10rα, HSPB1 and STAT1, whereas the expression of
NGFRAP1, ZFP110, ZFP91, NF1 and MAGED1 was
downregulated.
The changes in expression level of some genes were
similar in both the striatally lesioned and the R-apo-
morphine treated 6-OHDA striatally lesioned rats versus
the non-lesioned rats, such as the upregulation of GFRA3,
CD40, TFGb1, CX3CR1, IL-10rα, HSPB1 and STAT1,
and the downregulation of NGFRAP1, ZFP91, NF1 and
MAGED1.
The upregulation of GFRA1, CCKAR, IL-6rα and
STAT2 in the striatum of striatally lesioned rats was ei-
ther prevented or not different in the R-apomorphine
treated striatally lesioned rats versus the non-lesioned
rats.
Furthermore, a downregulation was observed for
NTRK1, GRPR and NPY2R in the striatally lesioned rats,
while the expression levels of these genes in the R-apo-
morphine treated striatally lesioned rats were not differ-
ent from the non-lesioned rats. ZFP110 was significantly
downregulated in the treated striatally lesioned rats ver-
sus the controls, whereas its expression in the striatally
lesioned rats was not different from the non-lesioned rats.

Involvement of CRH Receptors in the Neuroprotective Action of R-Apomorphine
in the Striatal 6-OHDA Rat Model
311
Based on the global overview of the gene expression
alterations, we chose to focus on the neuropeptide recap-
tors CRHR1, CRHR2, NPY1R and NPY2R. These 4
receptors are all G protein coupled receptors (GPCRs)
and have been implicated as potential drug targets in the
treatment of Parkinson’s disease, and are more exten-
sively described in the discussion section. Regarding
these neuropeptide receptors the following findings were
observed. The expression level of CRHR1 was not sig-
nificantly different between non-lesioned and striatally
lesioned rats. However, in the R-apomorphine treated
striatally lesioned rats an upregulation of the expression
of CRHR1 (p = 0.06) was observed when compared to
non-lesioned rats. Although this upregulation was near-
ing significance, there was no difference when compared
to striatally lesioned rats (Figure 4).
In the same way, a significant upregulation was found for
CRHR2 in the striatally lesioned rats versus the non-
lesioned and the treated striatally lesioned rats, whereas
no significant difference was found between the R-apo-
morphine treated striatally lesioned rats and the non-
lesioned rats, suggesting that the treatment normalized or
prevented the upregulation of CRHR2 (Figure 4).
Regarding the NPY1R, there was a downregulation in
the striatally lesioned rats versus the non-lesioned rats,
but this was not statistically significant. However, a sig-
nificant downregulation was observed in the striatally
lesioned rats compared to the R-apomorphine treated
striatally lesioned rats. There was no significant differ-
ence between non-lesioned and R-apomorphine treated
striatally lesioned rats, suggesting that R-apomorphine
normalized or prevented changes in the expression level
of NPY1R (Figure 5).
NPY2R was significantly downregulated in the stri-
atally lesioned rats when compared to the non-lesioned
rats. This downregulation was either prevented or nor-
malized by R-apomorphine, as no significant differences
were observed between the R-apomorphine treated stri-
atally lesioned and non-lesioned rats (Figure 5).
3.3. Protein Expression of CRHR1, CRHR2,
NPY1R and NPY2R
CRHR1 and CRHR2 protein levels, quantified with
ELISA, confirmed the results of the gene expression
analysis. The CRHR1 protein content in the denervated
Figure 4. CRHR1 and CRHR2 mRNA and protein expression. CRHR1 (upper left panel) and CRHR2 (lower left panel)
mRNA expression in the left striatum of control (n = 3), saline treated 6-OHDA striatal lesioned (n = 4) and R-apomorphine
treated 6-OHDA striatally lesioned rats (n = 4) was determined using quantitative real-time PCR as described in the Methods.
A fold-change greater than 1 is reported as up regulation, while lower than 1 is reported as down regulation. The protein
expression of CRHR1 (upper right panel) and CRHR2 (lower right panel) in the left striatum in all the groups [(control (n =
6), saline treated 6-OHDA striatal lesioned (n = 6) and R-apomorphine treated 6-OHDA striatally lesioned rats (n = 6)] was
determined using ELISA as described in the Methods. For each protein, the average control was set to 100%. Data are ex-
pressed as mean ± S.E.M. *Significantly different (one-way ANOVA followed by a Bonferroni’s Multiple Comparison Test: p
< 0.05).
Open Access NM

Involvement of CRH Receptors in the Neuroprotective Action of R-Apomorphine
in the Striatal 6-OHDA Rat Model
312
Figure 5. NPY1R and NPY2R mRNA expression. NPYR1 (left panel) and NPYR2 (right panel) mRNA expression in the left
striatum of control (n = 3), saline treated 6-OHDA striatal lesioned (n = 4) and R-apomorphine treated 6-OHDA striatally
lesioned rats (n = 4) was determined using quantitative real-time PCR as described in the Methods. A fold-change greater
than 1 is reported as up regulation, while lower than 1 is reported as down regulation. Data are expressed as mean ± S.E.M.
*Significantly different (one-way ANOVA followed by a Bonferroni’s Multiple Comparison Test: p < 0.05).
striata of the R-apomorphine treated striatally lesioned
rats was significantly higher when compared to the left
striata of the non-lesioned and the denervated striata of
the striatally lesioned rats, while the CRHR1 protein
content in the striata of non-lesioned rats and the dener-
vated striata of striatally lesioned rats was not signifi-
cantly different (Figure 4). CRHR2 protein levels of the
denervated striata of the striatally lesioned rats were sig-
nificantly higher when compared to the left striata of
non-lesioned rats and the denervated striata of the R-
apomorphine treated striatally lesioned rats, with no sta-
tistical difference between the R-apomorphine group and
the control group (Figure 4).
No significant changes in NPY1R and NPY2R protein
levels were detected in the left striata between the three
groups (data not shown), suggesting that these receptors
might not be involved in the biological action of R-
apomorphine. However, this method does not monitor
post-transcriptional and post-translational modifications,
and it provides only a partial picture of the biological
events [35].
4. Discussion
Our data show that R-apomorphine treatment (10 mg/kg/
day, s.c., during 11 days) of rats striatally lesioned with
6-OHDA is associated with a partial restoration of the
DA levels and the DOPAC:DA ratios in the ipsilateral
striatum. Furthermore, in the open field test an improve-
ment in distance moved and attenuation of the relative
meander was observed. The decreased locomotor activity,
such as in the lesioned rats, has been attributed to the loss
of DA neurotransmission [36]. The reason that not all the
investigated behavioural parameters assessed during the
open field test normalised or improved, might be due to
the fact that R-apomorphine does not fully restore the
neuronal degeneration caused by 6-OHDA, which is in
line with the observed partial neuroprotection. These data
confirm our previous findings where the same treatment
with R-apomorphine significantly reduced the ampheta-
mine-induced rotations, attenuated the DA levels and the
DOPAC:DA ratios, and subsequently suggest that the
functionality and the integrity of the nigrostriatal dopa-
minergic system have at least partially been preserved by
a treatment with R-apomorphine [14].
The gene expression analysis demonstrated that dif-
ferent genes are altered in the lesioned striatum of the
saline treated striatally lesioned rats and the R-apomor-
phine treated striatally lesioned rats. Genes that were
altered in the saline treated striatally lesioned rats, are
related to the regulation of neurotrophins, neuropeptides,
immune response, neurogenesis, growth and differentia-
tion, cell cycle, cell proliferation, apoptosis and tran-
scription factors and regulators, suggesting that different
major events occur which might be involved in the mo-
lecular pathways of the pathogenesis of Parkinson’s dis-
ease and confirming the complexity of the disorder.
Treatment with R-apomorphine of the striatally lesioned
rats did not reverse all the gene alterations that were ob-
served in the saline treated striatally lesioned rats.
A role for inflammation in the striatal 6-OHDA rat
model can be suggested from our data as several inflam-
matory-related genes were upregulated, such as CX3CR1,
IL6R, IL10RA, TGFB1, and CD40. The concept of
neuroinflammation as a primary response to 6-OHDA in
both the striatum and the substantia nigra has been con-
firmed by Na et al., 2010 [37]. Furthermore, the DA-
ergic neuronal loss within the striatal 6-OHDA rat model
is preceded by microglial activation [38,39], suggesting a
temporal relationship between neurodegeneration and
neuroinflammation [39]. Similarly, in vivo and postmor-
tem observations from Parkinson’s disease patients show
that indeed activated microglia are present and that the
Open Access NM

Involvement of CRH Receptors in the Neuroprotective Action of R-Apomorphine
in the Striatal 6-OHDA Rat Model
313
levels of pro-inflammatory mediators, such as TNF-α,
IL-6, IL-1β, are increased [40]. All these findings sub-
stantiate the hypothesis of a fundamental role of inflam-
mation in neurodegeneration.
Similarly, the brain alteration in the MPTP mouse
model of Parkinson’s disease has been investigated by
others using a cDNA expression array including 1200
genes fragments and identified 51 genes in the MPTP
mouse model related to similar major events and altera-
tions in genes, such as a general increase in IL-1β, IL-6
and IL-7, as well as in IL-1R, IL-2R, IL-3R and IL-4R,
which confirms the concept of inflammation in neurode-
generation [9]. Pretreatment with R-apomorphine (10
mg/kg/day, s.c.) for 5 days before MPTP (24mg/kg/day)
injections, reversed most of the gene alterations, sug-
gesting a possible anti-inflammatory action. Furthermore,
an increase in anti-inflammatory cytokine IL-10 mRNA
might reflect an attempt to protect the neurons from fur-
ther degeneration [9]. Although, the pharmacogenetic
profile of the drug R-apomorphine was already investi-
gated in the above mentioned study, major differences
with our experimental set-up are the rodent model, the
administration mode of R-apomorphine and some of the
selected genes for the screening. For instance, R-apo-
morphine was administrated before the MPTP injections
for 5 consecutive days by Grunblatt et al. (2001), while
in our study the treatment started 15 min before the le-
sion induction and continued for 10 days [9].
Based on the global overview of the gene expression
alterations (Table 1), we chose to focus on the GPCR
neuropeptide receptors CRHR1, CRHR2, NPY1R and
NPY2R. GPCRs represent a class of proteins with sig-
nificant clinical importance, as approximately 30% of all
modern therapeutic treatments target these receptors [41].
Furthermore, literature reports, as discussed below, sug-
gest that these four receptors might have relevance as
potential drug targets in the treatment of Parkinson’s
disease.
It has been demonstrated that activation of CRHR1 is
anti-inflammatory [42] and that urocortin, a CRH-like
peptide, restores key deficits, such as motor behavior,
striatal DA levels and dopaminergic cell death in a LPS
rat model of Parkinson’s disease via CRHR1 [43-45] and
protects against excitotoxic cell death via the same re-
ceptor [46]. Furthermore, the neuroprotective and neu-
rorestorative effects of urocortin in the 6-OHDA rat
model are mediated via CRHR1 [43-45]. The anti-in-
flammatory properties of urocortin in the periphery were
previously reported by Gonzalez-Rey [47]. Huang et al.
have also shown that urocortin modulates dopaminergic
neuronal survival via inhibition of glycogen synthase
kinase-3β and histone deacetylase [48]. In addition, Kim
et al. confirmed the downstream properties of urocortin
on MPP+ treated neuroblastoma cells and its mediation
via the activation of CRHR1 [49]. Further findings dem-
onstrate that the activation of CRHR2 is pro-inflamma-
tory in the periphery as it mediates the inflammatory re-
sponses via release of pro-inflammatory mediators at the
colonocyte level [50,51]. Our data show that R-apo-
morphine treatment is associated with a downregulation
of the pro-inflammatory CRHR2, and an upregulation of
the anti-inflammatory CRHR1, both at mRNA and at
protein levels.
Regarding the NPY receptors, Decressac et al. demon-
strated that neuroproliferation in vivo is mediated by
NPY1R [52]. Furthermore, NPY has an anti-inflamma-
tory effect that is mediated by NPY1R in vivo [53]. More
recently, Decressac et al. have shown that NPY is neuro-
protective in in vivo models of Parkinson’s disease via
the NPY2 receptor via activation of both MAPK and Akt
pathways [54]. Similarly, Thiriet et al. have shown that
the intracerebral administration of NPY in mice blocked
methamphetamine induced apoptosis and that this effect
was mainly mediated by the stimulation of the NPY2
receptor and to a lesser extent by NPY1 receptors [55].
Moreover, it has been shown that the NPY receptors are
involved in the attenuation of DA release in vitro [36,56]
and in vivo [36,57,58] and that the NPY2 receptor is in-
volved in DA synthesis in the rat striatum [36] and the
inhibition of glutamate release [59]. Regarding these
NPY receptors, our data demonstrates that R-apomor-
phine treatment is associated with an upregulation of the
NPY1R and NPY2R at mRNA level, but not at the pro-
tein level. However, it is important to note that changes
in mRNA levels are not always reflected by changes in
protein expression since proteins can be synthesized in
one brain area and afterwards transported to another [60].
Furthermore, post-transcriptional and post-translational
modifications may occur [61].
R-apomorphine has been already tested in vitro and
various different observations have been registered [21,
26,27,62]. It has been shown previously that R-apo-
morphine stimulates the synthesis and release of multiple
trophic factors, such as brain-derived neurotrophic factor
(BDNF) and glial cell line-derived neurotrophic factor
(GDNF), in both mesencephalic and striatal neurons,
thereby effectively preventing dopaminergic neuron loss
in vitro [27,62]. In another study, it was shown that fi-
broblast growth factor-2 expression is robustly induced
in cultured astrocytes in response to R-apomorphine [26].
It is worth noting that the study was performed on cul-
tured astrocytes derived from newborn animals, while the
astrocytes from aged brains of Parkinson’s disease may
react differently in vitro. The aforementioned findings
were not confirmed by our gene expression studies. Fur-
thermore, if astrocytes in vivo will respond to R-apo-
Open Access NM

Involvement of CRH Receptors in the Neuroprotective Action of R-Apomorphine
in the Striatal 6-OHDA Rat Model
314
morphine treatment in the same way as reported in the
aforementioned study needs to be investigated, as in vitro
and in vivo findings do not always correlate.
The absence of sham lesioned rats within our experi-
mental design may be considered by others as a study
limitation. However, various publications indicate that
sham lesioning of rodents has no effect on the main
pathological hallmarks of Parkinson’s disease, such as
DA loss [30,37,43,63,64]. Furthermore, Na et al. [37]
assessed the number of gene expression changes in vehi-
cle and 6-OHDA lesioned striatum and substantia nigra
at various time points by using cDNA microarray. They
found that three days after vehicle administration there
are profound changes in relative striatal gene expression
levels (<50 genes). However, this gene expression pat-
tern began to wane as a function of time, and fourteen
days after vehicle administration, the number of altered
genes was almost equal to zero. A similar pattern was
observed for 6-OHDA striatally lesioned rats. However,
within this group the number of altered genes was much
higher after 3 days (>150 genes) and after 14 days (>50
genes) compared to the vehicle lesioned rats. These
findings suggest that sham lesioning has an effect on
mRNA expression, but that it disappears very quickly as
a function of time [37]. In addition, needle insertion and
vehicle injection does not alter functional protein levels
of BDNF and CNTF [63,65]. However, some astrocytic
reaction in the neostriatum of solvent injected rats close
to the needle track has been observed, while in the neu-
rotoxin lesioned animals this reaction was much more
widespread [66-68]. Our own findings also show that
neurodegeneration caused by a unilateral injection of
6-OHDA in the medial forebrain bundle or the striatum,
has no effect on the striatal protein expression of GDNF
at different time points after lesion in the intact and the
denervated striata of the lesioned animals and that of
control animals [60]. The aforementioned findings sug-
gest that the insertion of the needle for lesioning does not
cause significant alterations in terms of striatal neuro-
transmitters and behaviour. However, some acute, minor
and temporary effects have been observed at the gene
and protein level, but this is limited to the needle tract. In
our study, we assessed the gene and protein expression
fourteen days after surgery. Even though, there would be
an effect of the needle insertion, the combination with
the neurotoxin is much more invasive and destructive.
Similarly, we did not include intact rats which have
been treated with R-apomorphine, as we wanted to in-
vestigate the effects of R-apomorphine in the striatum of
diseased, parkinsonian animals. Previously, we have
shown that intact rats treated with R-apomorphine do not
have any significant differences in striatal DA and
DOPAC content, and DA turnover when compared to
intact rats that didn’t receive any treatment [14]. Fur-
thermore, the number of tyrosine hydroxylase immuno-
reactive neurons in the SNpc were not different between
intact rats and intact rats treated with R-apomorphine.
5. Conclusion
Our data confirm the neuroprotective effects of R-apo-
morphine in the unilateral striatal 6-OHDA parkinsonian
rat model and suggest that they involve the alteration of
the striatal gene and the protein expression levels of the
anti-inflammatory CRHR1 receptor and the pro-inflam-
matory CRHR2 receptor. Furthermore, treatment with R-
apomorphine led to an upregulation of the NPY1R and
NPY2R at the mRNA level. These results provide a bet-
ter insight for understanding our previously observed
neuroprotective effects of R-apomorphine in the unilat-
eral striatal 6-OHDA parkinsonian rat model and confirm
its potential anti-inflammatory action. However, addi-
tional investigations, e.g. functional studies, are required
in order to confirm these findings. Despite the value of
the 6-OHDA rat model, as illustrated in this work, one
should be aware that the mechanism of 6-OHDA proba-
bly only reflects a small fraction of the events occurring
in human Parkinson’s disease. 6-OHDA induces rather
acute effects, which differ significantly from the slowly
progressive pathology of human Parkinson’s disease.
Moreover, Lewy body pathology is not present in the
surviving neurons, and no other brain areas involved in
Parkinson’s disease are affected, such as the olfactory
bulb and the locus coeruleus [69-71]. Therefore, further
work would be valuable in understanding the potential
therapeutic value of targeting the CRH and NPY recap-
tors in Parkinson’s disease.
6. Acknowledgements
The authors wish to acknowledge the animal care by Mrs
M. Cresens and technical assistance of Mrs. P. Verdood,
Mr. E. Questier, Mr. S. Branson, Mrs R. Berckmans, Mr
G. De Smet, Mrs C. De Rijck, and Mrs R.-M. Geens. We
also thank Prof. Dr. Pharm. V. Rogiers, Prof. Dr. H.
Heimberg and Prof. Dr. R Kooijman for the use of their
laboratory facilities. This work was conducted with fi-
nancial support of the Institute for the promotion of In-
novation by Science and Technology in Flanders (IWT)
(IWT420), National Fund for Scientific Research (FWO-
Vlaanderen) (G.0071.05) and of the Research Council of
the Vrije Universiteit Brussel. M. Varçin is holder of a
research grant from the IWT (IWT420). E. Bentea is a
research fellow of the FWO Vlaanderen.
REFERENCES
[1] A. H. Schapira, “Neurobiology and Treatment of Parkin-
Open Access NM

Involvement of CRH Receptors in the Neuroprotective Action of R-Apomorphine
in the Striatal 6-OHDA Rat Model
315
son’s Disease,” Trends in Pharmacological Sciences, Vol.
30, No. 1, 2009, pp. 41-47.
http://dx.doi.org/10.1016/j.tips.2008.10.005
[2] M. B. Youdim, W. J. Geldenhuys and C. J. Van der Schyf,
“Why Should We Use Multifunctional Neuroprotective
and Neurorestorative Drugs for Parkinson’s Disease?”
Parkinsonism & Related Disorders, Vol. 13, No. Suppl 3,
2007, pp. S281-S291.
http://dx.doi.org/10.1016/S1353-8020(08)70017-8
[3] P. A. LeWitt, “Subcutaneously Administered Apomor-
phine: Pharmacokinetics and Metabolism,” Neurology,
Vol. 62, No. 6, 2004, pp. S8-S11.
http://dx.doi.org/10.1212/WNL.62.6_suppl_4.S8
[4] I. U. Haq, P. A. Lewitt and H. H. Fernandez, “Apomor-
phine Therapy in Parkinson’s Disease: A Review,” Expert
Opinion on Pharmacotherapy, Vol. 8, No. 16, 2007, pp.
2799-2809.
http://dx.doi.org/10.1517/14656566.8.16.2799
[5] P. A. LeWitt, et al., “Open-Label Study Assessment of
Safety and Adverse Effects of Subcutaneous Apomor-
phine Injections in Treating ‘off’ Episodes in Advanced
Parkinson Disease,” Clinical Neuropharmacology, Vol.
32, No. 2, 2009, pp. 89-93.
http://dx.doi.org/10.1097/WNF.0b013e31816d91f9
[6] F. Stocchi, “Use of Apomorphine in Parkinson’s Dis-
ease,” Neurological Sciences, Vol. 29, No. Suppl 5, 2008,
pp. S383-S386.
http://dx.doi.org/10.1007/s10072-008-1053-8
[7] M. Kyriazis, “Neuroprotective, Anti-Apoptotic Effects of
Apomorphine,” Journal of Anti-Aging Medicine, Vol. 6,
No. 1, 2003, pp. 21-28.
http://dx.doi.org/10.1089/109454503765361551
[8] S. Ribaric, “The Pharmacological Properties and Thera-
peutic Use of Apomorphine,” Molecules, Vol. 17, No. 5,
2012, pp. 5289-5309.
http://dx.doi.org/10.3390/molecules17055289
[9] E. Grunblatt, et al., “Gene Expression Analysis in N-
methyl-4-phenyl-1,2,3,6-tetrahydropyridine Mice Model
of Parkinson’s Disease Using cDNA Microarray: Effect
of R-Apomorphine,” Journal of Neurochemistry, Vol. 78,
No. 1, 2001, pp. 1-12.
http://dx.doi.org/10.1046/j.1471-4159.2001.00397.x
[10] E. Grunblatt, et al., “Apomorphine Protects against MPTP-
Induced Neurotoxicity in Mice,” Movement Disorders,
Vol. 14, No. 4, 1999, pp. 612-618.
http://dx.doi.org/10.1002/1531-8257(199907)14:4<612::
AID-MDS1010>3.0.CO;2-6
[11] G. Battaglia, et al., “Continuous Subcutaneous Infusion
of Apomorphine Rescues Nigro-Striatal Dopaminergic
Terminals Following MPTP Injection in Mice,” Neuro-
pharmacology, Vol. 42, No. 3, 2002, pp. 367-373.
http://dx.doi.org/10.1016/S0028-3908(01)00178-2
[12] G. Battaglia, et al., “Morphological and Biochemical
Evidence That Apomorphine Rescues Striatal Dopamine
Terminals and Prevents Methamphetamine Toxicity,”
Annals of the New York Academy of Sciences, Vol. 965,
2002, pp. 254-266.
http://dx.doi.org/10.1111/j.1749-6632.2002.tb04167.x
[13] E. E. Sam and N. Verbeke, “Free Radical Scavenging
Properties of Apomorphine Enantiomers and Dopamine:
Possible Implication in Their Mechanism of Action in
Parkinsonism,” Journal of Neural Transmission Parkin-
son’s Disease and Dementia Section, Vol. 10, No. 2-3,
1995, pp. 115-27. http://dx.doi.org/10.1007/BF02251227
[14] H. Yuan, et al., “Neuroprotective and Neurotrophic Effect
of Apomorphine in the Striatal 6-OHDA-Lesion Rat Mo-
del of Parkinson’s Disease,” Brain Research, Vol. 1026,
No. 1, 2004, pp. 95-107.
http://dx.doi.org/10.1016/j.brainres.2004.08.015
[15] Y. K. Chen, et al., “Potent, Hydroxyl Radical-Scavenging
Effect of Apomorphine with Iron and Dopamine Perfu-
sion in Rat Striatum,” Brain Research, Vol. 896, No. 1-2,
2001, pp. 165-168.
http://dx.doi.org/10.1016/S0006-8993(01)02081-9
[16] F. Fornai, et al., “Dose-Dependent Protective Effects of
Apomorphine against Methamphetamine-Induced Nigro-
striatal Damage,” Brain Research, Vol. 898, No. 1, 2001,
pp. 27-35.
http://dx.doi.org/10.1016/S0006-8993(01)02125-4
[17] E. Himeno, et al., “Apomorphine Treatment in Alzheimer
Mice Promoting Amyloid-Beta Degradation,” Annals of
Neurology, Vol. 69, No. 2, 2011, pp. 248-256.
http://dx.doi.org/10.1002/ana.22319
[18] M. Gassen, et al., “Apomorphine Is a Highly Potent Free
Radical Scavenger in Rat Brain Mitochondrial Fraction,”
European Journal of Pharmacology, Vol. 308, No. 2,
1996, pp. 219-225.
http://dx.doi.org/10.1016/0014-2999(96)00291-9
[19] G. Williamson, K. Faulkner and G. W. Plumb, “Glucosi-
nolates and Phenolics as Antioxidants from Plant Foods,”
European Journal of Cancer Prevention, Vol. 7, No. 1,
1998, pp. 17-21.
[20] A. Ubeda, et al., “Iron-Reducing and Free-Radical-
Scavenging Properties of Apomorphine and Some Relat-
ed Benzylisoquinolines,” Free Radical Biology & Medi-
cine, Vol. 15, No. 2, 1993, pp. 159-167.
http://dx.doi.org/10.1016/0891-5849(93)90055-Y
[21] H. Hara, M. Ohta and T. Adachi, “Apomorphine Protects
against 6-Hydroxydopamine-Induced Neuronal Cell
Death through Activation of the Nrf2-ARE Pathway,”
Journal of Neuroscience Research, Vol. 84, No. 4, 2006,
pp. 860-866. http://dx.doi.org/10.1002/jnr.20974
[22] E. Grunblatt, et al., “Effects of R- and S-Apomorphine on
MPTP-Induced Nigro-Striatal Dopamine Neuronal Loss,”
Journal of Neurochemistry, Vol. 77, No. 1, 2001, pp.
146-156.
http://dx.doi.org/10.1046/j.1471-4159.2001.t01-1-00227.
x
[23] L. Ma, et al., “Activation of Glutathione Peroxidase and
Inhibition of p53-Related Apoptosis by Apomorphine,”
Journal of Alzheimer’s Disease, Vol. 27, No. 1, 2011, pp.
225-237.
[24] O. Weinreb, S. Mandel and M. B. Youdim, “Gene and
Protein Expression Profiles of Anti- and Pro-Apoptotic
Actions of Dopamine, R-Apomorphine, Green Tea Poly-
phenol (-)-Epigallocatechine-3-Gallate, and Melatonin,”
Open Access NM

Involvement of CRH Receptors in the Neuroprotective Action of R-Apomorphine
in the Striatal 6-OHDA Rat Model
316
Annals of the New York Academy of Sciences, Vol. 993,
No. , 2003, pp. 351-361; Discussion 387-393.
http://dx.doi.org/10.1111/j.1749-6632.2003.tb07544.x
[25] V. Coronas, et al., “Dopamine D3 Receptor Stimulation
Promotes the Proliferation of Cells Derived from the
Post-Natal Subventricular Zone,” Journal of Neurochem-
istry, Vol. 91, No. 6, 2004, pp. 1292-1301.
http://dx.doi.org/10.1111/j.1471-4159.2004.02823.x
[26] A. Li, et al., “Apomorphine-Induced Activation of Dopa-
mine Receptors Modulates FGF-2 Expression in Astro-
cytic Cultures and Promotes Survival of Dopaminergic
Neurons,” FASEB Journal, Vol. 20, No. 8, 2006, pp.
1263-1265. http://dx.doi.org/10.1096/fj.05-5510fje
[27] H. Guo, et al., “Apomorphine Induces Trophic Factors
That Support Fetal Rat Mesencephalic Dopaminergic
Neurons in Cultures,” European Journal of Neuroscience,
Vol. 16, No. 10, 2002, pp. 1861-1870.
http://dx.doi.org/10.1046/j.1460-9568.2002.02256.x
[28] M. Roceri, et al., “Stimulatory Role of Dopamine on Fi-
broblast Growth Factor-2 Expression in Rat Striatum,”
Journal of Neurochemistry, Vol. 76, No. 4, 2001, pp.
990-997.
http://dx.doi.org/10.1046/j.1471-4159.2001.00088.x
[29] F. Vaglini, et al., “Apomorphine Offers New Insight into
Dopaminergic Neuron Vulnerability in Mesencephalic
Cultures,” Neuropharmacology, Vol. 55, No. 5, 2008, pp.
737-742.
http://dx.doi.org/10.1016/j.neuropharm.2008.06.041
[30] M. Varcin, et al., “Acute versus Long-Term Effects of 6-
Hydroxydopamine on Oxidative Stress and Dopamine
Depletion in the Striatum of Mice,” Journal Of Neuro-
science Methods, Vol. 202, No. 2, 2011, pp. 128-136.
http://dx.doi.org/10.1016/j.jneumeth.2011.07.004
[31] S. Taylor, et al., “A Practical Approach to RT-qPCR-
Publishing Data That Conform to the MIQE Guidelines,”
Methods, Vol. 50, No. 4, 2010, pp. S1-S5.
http://dx.doi.org/10.1016/j.ymeth.2010.01.005
[32] G. Paxinos and C. Watson, “The Rat Brain in Stereotaxic
Coordinates,” 2nd Edition, Academic Press, Waltham,
1986.
[33] M. J. Millan, et al., “The Role of Dopamine D3 Compar-
ed with D2 Receptors in the Control of Locomotor Activ-
ity: A Combined Behavioural and Neurochemical Analy-
sis with Novel, Selective Antagonists in Rats,” Psycho-
pharmacology (Berlin), Vol. 174, No. 3, 2004, pp. 341-
357. http://dx.doi.org/10.1007/s00213-003-1770-x
[34] B. Winner, et al., “Dopamine Receptor Activation Pro-
motes Adult Neurogenesis in an Acute Parkinson Model,”
Experimental Neurology, Vol. 219, No. 2, 2009, pp. 543-
552. http://dx.doi.org/10.1016/j.expneurol.2009.07.013
[35] S. Mandel, O. Weinreb and M. B. Youdim, “Using cDNA
Microarray to Assess Parkinson’s Disease Models and the
Effects of Neuroprotective Drugs,” Trends in Pharma-
cological Sciences, Vol. 24, No. 4, 2003, pp. 184-191.
http://dx.doi.org/10.1016/S0165-6147(03)00067-1
[36] A. S. Adewale, H. Macarthur and T. C. Westfall, “Neuro-
peptide Y Induced Modulation of Dopamine Synthesis in
the Striatum,” Regulatory Peptides, Vol. 129, No. 1-3,
2005, pp. 73-78.
http://dx.doi.org/10.1016/j.regpep.2005.01.005
[37] S. J. Na, A. G. DiLella, E. V. Lis, K. Jones, D. M. Levine,
D. J. Stone and J. F. Hess, “Molecular Profiling of a 6-
Hydroxydopamine Model of Parkinson’s Disease,” Neu-
rochemical Research, Vol. 35, No. 5, 2010, pp. 761-772.
http://dx.doi.org/10.1007/s11064-010-0133-3
[38] L. Marinova-Mutafchieva, M. Sadeghian, L. Broom, J. B.
Davis, A. D. Medhurst and D. T. Dexter, “Relationship
between Microglial Activation and Dopaminergic Neu-
ronal Loss in the Substantia Nigra: A Time Course Study
in a 6-Hydroxydopamine Model of Parkinson’s Disease,”
Journal of Neurochemistry, Vol. 110, No. 3, 2009, pp.
966-975.
http://dx.doi.org/10.1111/j.1471-4159.2009.06189.x
[39] S. Walsh, D. P. Finn and E. Dowd, “Time-Course of Ni-
grostriatal Neurodegeneration and Neuroinflammation in
the 6-Hydroxydopamine-Induced Axonal and Terminal
Lesion Models of Parkinson’s Disease in the Rat,” Neu-
roscience, Vol. 175, 2009, pp. 251-261.
http://dx.doi.org/10.1016/j.neuroscience.2010.12.005
[40] L. M. Collins, A. Toulouse, T. J. Connor and Y. M. No-
lan, “Contributions of Central and Systemic Inflammation
to the Pathophysiology of Parkinson’s Disease,” Neuro-
pharmacology, Vol. 62, No. 7, 2012, pp. 2154-2168.
http://dx.doi.org/10.1016/j.neuropharm.2012.01.028
[41] V. A. Doze and D. M. Perez, “G-Protein-Coupled Recep-
tors in Adult Neurogenesis,” Pharmacological Reviews,
Vol. 64, No. 3, 2012, pp. 645-675.
http://dx.doi.org/10.1124/pr.111.004762
[42] J. Ock, H. Lee, S. Kim, W. H. Lee, D. K. Choi, E. J. Park,
S. H. Kim, I. K. Kim and K. Suk, “Induction of Micro-
glial Apoptosis by Corticotropin-Releasing Hormone,” Jour-
nal of Neurochemistry, Vol. 98, No. 3, 2006, pp. 962-972.
http://dx.doi.org/10.1111/j.1471-4159.2006.03933.x
[43] A. Abuirmeileh, A. Harkavyi, A. Kingsbury, R. Lever and
P. S. Whitton, “The CRF-Like Peptide Urocortin Greatly
Attenuates Loss of Extracellular Striatal Dopamine in Rat
Models of Parkinson’s Disease by Activating CRF(1)
Receptors,” European Journal of Pharmacology, Vol. 604,
No. 1-3, 2009, pp. 45-50.
http://dx.doi.org/10.1016/j.ejphar.2008.11.009
[44] A. Abuirmeileh, A. Harkavyi, R. Lever, C. S. Biggs and P.
S. Whitton, “Urocortin, a CRF-Like Peptide, Restores Key
Indicators of Damage in the Substantia Nigra in a Neuro-
inflammatory Model of Parkinson’s Disease,” Journal of
Neuroinflammation, Vol. 4, 2007, p. 19.
http://dx.doi.org/10.1186/1742-2094-4-19
[45] A. Abuirmeileh, R. Lever, A. E. Kingsbury, A. J. Lees, I.
C. Locke, R. A. Knight, H. S. Chowdrey, C. S. Biggs and
P. S. Whitton, “The Corticotrophin-Releasing Factor-Like
Peptide Urocortin Reverses Key Deficits in Two Rodent
Models of Parkinson’s Disease,” European Journal of
Neuroscience, Vol. 26, No. 2, 2007, pp. 417-423.
http://dx.doi.org/10.1111/j.1460-9568.2007.05653.x
[46] W. A. Pedersen, R. Wan, P. Zhang and M. P. Mattson,
“Urocortin, but not Urocortin II, Protects Cultured Hip-
Open Access NM

Involvement of CRH Receptors in the Neuroprotective Action of R-Apomorphine
in the Striatal 6-OHDA Rat Model
317
pocampal Neurons from Oxidative and Excitotoxic Cell
Death via Corticotropin-Releasing Hormone Receptor Type
I,” Journal of Neuroscience, Vol. 22, No. 2, 2002, pp. 404-
412.
[47] E. Gonzalez-Rey, A. Fernandez-Martin, A. Chorny and M.
Delgado, “Therapeutic Effect of Urocortin and Adreno-
medullin in a Murine Model of Crohn’s Disease,” Gut,
Vol. 55, No. 6, 2006, pp. 824-832.
http://dx.doi.org/10.1136/gut.2005.084525
[48] H. Y. Huang, S. Z. Lin, W. F. Chen, K. W. Li, J. S. Kuo
and M. J. Wang, “Urocortin Modulates Dopaminergic Neu-
ronal Survival via Inhibition of Glycogen Synthase Kinase-
3β and Histone Deacetylase,” Neurobiology of Aging, Vol.
32, No. 9, 2011, pp. 1662-1677.
http://dx.doi.org/10.1016/j.neurobiolaging.2009.09.010
[49] Y. Kim, M. K. Park and S. Chung, “Protective Effect of
Urocortin on 1-Methyl-4-Phenylpyridinium-Induced Dopa-
minergic Neuronal Death,” Molecules and Cells, Vol. 30,
No. 5, 2010, pp. 427-433.
http://dx.doi.org/10.1007/s10059-010-0132-x
[50] A. C. Moss, P. Anton, T. Savidge, et al., “Urocortin II Me-
diates Pro-Inflammatory Effects in Human Colonocytes via
Corticotropin-Releasing Hormone Receptor 2α,” Gut, Vol.
56, No. 9, 2007, pp. 1210-1217.
http://dx.doi.org/10.1136/gut.2006.110668
[51] E. Kokkotou, D. Torres, A. C. Moss, M. O'Brien, D. E.
Grigoriadis, K. Karalis and C. Pothoulakis, “Corticotropin-
Releasing Hormone Receptor 2-Deficient Mice Have Re-
duced Intestinal Inflammatory Responses,” Journal of Im-
munology, Vol. 177, No. 5, 2006, pp. 3355-3361.
[52] M. Decressac, L. Prestoz, J. Veran, A. Cantereau, M. Ja-
ber and A. Gaillard, “Neuropeptide Y Stimulates Prolif-
eration, Migration and Differentiation of Neural Precur-
sors from the Subventricular Zone in Adult Mice,” Neu-
robiology of Disease, Vol. 34, No. 3, 2009, pp. 441-449.
http://dx.doi.org/10.1016/j.nbd.2009.02.017
[53] M. Dimitrijevic, S. Stanojevića, K. Mitića, N. Kuštrimo-
vića, V. Vujićb, T. Miletića and V. Kovačević-Jovanovića,
“The Anti-Inflammatory Effect of Neuropeptide Y (NPY)
in Rats Is Dependent on Dipeptidyl Peptidase 4 (DP4)
Activity and Age,” Peptides, Vol. 29, No. 12, 2008, pp.
2179-2187.
http://dx.doi.org/10.1016/j.peptides.2008.08.017
[54] M. Decressac, B. Wright, B. David, P. Tyers, M. Jaber, R.
A. Barker and A. Gaillard, “Exogenous Neuropeptide Y
Promotes in Vivo Hippocampal Neurogenesis,” Hippo-
campus, Vol. 21, No. 3, 2011, pp. 233-238.
http://dx.doi.org/10.1002/hipo.20765
[55] N. Thiriet, X. L. Deng, M. Solinas, B. Ladenheim, W.
Curtis, S. R. Goldberg, R. D. Palmiter and J. L. Cadet,
“Neuropeptide Y Protects against Methamphetamine-In-
duced Neuronal Apoptosis in the Mouse Striatum,” Jour-
nal of Neuroscience, Vol. 25, No. 22 , 2005, pp. 5273-
5279.
http://dx.doi.org/10.1523/JNEUROSCI.4893-04.2005
[56] A. S. Adewale, H. Macarthur and T. C. Westfall, “Neu-
ropeptide Y-Induced Enhancement of the Evoked Release
of Newly Synthesized Dopamine in Rat Striatum: Media-
tion by Y2 Receptors,” Neuropharmacology, Vol. 52, No.
6, 2007, pp. 1396-1402.
http://dx.doi.org/10.1016/j.neuropharm.2007.01.018
[57] D. Quarta, C. P. Leslie, R. Carletti, E. Valerio and L. Ca-
berlotto, “Central Administration of NPY or an NPY-Y5
Selective Agonist Increase in Vivo Extracellular Mono-
amine Levels in Mesocorticolimbic Projecting Areas,”
Neuropharmacology, Vol. 60, No. 2-3, 2011, pp. 328-335.
http://dx.doi.org/10.1016/j.neuropharm.2010.09.016
[58] E. Zambello, L. Zanetti, G. F. Hédou, O. Angelici, R. Ar-
ban, R. O. Tasan, G. Sperk and L. Caberlotto, “Neuro-
peptide Y-Y2 Receptor knockout Mice: Influence of Ge-
netic Background on Anxiety-Related Behaviors,” Neu-
roscience, Vol. 176, No. , 2010, pp. 420-430.
http://dx.doi.org/10.1016/j.neuroscience.2010.10.075
[59] M. Smialowska, H. Domin, B. Zieba, E. Koźniewska, R.
Michalik, P. Piotrowski and M. Kajta, “Neuroprotective
Effects of Neuropeptide Y-Y2 and Y5 Receptor Agonists
in Vitro and in Vivo,” Neuropeptides, Vol. 43, No. 3,
2009, pp. 235-249.
http://dx.doi.org/10.1016/j.npep.2009.02.002
[60] B. Mertens, A. Massie, Y. Michotte and S. Sarre, “Effect
of Nigrostriatal Damage Induced by 6-Hydroxydopamine
on the Expression of Glial Cell Line-Derived Neurotro-
phic Factor in the Striatum of the Rat,” Neuroscience, Vol.
162, No. 1, 2009, pp. 148-154.
http://dx.doi.org/10.1016/j.neuroscience.2009.04.036
[61] S. Mandel, E. Grünblatt, P. Riederer and M. B. Youdim,
“Genes and Oxidative Stress in Parkinsonism: cDNA Mi-
croarray Studies,” Advances in Neurology, Vol. 91, 2003,
pp. 123-132.
[62] M. Ohta, I. Mizuta, K. Ohta, M. Nishimura, E. Mizuta, K.
Hayashid and S. Kuno, “Apomorphine Up-Regulates NGF
and GDNF Synthesis in Cultured Mouse Astrocytes,”
Biochemical and Biophysical Research Communications,
Vol. 272, No. 1, 2000, pp. 18-22.
http://dx.doi.org/10.1006/bbrc.2000.2732
[63] I. Branchi, I. D’Andreaa, M. Armida, et al., “Striatal 6-
OHDA Lesion in Mice: Investigating Early Neurochemical
Changes Underlying Parkinson’s Disease,” Behavioural
Brain Research, Vol. 208, No. 1, 2010, pp. 137-143.
http://dx.doi.org/10.1016/j.bbr.2009.11.020
[64] V. Francardo, A. Recchia, N. Popovic, D. Andersson, H.
Nissbrandt and M. A. Cenci, “Impact of the Lesion Pro-
cedure on the Profiles of Motor Impairment and Molecu-
lar Responsiveness to L-DOPA in the 6-Hydroxydopa-
mine Mouse Model of Parkinson’s Disease,” Neurobiol-
ogy of Disease, Vol. 42, No. 3, 2011, pp. 327-340.
http://dx.doi.org/10.1016/j.nbd.2011.01.024
[65] S. J. Haas, A. Ahrens, S. Petrov, O. Schmitt and A. Wree,
“Quinolinic Acid Lesions of the Caudate Putamen in the
Rat Lead to a Local Increase of Ciliary Neurotrophic Fac-
tor,” Journal of Anatomy, Vol. 204, No. 4, 2004, pp. 271-
281. http://dx.doi.org/10.1111/j.0021-8782.2004.00279.x
[66] R. W. Rodrigues, V. C. Gomide and G. Chadi, “Astro-
glial and Microglial Activation in the Wistar Rat Ventral
Tegmental Area after a Single Striatal Injection of 6-Hy-
droxydopamine,” International Journal of Neuroscience,
Vol. 114, No. 2, 2004, pp. 197-216.
Open Access NM

Involvement of CRH Receptors in the Neuroprotective Action of R-Apomorphine
in the Striatal 6-OHDA Rat Model
Open Access NM
318
http://dx.doi.org/10.1080/00207450490249338
[67] V. C. Gomide, G. A. Silveira and G. Chadi, “Transient
and Widespread Astroglial Activation in the Brain after a
Striatal 6-OHDA-Induced Partial Lesion of the Nigrostri-
atal System,” International Journal of Neuroscience, Vol.
115, No. 1, 2005, pp. 99-117.
http://dx.doi.org/10.1080/00207450490512696
[68] J. Henning, U. Strauss, A. Wree, J. Gimsa, A. Rolfs, R.
Benecke and U. Gimsa, “Differential Astroglial Activation
in 6-Hydroxydopamine Models of Parkinson’s Disease,”
Neuroscience Research, Vol. 62, No. 4, 2008, pp. 246-
253. http://dx.doi.org/10.1016/j.neures.2008.09.001
[69] M. E. Emborg, “Evaluation of Animal Models of Parkin-
son’s Disease for Neuroprotective Strategies,” Journal of
Neuroscience Methods, Vol. 139, No. 2, 2004, pp. 121-
143. http://dx.doi.org/10.1016/j.jneumeth.2004.08.004
[70] F. Blandini, M. T. Armentero and E. Martignoni, “The
6-Hydroxydopamine Model: News from the Past,” Park-
insonism & Related Disorders, Vol. 14, Suppl. 2, 2008,
pp. S124-S129.
http://dx.doi.org/10.1016/j.parkreldis.2008.04.015
[71] S. Duty and P. Jenner, “Animal Models of Parkinson’s
Disease: A Source of Novel Treatments and Clues to the
Cause of the Disease,” British Journal of Pharmacology,
Vol. 164, No. 4, 2011, pp. 1357-1391.
http://dx.doi.org/10.1111/j.1476-5381.2011.01426.x