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Temporal Dissociation of Striatum and Prefrontal Cortex
Uncouples Anhedonia and Defense Behaviors Relevant
to Depression in 6-OHDA-Lesioned Rats
Filipe C. Matheus
1
&Daniel Rial
1,2
&Joana I. Real
2
&Cristina Lemos
2
&
Reinaldo N. Takahashi
1
&Leandro J. Bertoglio
1
&
Rodrigo A. Cunha
2,3
&Rui D. Prediger
1
Received: 2 February 2015 /Accepted: 1 July 2015
#Springer Science+Business Media New York 2015
Abstract The dorsolateral striatum (DLS) processes motor
and non-motor functions and undergoes extensive dopaminer-
gic degeneration in Parkinson’s disease (PD). The nigrostriatal
dopaminergic degeneration also affects other brain areas in-
cluding the pre-frontal cortex (PFC), which has been associ-
ated with the appearance of anhedonia and depression at pre-
motor phases of PD. Using behavioral, neurochemical, and
electrophysiological approaches, we investigated the temporal
dissociation between the role of the DLS and PFC in the
appearance of anhedonia and defense behaviors relevant to
depression in rats submitted to bilateral DLS lesions with 6-
hydroxydopamine (6-OHDA; 10 μg/hemisphere). 6-OHDA
induced partial dopaminergic nigrostriatal damage with no
gross motor impairments. Anhedonic-like behaviors were ob-
served in the splash and sucrose consumption tests only 7 days
after 6-OHDA lesion. By contrast, defense behaviors relevant
to depression evaluated in the forced swimming test and social
withdrawal only emerged 21 days after 6-OHDA lesion when
anhedonia was no longer present. These temporally dissociat-
ed behavioral alterations were coupled to temporal- and
structure-dependent alterations in dopaminergic markers such
as dopamine D
1
and D
2
receptors and dopamine transporter,
leading to altered dopamine sensitivity in DLS and PFC cir-
cuits, evaluated electrophysiologically. These results provide
the first demonstration of a dissociated involvement of the
DLS and PFC in anhedonic-like and defense behaviors rele-
vant to depression in 6-OHDA-lesioned rats, which was
linked with temporal fluctuations in dopaminergic receptor
density, leading to altered dopaminergic system sensitivity in
these two brain structures. This sheds new light to the duality
between depressive and anhedonic symptoms in PD.
Keywords Parkinson’s disease .Depression .Anhedonia .
Social isolation .Dorsolateral striatum .Pre-frontal cortex
Introduction
Parkinson’s disease (PD) patients exhibit non-motor alter-
ations several years before the onset of classical motor symp-
toms (i.e., rigidity, resting tremor, and bradykinesia) [1].
Mood disorders such as anhedonia and depression are insidi-
ous non-motor symptoms of PD and very detrimental to the
quality of life of PD patients [2]. These pre-motor symptoms
may fluctuate in intensity and also may be dissociated from
each other over time during the progression of PD [3].
Depression has a heterogeneous symptomatology and re-
sponsiveness to treatment. Anhedonia, a decreased ability to
experience pleasure, is a core symptom of this mental illness
[4], and its occurrence seems to be variable in intensity and
duration depending on the subtype of depression affecting the
patients [5].Social isolation is present in PD patients [6]andis
also a symptom found in some cases of depression [4]. Impor-
tantly, non-motor symptoms of PD are underdiagnosed and
Electronic supplementary material The online version of this article
(doi:10.1007/s12035-015-9330-z) contains supplementary material,
which is available to authorized users.
*Rui D. Prediger
ruidsp@hotmail.com
1
Departamento de Farmacologia, Centro de Ciências Biológicas,
Universidade Federal de Santa Catarina,
Florianópolis, SC 88049-900, Brazil
2
Center for Neuroscience and Cell Biology (CNC), University of
Coimbra, 3004-517 Coimbra, Portugal
3
Faculty of Medicine, University of Coimbra,
3005-504 Coimbra, Portugal
Mol Neurobiol
DOI 10.1007/s12035-015-9330-z
consequently undertreated [3], and it is critical to improve the
knowledge about the neurobiology of these symptoms in PD
patients to offer adequate therapeutic strategies. For instance,
many PD patients experience apathy independently of depres-
sion [7], warring for a better comprehension of the relative
modifications of different brain structures involved in the
modulation of emotional responses to clarify this duality be-
tween depression and anhedonia in PD.
Dopaminergic degeneration of dorsolateral striatum (DLS)
is a cardinal feature of PD, but it also affects other brain struc-
tures such as the pre-frontal (PFC) [8]. Functional connectiv-
ity between cortical and striatal neuronal systems plays a piv-
otal role in sensory, emotion, and motor functions [9–11].
However, the neurobiology of anhedonia and depression in
PD is still poorly understood, and the possible different par-
ticipation of the DLS and PFC to process different non-motor
symptoms of PD remains unknown.
In this study, through the use of behavioral, neurochemical,
and electrophysiological approaches, we investigated the tem-
poral and the structural dissociation between the role of DLS
and PFC in the appearance of anhedonia and defense behav-
iors relevant to depression in rats submitted to bilateral 6-
hydroxydopamine (6-OHDA) lesions in the DLS. Our find-
ings provide the first evidence of a dissociative involvement
of the DLS and PFC, respectively, in anhedonic and defense
behaviors relevant to depression in 6-OHDA-lesioned rats.
These behavioral modifications were accompanied by tempo-
ral fluctuations in the dopaminergic receptor density, leading
to altered pre-synaptic control and dopaminergic sensitivity in
the DLS and in the PFC.
Methods and Materials
Animals
Male Wistar rats weighing 300–350 g, aged 12–16 weeks at
the time of testing, were used. All procedures were approved
by the Institutional Ethical Committee for the care and use of
laboratory animals of the Federal University of Santa Catarina
(CEUA PP00830/2013) and the Ethical Committee of the
Center for Neuroscience of Coimbra.
Drugs and Stereotaxic Surgery
The dose of 6-OHDA (Sigma-Aldrich, USA) was selected
(Supplementary data Fig. S1 and Fig. S2) at a dose of
10 μg/hemisphere and was bilaterally injected into the DLS
(AP, +0.2 mm; ML, ±3.5 mm; DV, −4.8 mm from bregma and
dura). All animals were administered intra-peritoneally (i.p.)
with desipramine (20 mg/kg) (Sigma-Aldrich, USA) 30 min
before surgery, in order to protect noradrenergic terminals
from 6-OHDA toxicity. The stereotaxic surgery was
performed under ketamine (75 mg/kg)/xylazine (8 mg/kg)
i.p. anesthesia. Sham-operated rats followed the same protocol
except for the fact that vehicle was injected instead of 6-
OHDA. Fluoxetine (Sigma-Aldrich, USA) and bupropion
(Sigma-Aldrich, USA) were dissolved in saline solution, and
the dose of these drugs was selected based on previous studies
[12,13]. The behavioral experiments were carried out 7 or
21 days after the surgery and either transcardially perfused
or killed by decapitation after halothane anesthesia following
behavioral tests.
Behavioral Tests
Assessment of Motor Function
Spontaneous locomotor activity was assessed in the open field
test and allowed to freely explore it during 15 min to measure,
using the ANY-maze software (Stoelting, USA), the total dis-
tance travelled and the average speed as indicators of sponta-
neous locomotor activity, as described previously [14]. The
balance and motor coordination of rats were tested in the ac-
celerated rotarod (Insight, Ribeirão Preto, Brazil) with auto-
matic increase speed of the cylinder rotation (phase 1–2,
16 rpm; phase 3–4, 20 rpm; phase 5–6, 25 rpm; phase 7–8,
28 rpm; phase 9–10, 37 rpm) to measure the latency to fall, as
previously described [14]. The measurement of grip force was
made using a computerized grip force meter (Grip Strength
Meters, Columbus Instruments, Columbus, OH, USA), simul-
taneously evaluating the right and left limbs and expressing
grip force as the difference in grams, as previously described
[14].
Assessment of Emotional Parameters
The sucrose consumption test, frequently used to measure
anhedonia [15], compared the consumption of water and of
a 0.8 % sucrose solution during 48 h, as described previously
[16]. Additionally, the splash test, which evaluates a form of
motivational behavior considered to parallel some symptoms
of anhedonia such as apathetic behavior [17], was carried out
to measure the time spent grooming which was recorded for a
period of 15 min after squirting a 10 % sucrose solution on the
dorsal coat of the animals, as previously described [18]. De-
fense behaviors relevant to depression were evaluated in a
forced swimming test, scoring the total duration of immobility
and swimming during a 5-min test session in glass cylinders
containing water, as previously described [14]. Social interac-
tion was evaluated as previously described [19,20]bymea-
suring the amount of time spent by each rat in a pair sniffing,
grooming, following, kicking, mounting, jumping on, wres-
tling/boxing, and crawling under/over the partner during a 10-
min period of interaction. Anxiety-like behavior was evaluat-
ed using an elevated plus-maze by measuring during 5 min the
Mol Neurobiol
percent open arm entries (% open arm entries: open entries /
total entries × 100) and percent time on open arms (% open
time), as previously described [14].
Immunohistochemistry
The density of tyrosine hydroxylase (TH) was evaluated in
free-floating sections containing the DLS and substantia nigra
(25 μm thick) stained with a rabbit anti-TH polyclonal anti-
body (1:1500, ab-112 Abcam, UK) followed by a goat anti-
rabbit secondary antibody (1:2000; ab60317, Chromeo
TM
546
Abcam, UK), and the fluorescent signals were detected using
a fluorescent microscope (BioZero8000, Keyence Corp., Ja-
pan), as previously described [21].
Wester n Blo tting
Striatum or PFC homogenates were separated by SDS-PAGE
and, after electrotransfer, protein and molecular weight
markers (BioRad, Mississauga, Canada) were revealed by
Ponceau Red staining Then, membranes were incubated over-
night at 4 °C with antibodies anti-D
1
R (sc-14001; Santa Cruz
Biotechnology, USA), anti-D
2
R (sc-5303; Santa Cruz Bio-
technology, USA), or anti-DAT (MAB369; Millipore, Germa-
ny) and, after washing, with horseradish peroxidase-
conjugated anti-rabbit, anti-mouse, and anti-rat antibodies
for 2 h. Blots were visualized using the PerkinElmer ECL
system, as previously described [22].
Extracellular Electrophysiological Recordings
Electrophysiological recordings were carried out as previous-
ly described [23] by extracellularly recording population
spikes (PSs) either in layer V of the medial prelimbic PFC
upon stimulation of layers II/III in coronal slices (250–
300 μm thick) containing the PFC or in the DLS upon stim-
ulation of white matter above the DLS inslices (400 μmthick)
containing the DLS. The intensity of stimulation was selected
to yield 40–50 % of the maximum response, which was quan-
tified by its population spike amplitude. Paired-pulse stimula-
tion consisted of two stimuli delivered with an inter-stimuli
interval of 50 or 250 ms for PFC slices or 20 or 160 ms for
DLS slices. The paired-pulse ratio was calculated as the ratio
of the second response to the first response. Dopamine (25,
50, 100 μM) was cumulatively added through the superfusion
solution, and its effect estimated by changes of PS amplitude.
Experimental Protocol
Rats were first stereotaxically injected with 6-OHDA (or ve-
hicle for controls) in the DLS (Supplementary data Fig. S3),
and independent groups of animals were tested only once in
the behavioral tasks after either 7 or 21 days: Motor function
was first characterized using the rotarod, the open field, and
the grip force tests; then, the rats were tested in the non-motor
behavioral tasks such as sucrose preference and splash tests
(anhedonia), forced swimming and social interaction (defense
behaviors relevant to depression), and elevated plus-maze
(anxiety-like behaviors). Some rats were then transcardially
perfused, and their brains were processed for immunohisto-
chemical quantification of tyrosine hydroxylase (TH) in the
DLS and substantia nigra (SN). Other rats were killed and
their brains were dissected for Western blotting quantification
of D
1
R, D
2
R, and dopamine transporter in the striatum and
PFC. A last set of rats were killed to carry out extracellular
electrophysiological recordings in DLS and PFC slices. Final-
ly, independent experiments were carried out following the
same protocol, to test the impact of the antidepressant drugs
fluoxetine or bupropion on the anhedonic-like and defense
behaviors relevant to depression 7 and 21 days after 6-
OHDA lesion.
Data Analysis and Statistics
Statistical analysis was performed by two-way analysis of
variance (ANOVA) factorial followed by Newman-Keuls post
hoc test, except the analysis of electrophysiological data that
was carried out using Student’sttests. The values are
expressed as mean± standard error of mean (s.e.m), and the
significance level was P<0.05.
Results
Intra-DLS 6-OHDA Injection Induces Partial Loss
of Dopaminergic Neurons in the Nigrostriatal Pathway
with No Motor Impairments
The intra-striatal administration of 6-OHDA caused a retro-
grade lesion of the nigrostriatal dopaminergic pathway
(Fig. 1). In fact, both at 7 and at 21 days after 6-OHDA injec-
tion, the number of TH-positive cells was reduced by 6-
OHDA compared to control in both the DLS (7 days, 63.0±
2.5 %, F
(2, 21)
=423.0,P<0.05; 21 days, 65.6±1.8 %, F
(2, 21)
=
399.5, P<0.05) (Fig. 1a, b) and SN (7 days, 50.0±4.5 %, F
(2,
21)
=20.26, P< 0.05; 21 days, 68.0± 4.4 %, F
(2, 21)
=20.26,
P<0.05) (Fig. 1c, d); thus, there was a progression over time
of the lesion only in the SN (Fig. 1c), and the same extent of
degeneration was observed in both hemispheres (left and
right) in the DLS and SN at both time points after 6-OHDA
administration (Fig. 1).
However, the extent of 6-OHDA-induced dopaminergic
injury was not sufficient to impair motor function, as assessed
in different behavioral tests, namely, in the open field (Fig. 1e,
f), rotarod (Fig. 1g), and grip force tests (Fig. 1h).
Mol Neurobiol
Decoupling Anhedonia and Defense Behaviors Relevant
to Depression in 6-OHDA-Lesioned Rats
When compared to the control group, rats lesioned with 6-
OHDA displayed a decreased sucrose consumption (F
(1,
33)
=7.35, P< 0.05) (Fig. 2a) and a decreased time of grooming
(F
(1, 33)
=8.57, P< 0.05) (Fig. 2c) only 7 days after 6-OHDA
injection (but not after 21 days), which is indicative of an
anhedonic-like behavior. Conversely, in the forced swimming
test, 6-OHDA-lesioned rats displayed an increased immobility
time (F
(1, 33)
=12.93,P<0.05) (Fig. 2d) and a decreased swim-
ming time (F
(1, 33)
=13.22, P<0.05) only at 21 days (but not
after 7 days) after 6-OHDA injection (Fig. 2e). At this later
time point after 6-OHDA injection (21 days), the lesioned rats
also exhibited a significant reduction of social interaction be-
haviors (sniffing, grooming, following, kicking, mounting,
jumping on, wrestling/boxing, and crawling under/over the
partner) (Fig. 2f), which was not observed 7 days after 6-
OHDA injection. On the other hand, 6-OHDA-lesioned rats
did not exhibit a modified anxiety-like behavior in the elevat-
ed plus-maze at neither 7 nor 21 days after 6-OHDA injection
(Supplementary data Fig. S4).
The 6-OHDA-induced anhedonia observed only at 7 days
was prevented by a treatment with bupropion (10 mg/kg, i.p.,
during 7 days) in both the sucrose preference task (F
(1, 27)
=
7.73, P<0.05) (Fig. 3a) and in the splash test (F
(1, 26)
=62.52,
P<0.05) (Fig. 3c). The treatment with fluoxetine (10 mg/kg,
i.p., during 7 days) prevented the impairments induced by 6-
OHDA only in splash test (F
(1, 28)
=46.39, P<0.05) (Fig. 3d)
but not in sucrose preference task (F
(1, 27)
=1.14, P>0.05)
(Fig. 3b). Regarding the 6-OHDA-induced defense behaviors
relevant to depression observed only at 21 days, a chronic i.p.
treatment during 21 days with both fluoxetine (10 mg/kg) or
bupropion (10 mg/kg) prevented these alterations in immobil-
ity time (bupropion, F
(1, 28)
=18.05, P<0.05; fluoxetine, F
(1,
27)
=19.85, P<0.05) (Fig. 3e, f). Likewise, the 6-OHDA-
induced decreased investigation time in the social interaction
test observed only at 21 days was also prevented by a chronic
i.p. treatment during 21 days with both fluoxetine (10 mg/kg;
F
(1, 26)
=33.21, P<0.05) or bupropion (10 mg/kg; F
(1, 28)
=8.4
1, P<0.05) (Fig. 3g, h).
6-OHDA Induces Time-Dependent Changes of Dopamine
Receptors (D
1
RandD
2
R) and Transporter (DAT)
in the Striatum and PFC
The observed anhedonic and defense behaviors relevant to
depression caused by 6-OHDA administration were accompa-
nied by temporal fluctuations of the density of dopaminergic
markers. Thus, 6-OHDA increased the density of D
1
R(F
(1,
18)
=8.99, P< 0.05) (Fig. 4a)andD
2
R(F
(1, 18)
=3.68, P<0.05)
(Fig. 4b) in the striatum after 7 days, which returned to control
levels at 21 days after 6-OHDA administration (Fig. 4a, b). By
contrast, 6-OHDA did not modify the density of either D
1
Ror
D
2
R in the PFC either after 7 days or after 21 days (Fig. 4d, e).
Finally, we report that 6-OHDA decreased DAT density both
in the striatum (F
(1, 18)
=10.58,P< 0.05) (Fig. 4c)aswellasin
the PFC (F
(1, 17)
=13.65, P<0.05) (Fig. 4f)onlyat21days.
Fig. 1 6-Hydroxydopamine (6-OHDA) decreased the optical density of
tyrosine hydroxylase (TH) in the dorsolateral striatum (DLS) and the
number of TH-positive cells in the substantia nigra (SN) to a similar
extent in both hemispheres left (L) and right (R) with no motor impair-
ments. aImmunohistochemical quantification of TH staining in the DLS.
bRepresentative coronal sections of the DLS. cImmunohistochemical
quantification of TH-positive cells in the SN. dRepresentative coronal
sections of the SN. eTotal distance travelled during 15 min in the open
field apparatus. fAverage speed in the open field. gLatencytofallinthe
accelerated rotarod test. hGrip force test to evaluate the strength of the
forelimbs. (*P<0.05 vs. control group;
#
P<0.05 vs. 6-OHDA group
7 days after injection, two-way ANOVA followed by the Newman-
Keuls post hoc tests; N=5–10 rats/group)
Mol Neurobiol
Fig. 2 Temporal dissociation between anhedonic-like and defense be-
haviors in 6-hydroxydopamine (6-OHDA)-lesioned rats. 6-OHDA de-
creased sucrose preference when compared to control group 7 days after
6-OHDA injection, (a) and this anhedonic-like behavior was not present
21 days after the 6-OHDA injection (b). The same pattern of response
was observed in the splash test, confirming that anhedonic-like behavior
was present only after 7 days (c). 6-OHDA increased the immobility time
(d) and decreased the swimming time (e) in the forced swimming test
only 21 days after 6-OHDA injection. In the social interaction test, 6-
OHDA increased the social isolation when compared to control group
only 21 days after 6-OHDA injection (f). (*P< 0.05 vs. control group,
two-way ANOVA followed by the Newman-Keuls post hoc tests; N=8–
10 rats/group)
Fig. 3 The effects of bupropion (10 mg/kg, i.p.) and fluoxetine
(10 mg/kg, i.p.) on the anhedonic-like and defense behaviors of 6-
hydroxydopamine (6-OHDA)-lesioned rats. Treatment with bupropion
(a), but not with fluoxetine (b), during 7 days reversed the anhedonic-
like behaviors induced by 6-OHDA lesion. Both bupropion (c)andflu-
oxetine (d) treatments during 7 days reversed the 6-OHDA-induced re-
duction of grooming time in splash test. Chronic treatments during
21 days with bupropion or fluoxetine reversed the helpless behavior in
the forced swimming test (e,f) and social isolation (g,h) induced by 6-
OHDA. (Sucrose preference test, *P< 0.05 vs. when compared sucrose
consumption with water consumption in the test phase by each group;
#
P<0.05 vs. sucrose consumption of control group, two-way ANOVA.
Splash, forced swimming, and social interaction tests, *P<0.05 vs. con-
trol group;
#
P<0.05 vs. 6-OHDA group, two-way ANOVA followed by
the Newman-Keuls post hoc tests; N=8–10 rats/group)
Mol Neurobiol
6-OHDA Induces Temporal Dissociation of Dopamine
Sensitivity in the DLS and PFC
Electrophysiological recordings in the DLS or in PFC re-
vealed that the 6-OHDA administration did not alter the
stimulus-sensitivity curve at either 7 or 21 days after lesion
(Supplementary data Fig. S5). Paired-pulse stimulation
showed a significant decrease of paired-pulse facilitation in
the DLS only at 7 (but not 21) days after 6-OHDA lesion
(Fig. 5a), indicative of an early striatal pre-synaptic disruption,
whereas paired-pulse stimulation was not modified in the PFC
either 7 or 21 days after 6-OHDA administration (Fig. 5e).
Since 6-OHDA induces different time-dependent changes
of dopamine receptors (D
1
RandD
2
R) and DAT in the stria-
tum and PFC, we next investigated possible 6-OHDA-
induced changes of dopamine sensitivity on synaptic trans-
mission in DLS and PFC slices. In contrast to slices from
control rats, dopamine increased synaptic transmission in the
DLS at concentrations of 25 μM (75±31 % over baseline, n=
4, P<0.05) and 50 μM (64±35 % over baseline, n=4,
P<0.05) at 7 days (Fig. 5b)butnotat21days(Fig.5c)after
6-OHDA lesion. By contrast, in the PFC, there was no alter-
ations of dopamine sensitivity at 7 days (Fig. 5f), but we
observed a decreased dopamine sensitivity selectively at
21 days after the 6-OHDA lesion (21±6 % inhibition by
25 μM dopamine in 6-OHDA-lesioned vs. 62± 5 % inhibition
in control, n=4, P< 0.05; 35± 5 % inhibition by 50 μMdopa-
mine in 6-OHDA-challenged vs. 67±7 % inhibition in con-
trol, n=4, P<0.05 (Fig. 5g).
Discussion
The present study demonstrates that a mild bilateral dopaminer-
gic lesion of the rat DLS, which was insufficient to trigger motor
deficits, mimicked some core non-motor symptoms of PD such
as anhedonia and defense behaviors relevant to depression, as
occurs in the prodrome phase of PD. Most importantly, we
observed a temporal dissociation between anhedonia and de-
fense behaviors relevant to depression that was coupled with a
parallel temporal dissociation of neurochemical and electro-
physiological alterations in the dorsolateral striatum and in the
pre-frontal cortex. This indicates a dissociated neurobiological
basis for the depressive and anhedonic symptoms in early PD.
The present model of 6-OHDA administration together
with desipramine triggered a stable decrease of TH immuno-
reactivity in the striatum but an evolving dopaminergic lesion
in the substantia nigra (40 % at 7 days and 68 % at 21 days), as
occurs in PD patients [1]. Importantly, this observed extent of
dopaminergic degeneration was not sufficient to impair motor
Fig. 4 6-Hydroxydopamine (6-OHDA) differently altered the density of
dopamine receptors (D
1
R and D
2
R) and dopamine transporter (DAT)
with time (7 and 21 days after 6-OHDA injection) in the striatum and
pre-frontal cortex (PFC). Western blot quantification of D
1
R(a), D
2
R(b),
and DAT (c) in striatum; D
1
R(d), D
2
R(e), and DAT (f)inPFC.(*P<0.05
vs. control group,
#
P<0.05 vs. 6-OHDA group 7 days after injection,
two-way ANOVA followed by the Newman-Keuls post hoc tests; N=5
rats/group)
Mol Neurobiol
function, as gauged by the lack of modification of the perfor-
mance in the open field (horizontal activity), rotarod (balance
and coordination), and grip force (strength of the forelimbs)
tests. However, this mild dopaminergic damage triggered
mood-related behavioral alterations, such as anhedonia, de-
fense behaviors, and impaired social interaction, as also ob-
served in previous studies [24–26]; thus, this 6-OHDA-based
model is an experimental model well-suited to evaluate be-
haviors related to symptoms occurring in the PD prodrome [3,
27,28], without affecting motor functions that could confound
the interpretation of emotional parameters associated to de-
fense behaviors relevant to depression and anhedonia. Fur-
thermore, we now confirmed that fluoxetine and the atypical
antidepressant drug bupropion prevented the appearance of
these behavioral alterations, further validating the pertinence
of these alterations to the PD-related depressive status [29,
30]. This is particularly relevant since PD patients exhibit a
wide range of non-motor symptoms such as anhedonia, de-
pression, and anxiety that affect their quality of life and rep-
resent a major unmet therapeutic need [2,3,6].
A main finding of the present study was the temporal dis-
sociation of the appearance of these different behavioral alter-
ations: thus, anhedonic-like behaviors, evaluated in sucrose
preference and in splash tests, were observed at early
(7 days) but not at later (21 days) time points after 6-OHDA
injection; by contrast, defense behaviors relevant to depres-
sion were present only at later periods (21 days) after 6-
OHDA injection. Interestingly, parkinsonian patients also ex-
hibit this dissociation of symptoms of anhedonia and depres-
sion [31,32]. Since 6-OHDA triggers functional alterations in
the striatum [33,34] as well as in the PFC [35,36] and these
two interconnected brain regions [37,38] are involved in the
control of motivation, reward, and defense behaviors relevant
to depression [reviewed in [39–42], we explored neurochem-
ical and electrophysiological alterations in these two brain
regions at 7 and at 21 days after 6-OHDA administration to
gain a mechanistic insight on this temporal dissociation be-
tween anhedonia and defense behaviors relevant to depres-
sion. We observed that the appearance of anhedonia at 7 days
was selectively associated with an increased density of both
D
1
and D
2
receptors in the striatum, together with a decreased
short-term plasticity and increased dopaminergic sensitivity in
corticostriatal synapses. This is in notable agreement with the
involvement in operant motivation of aberrant dopaminergic
signaling in the striatum [43–46], suggesting that abnormal
dopaminergic control of striatal function might be selectively
associated with the hedonic alterations observed 7 days after
6-OHDA administration. This is re-enforced by the observa-
tion that there was a parallel disappearance of anhedonic be-
havior as well as striatal dopaminergic alterations at 21 days
after 6-OHDA administration. By contrast, at 21 days after 6-
OHDA administration, we observed the emergence of defense
behaviors relevant to depression that paralleled the appearance
of changes now in the PFC, typified by a reduction of the
density of DAT and a decreased inhibition by dopamine of
excitatory transmission in the PFC. This is in agreement with
the involvement of the PFC in the control of emotional re-
sponses [reviewed in [39–42] and in the appearance of non-
motor symptoms in PD [27,28]. Also, PFC dysfunction un-
derlies major depressive disorders [40,47,48]withpreserved
or disrupted hedonic function [49] and is also involved in the
emergence of social phobia and their comorbidities [50]. This
tentative association of PFC modifications with helpless and
social impaired behaviors is re-enforced by the observation
Fig. 5 6-Hydroxydopamine (6-OHDA) induces a temporal and structur-
al dissociation of dopamine sensitivity in excitatory synapses of the dor-
solateral striatum (DLS) and in the medial prefrontal cortex (PFC). In
population spikes recorded in the DLS (a), the paired-pulse facilitation
(ratio of the second vs. first pulse, P2/P1 ratio, with an inter-pulse interval
of 20 ms) was decreased at 7 but not at 21 days after injection of 6-OHDA
(b). Dopamine (25 and 50 μM) increased synaptic transmission in the
DLS, when compared to control group at 7 days after the injection of 6-
OHDA (c) but was devoid of effects at 21 days after 6-OHDA injection
(d). In population spikes recorded in layer 5 of the PFC (e), the paired-
pulse facilitation (ratio of the second vs. first pulse, P2/P1 ratio, with an
inter-pulse interval of 50 ms) was not altered either at 7 but not at 21 days
after 6-OHDA injection (f). Dopamine sensitivity was not affected 7 days
after 6-OHDA injection (g), but there was a decreased sensitivity (25 and
50 μM) to inhibit synaptic transmission 7 days after the injection of 6-
OHDA when compared to control group at (h). (*P<0.05 vs. control
group, Student’sttest; N=4 rats/group)
Mol Neurobiol
that there was a lack of neurochemical and electrophysiolog-
ical modifications at 7 days after 6-OHDA administration,
which paralleled the lack of modification of defense behaviors
relevant to depression. These observations lead us to propose
that the behavioral dissociation between anhedonia and de-
fense behaviors relevant to depression could be related to
time-dependent alternations in these two different brain re-
gions, namely, the striatum and PFC, respectively. This is line
with the complex impact [51,52] of the dopaminergic system
in the control of anhedonia and other defense behaviors
relevant to depression [reviewed in [53,54] and with the
different levels of dopaminergic denervation causing differ-
ent changes in dopaminergic signaling in different brain
regions [55]. This is also in notable agreement with some
clinical studies suggesting a different involvement of dif-
ferent brain areas in the relationship of anhedonia and
depression in PD patients [32,56].
Overall, these results provide pioneering evidence of a tem-
poral and structural dissociation of the role of DLS and PFC
related to different non-motor behavioral impairments in rats
submitted to 6-OHDA. These behavioral modifications are
accompanied by different temporal fluctuations in the striatum
and PFC of the dopaminergic receptor density, leading to al-
tered pre-synaptic control and dopaminergic system sensitiv-
ity. This sheds new light on the neurobiological basis under-
lying the dissociation of anhedonia and defense behaviors
relevant to depression in early PD, although the exact patho-
physiological mechanisms underlying these conditions still
need to be clarified.
Acknowledgments This work was supported by grants from Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq),
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES-FCT), Programa de Apoio aos Núcleos de Excelência
(PRONEX - Project NENASC), Fundação de Apoio à Pesquisa do
Estado de Santa Catarina (FAPESC), Ciência sem Fronteiras, DARPA
(09-68-ESR-FP-010), NARSAD, QREN (09-68-ESR-FP-010), and
Santa Casa da Misericórdia. F.C.M and D.R received scholarships from
CNPq. R.N.T. and R.D.P. are supported by research fellowship from
CNPq.
Conflict of interest All authors reported no biomedical financial inter-
ests or potential conflicts of interest.
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