Neurobiology of Disease 66 (2014) 19–27
Contents lists available at ScienceDirect
Neurobiology of Disease
journal homepage: www.elsevier.com/locate/ynbdi
Dopaminergic manipulations and its effects on neurogenesis and motor
function in a transgenic mouse model of Huntington's disease
M.L. Choi a,1, F. Begeti a,b,1, J.H. Oh c, S.Y. Lee c, G.C. O'Keeffe a, C.D. Clelland a, P. Tyers a, Z.H. Cho a,
Y.B. Kim a, R.A. Barker a,d,⁎
a
Department of Clinical Neurosciences, John van Geest Centre for Brain Repair, University of Cambridge, Cambridge CB2 0PY, UK
School of Clinical Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge CB2 0SP, UK
Neuroscience Research Institute, Gachon University, Incheon 405-760, Republic of Korea
d
Department of Neurology, Addenbrooke's Hospital, Cambridge CB2 0QQ, UK
b
c
a r t i c l e
i n f o
Article history:
Received 17 October 2013
Revised 29 January 2014
Accepted 10 February 2014
Available online 19 February 2014
Keywords:
Huntington's disease
Dopamine
Adult hippocampal neurogenesis
a b s t r a c t
Huntington's disease (HD) is an inherited neurodegenerative disorder that is classically defined by a triad of
movement and cognitive and psychiatric abnormalities with a well-established pathology that affects the
dopaminergic systems of the brain. This has classically been described in terms of an early loss of dopamine D2
receptors (D2R), although interestingly the treatments most effectively used to treat patients with HD block
these same receptors. We therefore sought to examine the dopaminergic system in HD not only in terms of
striatal function but also at extrastriatal sites especially the hippocampus, given that transgenic (Tg) mice also
exhibit deficits in hippocampal-dependent cognitive tests and a reduction in adult hippocampal neurogenesis.
We showed that there was an early reduction of D2R in both the striatum and dentate gyrus (DG) of the hippocampus in the R6/1 transgenic HD mouse ahead of any overt motor signs and before striatal neuronal loss.
Despite downregulation of D2Rs in these sites, further reduction of the dopaminergic input to these sites by
either medial forebrain bundle lesions or receptor blockade using sulpiride was able to improve both deficits
in motor performance and adult hippocampal neurogenesis. In contrast, a reduction in dopaminergic innervation
of the neurogenic niches resulted in impaired neurogenesis in healthy WT mice. This study therefore provides
evidence that D2R blockade improves hippocampal and striatal deficits in HD mice although the underlying
mechanism for this is unclear, and suggests that agents working within this network may have greater effects
than previously thought.
© 2014 Elsevier Inc. All rights reserved.
Introduction
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder that is clinically defined by a movement disorder (typically
chorea) together with cognitive and psychiatric disturbances. As in
other neurodegenerative disorders, the functional abnormalities in HD
have been proposed to result from a compromise of neural circuitry
associated with cellular dysfunction or death (Ross and Tabrizi, 2011).
The neuronal loss in HD is extensive, and in particular, many areas of
the CNS including the hippocampus are affected by the disease process
Abbreviations: 6-OHDA, 6-hydroxyldopamine; D1R, dopamine D1 receptor; D2R,
dopamine D2 receptor; DCX, doublecortin; DG, dentate gyrus; HD, Huntington's disease;
L-DOPA, 3,4-dihydroxy-L-phenylalanine; micro-PET, micro-positron emission tomography;
MFB, medial forebrain bundle; SVZ, subventricular zone; TH, tyrosine hydroxylase; VTA,
ventral tegmental area.
⁎ Corresponding author at: Department of Clinical Neurosciences, John van Geest
Centre for Brain Repair, University of Cambridge, Cambridge CB2 0PY, UK.
E-mail address: [email protected] (R.A. Barker).
Available online on ScienceDirect (www.sciencedirect.com).
1
M. L. Choi and F. Begeti are the first authors.
http://dx.doi.org/10.1016/j.nbd.2014.02.004
0969-9961/© 2014 Elsevier Inc. All rights reserved.
even in the earliest stages (Ross and Tabrizi, 2011). Indeed, HD transgenic (Tg) mouse models exhibit a range of hippocampal-dependent
impairments, one of the best described being a reduction in adult
neurogenesis in the dentate gyrus (DG) (Gil et al., 2005; Lazic et al.,
2004; Phillips et al., 2005). During this process, endogenous neural
precursor cells give rise to immature neurons which eventually mature
and functionally integrate into the circuitry (Ming and Song, 2011), a
process which is critical to some aspects of cognition (Clelland et al.,
2009; Sahay et al., 2011).
Dopaminergic disturbances are also a common feature of the disease
particularly a progressive reduction in striatal and extrastriatal D2
receptor (D2R) binding which begins before clinical manifestations
and correlates with disease progression and frontostriatal cognitive
impairment (Bäckman and Farde, 2001; Ginovart et al., 1997; Pavese
et al., 2003, 2010; Ramos et al., 2013; van Oostrom et al., 2009). In line
with human HD studies, there is evidence of an early reduction in
striatal D2R expression in HD Tg mice (Benn et al., 2010; Cha et al.,
1998; Cummings et al., 2006; Glass et al., 2004) although there have
been limited approaches to demonstrate this in vivo using PET imaging
as has been done in human HD patients. Furthermore, whether similar
20
M.L. Choi et al. / Neurobiology of Disease 66 (2014) 19–27
(9.25–12.95 MBq, 200 μl) via the tail vein. The acquired data was reconstructed using a 2D-filtered back-projection algorithm (microPET
ManagerTM, Siemens Medical Solutions, Knoxville, USA). Regions
of interest (ROIs) for the striatum were drawn on the reconstructed
PET images using a mouse brain atlas (Paxinos and Franklin, 2001).
Regional radioactivity was determined for each dynamic frame in the
ROIs. Tracer binding was calculated on the basis of the binding potential
(BP) derived from a Logan plot graphical analysis technique (Logan
et al., 1996).
changes occur in the dentate gyrus is unknown. Such questions have
significant clinical interest as the majority of drugs used to effectively
treat this disease block these receptors and/or deplete presynaptic
dopamine (Mason and Barker, 2009; Priller et al., 2008).
We therefore sought to examine the dopaminergic system in HD not
only in terms of striatal function but also at extrastriatal sites, especially
the hippocampus. We used the R6/1 HD Tg mouse model which
expresses exon 1 of the human huntingtin (htt) gene carrying 110–
120 CAG repeats (Mangiarini et al., 1996). This line of HD mice has
been well studied in terms of both the dopaminergic system and adult
neurogenesis and also has a relatively slow evolution of progressive
motor signs which makes them amenable especially to studies looking
at the premanifest motor phase of the illness (Lazic et al., 2004; Ortiz
et al., 2011; Petersen et al., 2002; Walker et al., 2011). We now demonstrate that in these HD Tg mice, using in vivo micro-PET imaging and
mRNA expression, there is a significant reduction of D2R expression in
the striatum and DG respectively which begins ahead of overt disease
manifestations and neuronal loss in agreement with previous studies.
Despite this reduction of D2R, further reduction of the dopaminergic
input from a partial bilateral medial forebrain bundle (MFB) lesion
paradoxically improved motor behavior and partially ameliorated
adult hippocampal neurogenesis in the R6/1 mouse model of HD
while having opposite effects in wild-type (WT) mice. Using the D2R
antagonist which is commonly used to treat chorea and behavioral
symptoms in HD, we observed a partial increase in hippocampal
neurogenesis while there was no improvement in motor impairment.
These findings have important clinical implications especially given
that dopaminergic drugs are widely used in the treatment of patients
with HD.
The DG was micro-dissected according to a previously described
protocol (Hagihara et al., 2009) and samples were stored at − 80 °C
until use. RNA was extracted using an RNeasy Mini Kit (Qiagen,
74106) according to the manufacturer instructions and eluted in 30 μl
of RNase-free water, the concentration of which was determined
using a Nanodrop 2000c spectrophotometer (Thermo Scientific) and
stored at − 80 °C. cDNA was reverse transcribed using a Transcriptor
First Strand cDNA Synthesis Kit (Roche) with anchored-oligo(dT)
primers according to the manufacturer instructions. Quantitative
Polymerase Chain Reactions (qPCR) were subsequently undertaken
using Solaris Mouse qPCR Gene Expression Assays for D2DR. ATP5b
was chosen as the reference gene due to its stable level of expression
in HD mouse models when compared to other commonly used housekeeping genes (Benn et al., 2008). All qPCR reactions were performed
in triplicates. Each dissection contained 3–5 R6/1 mice and 3–5 WT
littermates and results were repeated with three independent dissections at different ages: 8, 12 and 16 weeks.
Materials and method
Dopamine lesion surgery
Animals
12–13 week old WT (n = 30 from Harlan Laboratories) mice or R6/1
(n = 22) mice and their WT littermates (n = 19) received bilateral injections of 6-OHDA (Sigma, UK) into the medial forebrain bundle
(MFB). A stock of 6-OHDA was prepared in 0.01% ascorbic acid/saline
and stored at −20 °C. The working doses were diluted daily and then
carried on ice to the operating room. Mouse surgery was performed
under isoflurane anesthesia using a mouse stereotaxic frame. The stereotaxic coordinates for MFB lesions were A/P: − 1.1 (from bregma),
M/L: ±1.1, and D/V: −4.7 (from the dura surface). Drugs were injected
bilaterally at the rate of 0.5 μl/min through a Hamilton syringe (Hamilton Company, Switzerland) and the needle was left in situ for 5 min following delivery of the toxin to allow for its diffusion. For sham surgery,
the identical volume of saline was injected using the same protocols as
for the 6-OHDA injections.
C57BL/6 wild type (WT) mice were purchased from Harlan Laboratories (UK). R6/1 Tg mice were purchased from the Jackson Laboratory
(USA) and the colony was maintained by backcrossing to C57BL/6 females purchased from Harlan Laboratories (UK). After the mice were
weaned, tissue from ear biopsies was sent to Largen Inc. (USA) for
genotyping. All animals were kept in a temperature and humiditycontrolled (22 °C) room on a 12-hour light/dark cycle. The mice were
separately housed in single-sex cages of 3–4 mice per cage. Water and
food was made freely available in the home cage. All experiments
were performed using only female mice in the John van Geest Centre
for Brain Repair, University of Cambridge, UK and Neuroscience Research Institute, Gachon University, Republic of Korea in strict accordance with appropriate Home Office project and personal licenses.
Only female mice were used because of the need to avoid problems of
fighting and sex hormone influences on any behavioral tests. The protocols were approved by the ethical committees of the University of Cambridge (UK) and the University of Gachon (Republic of Korea).
Micro-PET imaging
For the micro-PET study, animals were shipped via an air courier to
NRI, Gachon University of Medicine & Science, Republic of Korea and
allowed to recover for 2 weeks prior to scanning. R6/1 (n = 4) and
WT littermate (n = 4) mice were scanned twice, at 12–13 weeks and
at 21–23 weeks of age. Anesthesia was induced and maintained with
passive oxygen/isoflurane at 1.5 l/min. Each mouse was positioned
prone on a bed with its brain centered in the gantry and its head fixed
by a nose bar. A PET scan was performed using a Focus 120 MicroPET
system (Concorde Microsystems, Knoxville, TN) with 1.18 mm (radial),
1.13 mm (tangential) and 1.45 mm full width at half maximum
(FWHM) resolution at the center (Kim et al., 2007). Dynamic scans
were performed for 90 min immediately after a [C] raclopride injection
mRNA expression
Drug administration
Quinpirole and S-(–) sulpiride were purchased from Tocris (UK) and
dissolved in saline with ethanol (quinpirole: to 100 mM and S-(–)
sulpiride: to 10 mM, all of which were soluble in saline) using a warm
bath (30 °C). Stocks of a constant volume were stored in a − 20 °C
freezer until use. 3,4-Dihydroxy-L-phenylalanine (L-DOPA, Sigma UK)
was dissolved in saline using a warm bath on the day of administration
(6 mg/kg) and was stabilized by co-injection with benserazide hydrochloride (12 mg/kg, Sigma UK). Stock drugs were dissolved in saline
to obtain final working concentrations (quinpirole: 5 ml/kg, sulpiride:
10 ml/kg). Either drug or saline was injected intraperitoneally (i.p.) at
10 ml/kg volume in R6/1 (n = 27) and their WT littermate mice (n =
24).
Immunohistochemistry and cell quantification
Animals were perfused with 4% paraformaldehyde (PFA) and brains
were post fixed overnight and then cryoprotected in 30% sucrose. Brains
M.L. Choi et al. / Neurobiology of Disease 66 (2014) 19–27
were cut into 40 μm free-floating coronal sections which were stored in
30% glycerol anti-freezing solution until use. The following immunohistochemistry was undertaken:
- Adult neurogenesis: a one in twelve series of sections was stained
with doublecortin (DCX) (goat anti-DCX, 1:250, Santa Cruz
Biotechnology) and visualized with a fluorescent secondary
antibody (Alexa donkey anti-goat, 1:250, Invitrogen). DCX positive
cells were counted throughout the SVZ of the LV and the
rostrocaudal extent of the granular cell layer (GCL) and SGZ within
the DG. All sections were double stained with neuronal nuclei
(NeuN).
- Striatal neuronal population: a one in twelve series of sections
was stained with NeuN (mouse anti-NeuN, 1:500, Millipore) and
visualized with fluorescent secondary antibodies (Alexa donkey
anti-mouse 647, 1:250, Invitrogen). NeuN positive cells were
counted in both the dorsal and the ventral striatum.
- Dopaminergic innervation: a one in six series of sections was
stained with tyrosine hydroxylase (TH, rabbit anti-TH, 1:4000,
Millipore) visualized by biotinylated secondary antibody (antirabbit, 1:200, Invitrogen) and the diaminobenzidine system
(Vectastain ABC kit, Vector Laboratories) in three areas, the
substantia nigra (SN), VTA and striatum. The number of TH positive
cells was counted in the SN and VTA and the intensity of TH fibers
was measured in the striatum using images of the whole striatum
captured by the Olympus Stereo Investigator. The degree of TH
fiber reduction by a 6-OHDA lesion was obtained by comparing
it to that recorded in the control group. The degree of TH immunoreactivity in the SVZ and SGZ was insufficiently intense to be
quantified.
21
Results
D2Rs are down-regulated early in the disease course of R6/1 mice in the
striatum and DG
We used [11C]raclopride PET imaging to examine D2R changes in
premanifest and manifest R6/1 mice compared to WT littermates.
Mice were scanned twice, between 11–13 weeks and 21–23 weeks of
age, and the whole striatum was used as the region of interest. Striatal
D2R binding was significantly decreased in the 11–13 week old R6/1
mice compared to their WT littermates (P = 0.006). This was also the
case in the 21–23 week old mice (P = 0.009) although there was no
further reduction in D2R binding at this time (Fig. 1B), despite the
animals having developed motor features (Mangiarini et al., 1996).
Histological analysis showed no significant difference in the number
of mature NeuN + neurons in the striatum of 11–13 week R6/1 mice
indicating that the reduction in D2R binding was not due to neuronal
loss (Fig. 1D). Manifest 21–23 week old R6/1 mice had a reduction in
the number of neurons in the striatum (P = 0.0009, Fig. 1D), despite
there being no further reduction in D2R binding. Since the current resolution of PET is too low to quantify D2R in the DG, we micro-dissected
this region in R6/1 mice aged 8, 12 and 16 weeks old, in order to
measure mRNA expression. We observed a reduction in D2R mRNA at
all age points studied (P = 0.0009, Fig. 1C). Again the loss of D2R
expression was not progressive in R6/1 mice.
Partial 6-OHDA lesions of the MFB lead to a decrease in adult neurogenesis
in both the SVZ and SGZ of WT mice
Motor balance and co-ordination was assessed using the rotarod
(Ugo basile, SRL) in a temperature and humidity-controlled (22 °C)
room and the test was performed during the light cycle (between
7 am and 7 pm). The speed of rotation was increased by 4 rpm every
30 s until it reached 44 rpm. One day prior to testing, all animals were habituated to the experimental room and the rotarod. On the test day mice
were individually placed on the rod and it was checked that they
were able to walk forward at 4 rpm for a minute before starting
the test. The test was conducted for one day and consisted of three
trials. The speed at which each mouse fell off the rotarod was
recorded and each mouse was tested three times with a 15-minute
interval between trials. Animals were returned to the home cage
during this interval. The average speed at which they fell off for the
three trials was used.
We next investigated the role of dopamine in WT mice by reducing
dopaminergic input to both constitutive neurogenic sites via selective
6-hydroxydopamine (6-OHDA) partial bilateral lesions of the MFB
(Borta and Höglinger, 2007). Since complete bilateral MFB lesions
have been associated with high morbidity and mortality (von Bohlen
und Halbach, 2011), we first determined the optimal dose of 6-OHDA
and found that doses greater than 2 μg/μl were associated with a significant mortality rate in WT mice (data not presented). A dose of 1.5 μg/μl
of 6-OHDA was therefore used and resulted in lesions with a 21% and
18% loss of dopaminergic cells in the SN (P = 0.011) and VTA (P =
0.017) respectively, and a 33% loss of dopaminergic fibers in the striatum (P = 0.0009) 4 weeks after the lesion (Fig A1).
We then examined the effect of this loss of a dopaminergic
input followed by dopamine replacement on adult neurogenesis. Two
weeks after the MFB lesions, animals received either saline or L-DOPA
(6 mg/kg) stabilized by benserazide hydrochloride (12 mg/kg) daily
for 7 days. All mice were perfused 2 weeks after the last drug administration in order to quantify adult neurogenesis through counting the
number of doublecortin (DCX) positive immature neurons in the SGZ
and SVZ (see Fig. 2A for experimental timeline). We found that partial
bilateral MFB lesions led to a significant decrease in the number of
DCX positive cells in the SGZ (P = 0.0009) and SVZ (P = 0.0009),
which was rescued by L-DOPA treatment (P = 0.021) at both sites
(Fig. 2C and D). This suggests that the dopaminergic input to the SVZ
and SGZ is essential in maintaining normal levels of adult neurogenesis
in mice.
Statistical analysis
6-OHDA lesions of the MFB ameliorate motor impairments and increased
adult hippocampal neurogenesis in the R6/1 mouse model of HD
Statistical comparisons were conducted using a two-way ANOVA
followed by post-hoc tests with a Bonferroni correction when statistical
significance was observed or independent-samples T-tests where
appropriate. Data was expressed as the mean ± SEM and the level
of statistical significance (alpha) was set at 0.05. All statistical
analyses were performed using SPSS 17.0 or 19.0 and Prism 5 for
Windows.
We then investigated the effects of similar dopaminergic lesions in
the R6/1 mouse model. We found that R6/1 mice exhibited greater
sensitivity to 6-OHDA than WT littermate mice in that doses greater
than 1 μg/μl were more likely to be fatal (data not presented). As a
result, we used a dose of 0.5 μg/μl 6-OHDA which resulted in lesions
with a 16.5% (P = 0.039) and 12.2% (P = 0.028) reduction in midbrain
dopaminergic cells in the SN and VTA respectively and a 6.3% reduction
After staining, cells were counted using either a 20 × or 40 ×
objective on a Leica fluorescence microscope or Olympus stereo system
and numbers counted were multiplied by either 12 or 6 depending on
the fraction of sections stained to get an estimate of the total number
of cells in the regions of study. Representative fluorescent images
were obtained using a Leica confocal microscope.
Motor-coordination assessment
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M.L. Choi et al. / Neurobiology of Disease 66 (2014) 19–27
Fig. 1. R6/1 HD mice showed an early reduction in D2R expression in both the striatum and DG. (A) PET imaging of RAC distribution in WT and R6/1 mouse brains. (B) There was a
significant difference in the binding potential (BP) between R6/1 and WT littermate mice at both premanifest (11–13 weeks of age) and manifest (21–23 weeks of age) time points
(an effect of genotype F (1, 8) = 25.27, P = 0.001) but there was no change over time (an effect of time F (1, 8) = 1.210, P = 0.3033, interaction of time × genotype F (1, 8) =
0.142, P = 0.715, n = 4 per group). (C) Microdissection of the DG showed reduced D2R mRNA expression at 8 (n = 3), 12 (n = 3) and 16 weeks (n = 3) of age in R6/1 mice where
n is the number of independent dissections (3–5 R6/1 and 3–5 WT littermates were used per dissection). The decrease in mRNA expression did not progress over time. There was an effect
of genotype (F (1, 2) = 81, P = 0.012) but not age (F (2, 4) = 0.568, P = 0.608) and no interaction between genotype and age (F (2, 4) = 0.568, P = 0.530). (D) While there was no
difference between the pre-manifest (11–13 weeks of age) R6/1 and WT mice in terms of the number of striatal neurons, the number was significantly decreased in R6/1 mice at the
later time point (n = 4–5 per group). There were effects of genotype (F (1, 16) = 5.74, P = 0.029) and time (F (1, 16) = 22.59, P = 0.0002) and an interaction of genotype × time
(F (1, 16) = 8.068, P = 0.011). (E) Representative images of NeuN stained sections. Blue: Hoechst positive cells, red: NeuN positive cells, and pink: cells co-expressing Hoechst and
NeuN. Note: Str: striatum, HG: Harderian gland, **P b 0.001, and #P b 0.05.
in dopaminergic fibers to the striatum in both WT and R6/1 mice
(Fig. A2).
This smaller dose of 0.5 μg/μl of 6-OHDA had no effect on rotarod
performance (Fig. 3B) and did not lead to a significant reduction in the
number of DCX positive cells in the DG or the SVZ in WT mice (Fig. 3E
and F). In contrast, this lesion strikingly improved rotarod performance
(P = 0.0009, Fig. 3C) and led to a significant increase in the number of
DCX positive cells in the SGZ in R6/1 mice (P = 0.007, Fig. 3E) indicating
that reducing the dopaminergic input had a beneficial effect on motor
performance and adult hippocampal neurogenesis. No effect was
observed in the SVZ (Fig. 3F), a site at which adult neurogenesis is not
reduced in these mice (Grote et al., 2005; Lazic et al., 2006).
Deleterious effects of dopamine in R6/1 mice are mediated through D2Rs
Since the majority of drugs used in the treatment of HD patients
block D2Rs (Mason and Barker, 2009), we subsequently explored
whether the deleterious effects of dopamine in HD are mediated via
these receptors. We used the D2R antagonist sulpiride which, although
commonly used to treat chorea and behavioral features in HD patients,
M.L. Choi et al. / Neurobiology of Disease 66 (2014) 19–27
23
Fig. 2. Bilateral partial MFB dopaminergic lesions alter neurogenesis in the adult mouse brain. (A) WT mice received 1.5 μg/μl of 6-OHDA bilaterally into the MFB. 2 weeks post-surgery, mice
were randomly divided into groups to receive either saline or L-DOPA treatment. All animals were sacrificed 2 weeks after the last drug treatment (n = 5–7 per group). (B) Representative
images of DCX positive cells in the SGZ and SVZ respectively. (C) Comparison of the number of DCX positive cells in the SGZ between sham and 6-OHDA lesioned groups in saline and L-DOPA
treated groups. There were effects of a 6-OHDA lesion (F (1, 16) = 23.94, P = 0.0002) and L-DOPA treatment (F (1, 16) = 4.867, P = 0.042) and an interaction of lesion × L-DOPA treatment
(F (1, 16) = 10.52, P = 0.0051). (D) The same comparisons in the SVZ showed that there were also effects of the 6-OHDA lesions at this site (F (1, 16) = 12.46, P = 0.002), with L-DOPA
treatment (F (1, 16) = 8.731, P = 0.009) and an interaction of lesion × L-DOPA treatment (F (1, 16) = 12.78, P = 0.002). Note. GCL: granular cell layer, LV: lateral ventricle, Str: striatum,
**P b 0.001, and #P b 0.05.
has yet to be investigated in a mouse model of HD and compared its
effects with L-DOPA and the D2R agonist quinpirole. Naïve R6/1 and WT
littermate mice were treated with these drugs for 7 days and then
were perfused 2 weeks after the last injection in order to study the
effects of these drug treatments on adult neurogenesis (see Fig. 4A for
experimental timeline). Neither D2R agonist nor L-DOPA treatment had
any effect in R6/1 mice (Figs. 4B and C) while treatment with the D2R
antagonist sulpiride showed a partial increase of the number of DCX
positive cells in the SGZ of R6/1 mice (P = 0.044, Fig. 4D) indicating
that blockade of D2R improves adult hippocampal neurogenesis in
these mice. However, sulpiride treatment had no effect on motor
performance (Fig. 4G) nor did L-DOPA or quinpirole treatment (Figs. 4E
and F). In WT littermates, 5 mg/kg quinpirole did enhance hippocampal
neurogenesis but no effect was observed with either 6 mg/kg L-DOPA or
10 mg/kg sulpiride.
Discussion
In this study we have demonstrated a seemingly “paradoxical role”
for dopamine in HD when compared with the normal brain. Although
D2Rs were downregulated early in the course of HD in both the striatum
and the DG of the R6/1 mice, a reduction of dopaminergic input via MFB
lesions or sulpiride treatment was beneficial, unlike that observed in
WT mice. Given that dopamine blocking/depleting drugs are widely
used in the management of HD, our observations may have wide
reaching implications in the treatment of these patients.
The normal role of dopamine in adult hippocampal neurogenesis
It is widely accepted that dopamine controls the proliferation of
neural precursor cells (NPCs) in the SVZ and that this is mediated
through D2Rs leading to the paracrine release of epidermal growth
factor (EGF) (O'Keeffe et al., 2009) and ciliary neurotrophic factor
(CNTF) (Yang et al., 2008). Although there is substantially less evidence
regarding the role of dopamine on the SGZ, there are some studies
implying that dopamine may play a similar role at this site. Firstly, the
DG receives a dopaminergic input from the VTA (Gasbarri et al., 1997;
Leranth and Hajszan, 2007) and the dopaminergic terminals contact
proliferating cells in the SGZ (Hoglinger et al., 2004). Secondly, Yang
and colleagues have demonstrated that there is an increase in the number of proliferating cells in both the SVZ and SGZ of WT mice when
D2Rs are pharmacologically stimulated along with up-regulation of
CNTF (Yang et al., 2008). Additionally, dopamine and its receptors are
significantly involved in synaptic transmission, long-term potentiation
(LTP) and memory function in the hippocampus all of which may
involve adult neurogenesis (Blackwell et al., 2008; Crook and Housman,
2012; Mu et al., 2011; Rossato et al., 2009).
24
M.L. Choi et al. / Neurobiology of Disease 66 (2014) 19–27
Fig. 3. Early manifest R6/1 mice with a partial 6-OHDA lesion showed improved motor performance and a rescue of hippocampal neurogenesis. (A) R6/1 and WT littermate mice received
either lesions (0.5 μg/μl 6-OHDA) or sham (saline) surgery bilaterally to the MFB (n = 5–7 per group). All animals were sacrificed for the neurogenesis study following the second rotarod
test. (B) There was no behavioral effect of the 6-OHDA lesion on motor performance at the two different time points in WT littermate mice (F (1, 6) = 0.300, P = 0.603). (C) There was
though an effect of such 6-OHDA lesions on motor performance (F (1, 9) = 9.176, P = 0.025) in R6/1 mice over time (F (1, 9) = 23.44, P = 0.0009). There was also an interactive effect of
lesion × time (F (1, 9) = 11.64, P = 0.008). (D) Representative images of DCX positive cells in the SGZ. A white color represents DCX positive cells in the DG. (E) Comparison of the number
of DCX positive cells in the SGZ between 6-OHDA and sham lesioned R6/1 and WT mice. There were effects of both the 6-OHDA lesion (F (1, 12) = 6.171, P = 0.023) and genotype (F (1,
12) = 53.91, P = 0.0001). There was also an interaction of lesion × genotype (F (1, 12) = 26.49, P = 0.0002). (F) The number of DCX positive cells was compared in the SVZ and no
statistical differences were found in any of the groups. Note. GCL: granular cell layer, **P b 0.001, and ##P b 0.001.
In this study, we have provided additional evidence for the role of
dopamine in maintaining adult hippocampal neurogenesis by demonstrating that lesioning the dopaminergic input to the DG in WT mice
leads to a decrease in normal adult hippocampal neurogenesis which
is rescued by administration of L-DOPA, in line with a previous MPTP
study (Hoglinger et al., 2004). In contrast studies that have tried to
block dopamine receptors have been less clear cut — for example haloperidol has been shown to decrease (Backhouse et al., 1982; Wakade
et al., 2002), increase (Dawirs et al., 1998; Kippin et al., 2005) or have
no effect (Malberg et al., 2000) on hippocampal neurogenesis. Similar
contradictory results have also been seen with regard to neurogenesis
in the SVZ (Backhouse et al., 1982; Wakade et al., 2002). However in
addition to dopamine receptor blockade, haloperidol has additional
pharmacological properties on the glutamatergic, noradrenergic and
serotoninergic systems which are all known to influence neurogenesis
(Grote and Hannan, 2007), which complicates the interpretation of
such studies.
The primary roles of the CNS dopaminergic system are in controlling
motor function, aspects of cognition and motivational behaviors as well
as endocrine functions (Wise, 2004). In this study we have shown that
inhibiting the dopaminergic system in R6 transgenic mice improved
motor performance as assessed using a rotarod test, a test which
seems to not be sensitive to motivational aspects of behavior (Brooks
et al., 2012; Jones and Roberts, 1968). It is though still possible that
this improvement related to a non-motor effect of the dopaminergic
lesions, such as an anti-depressive effect. The reason for this is that the
female R6 line mice have been reported to show early depression-like
phenotypes (Pang et al., 2009) and that the dopaminergic system has
also been implicated in the beneficial effects of some antidepressant
drugs in these same mice (Renoir et al., 2012). Indeed an improved
rotarod performance was reported in the N171-82Q mouse model of
HD when the Tg mice were treated with the SSRI antidepressant sertraline (Duan et al., 2008).
The “paradoxical” role of dopamine in HD
D2R downregulation is one of the earliest findings in HD patients
and has also been described in several mouse models. In this study,
we confirmed that D2Rs are downregulated in the striatum but have
also now shown for the first time that D2Rs are downregulated in the
DG in a HD mouse model from 8 weeks of age, in a non-progressive
fashion.
Despite this reduction in D2R, the majority of drugs currently used to
treat HD block D2Rs and/or deplete presynaptic dopamine (Mason and
M.L. Choi et al. / Neurobiology of Disease 66 (2014) 19–27
25
Fig. 4. D2R is implicated in the reduced SGZ neurogenesis seen in the R6/1 mouse model of HD. (A) R6/1 and WT littermate mice received dopaminergic agonists (quinpirole, L-DOPA) or
antagonist (sulpiride) for 7 days. All animals were sacrificed following the second rotarod test (n = 5 per group). (B) Quinpirole (5 mg/kg) treatment increased neurogenesis only in WT
littermate mice (effects of quinpirole: F (1, 12) = 5.311, P = 0.039 and genotype: F (1, 12) = 85.49, P b 0.0001). (C) There was no effect of L-DOPA treatment (6 mg/kg) in either WT or R6/1
mice. (D) The number of DCX positive cells was increased after sulpiride (10 mg/kg) treatment in R6/1 mice (effects of sulpiride: F (1, 16) = 4.736, P = 0.0449 and genotype: F (1, 16) =
136.4, P = 0.0001). (E, F, G) Neither quinpirole (5 mg/kg), L-DOPA (6 mg/kg) nor sulpiride (10 mg/kg) treatment affected motor performance in either R6/1 or WT mice. Note. *P b 0.05 and
##
P b 0.001.
Barker, 2009; Priller et al., 2008) indicating that a reduction of the
dopaminergic input appears to be of benefit. However, despite the
widespread use of such drugs in clinical practice, very few studies
have examined the effects of these agents in HD mouse models and
ours is the first study to investigate the effects of the D2R antagonist
sulpiride in such a model and show a beneficial effect on both motor
function and adult hippocampal neurogenesis. Similar beneficial effects
of dopamine reduction in vivo in transgenic HD mouse models have
previously been described with lesions of the substantia nigra (Stack
et al., 2007) or the administration of tetrabenazine (Tang et al., 2007),
whereas increased dopamine levels, via administration of L-DOPA, have
deleterious effects (Hickey et al., 2002). Although the mechanism by
which dopamine blockade is beneficial is unknown, and something
that is currently is under investigation, there is some evidence to suggest that dopaminergic abnormalities lead to excitotoxic cell death in
HD. One such piece of evidence is that in striatal lesions produced by
3-nitropropionic acid (3-NP), which were used as an early model of
HD, the neurotoxic effects were attenuated by reducing the dopaminergic input to the striatum using dopaminergic lesions (Jakel and Maragos,
2000; McLaughlin et al., 1998; Reynolds et al., 1998). Furthermore
while there is no difference in neuronal survival in striatal cultures of
WT and R6 mouse models, HD striatal neurons are more susceptible to
dopamine-induced stress with increased cell death (Charvin et al.,
2005; Petersén et al., 2001). One reason for this may be that dopamine
is sufficient to directly damage neurons via oxidative stress and the production of free radicals which leads to impaired autophagy (Petersén
et al., 2001). However, other studies have found that dopamine does
not directly cause cell death but may work synergistically with glutamate causing excitotoxicity (Tang et al., 2007). This may be important
since both the striatum and the DG are key sites of glutamatergic and
dopaminergic input (Leranth and Hajszan, 2007) hence explaining the
motor impairments and reduction in neurogenesis observed in a variety
of mouse models of HD.
The vulnerability of striatal neurons may also be directly linked to
reduced D2R levels given that overactivation of dopaminergic signaling
such as elevated dopamine release promotes apoptosis/degeneration of
striatal neurons (Cyr et al., 2003; Stephans and Yamamoto, 1996). D2R
is a main modulator of medium spiny neurons (MSNs), the main output
neuron in the striatum (Lalchandani et al., 2013), and elevated dopamine levels have been reported in both human and animal HD models
(Garrett and Soares-da-Silva, 1992; Jahanshahi et al., 2010). Thus in
HD there may be abnormal over-activation of the dopaminergic networks which target susceptible neuronal populations such as the striatal
MSNs, with secondary downregulation and loss of D2Rs.
In our study, we observe a partial increase of neurogenesis in the DG
after treatment with the D2R antagonist sulpiride which suggests that
at least some of the detrimental actions of dopamine in the DG are
mediated by D2R. However, we did not examine a role for D1R which
26
M.L. Choi et al. / Neurobiology of Disease 66 (2014) 19–27
has also been shown to be abnormal in HD (Andrews et al., 1999;
Ginovart et al., 1997; Turjanski et al., 1995). Indeed, there have been
emerging studies suggesting a role for D1R in HD. For example, mice
in which striatal D1Rs are genetically ablated have some of the neuropathological features of HD including reduced body weight, impaired
locomotor and striatal atrophy (Kim et al., 2014). Mutant huntingtin
has also been reported to promote striatal cell death through D1R
(Paoletti et al., 2008), and a D1R agonist has been reported to rescue
impaired long-term potentiation in a mouse model of HD (Dallerac
et al., 2011). We therefore cannot exclude the possibility that D1Rs
are also involved in the beneficial effects of dopaminergic lesions in
R6/1 mice and further studies on this are needed.
Implications for the treatment of human HD
Despite the recent advances in better understanding the pathogenesis of HD, there have been very few breakthroughs in terms of medical
treatments. At present this disease is incurable and treatment focuses
on symptom management mainly based on anecdotal observation
(Mason and Barker, 2009). Tetrabenazine, the only licensed drug for
use in HD, is a vesicular monoamine transporter blocker which depletes
the monoamine transmitters throughout the central nervous system
including dopamine and this has been shown to reduce chorea in HD
(Huntington Study Group, 2006). In addition to tetrabenazine, other
dopaminergic drugs such as sulpiride are also commonly used off label
to treat chorea and mood fluctuations in HD. This is the first study to
investigate the effects of sulpiride in a mouse model of HD, and we
found that it led to an increase in adult hippocampal neurogenesis, a
process which we have previously shown to be important in rodent
cognition (Clelland et al., 2009). This gives rise to the possibility that
these drugs could have more than just symptomatic benefits on motor
aspects of the disorder. Nevertheless, further studies are needed to
establish whether these agents have significant cognitive benefits in
patients.
Conclusions
Overall our findings reveal an unexpected role for dopamine in HD
mice, and that despite a decrease in D2Rs in the striatum and DG, further
reducing this dopaminergic input is beneficial and opposite to that
observed in WT mice. These findings may have significant clinical
relevance given that currently dopamine blockers and D2R antagonists
are used as symptomatic treatments of chorea and mood fluctuations in
human HD. It is therefore imperative that we better understand how
dopamine regulates adult neurogenesis and neuronal function in
the HD brain given that manipulation of this system may have consequences on disease progression and expression.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.nbd.2014.02.004.
Acknowledgments
We especially appreciated the help of Dr William Kuan in
maintaining the R6/1 mouse colony and Chaeho Hwang for his useful
input and discussions as well as technical assistance for this work. We
also thank Professor Tim Bussey and Dr Lisa Saksida at the Department
of Experimental Psychology, University of Cambridge for their useful
comments. This work was supported by generous donations from
patients and their families to the Huntington's Disease Research Clinic
(Professor Roger Barker) at the John van Geest Centre for Brain Repair,
University of Cambridge and especially the Cathy Aizlewood family.
This work was also supported in part by a Cambridge Overseas Trust
award to Minee Choi and by a Medical Research Council studentship
and James Baird Fund award to Faye Begeti.
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