Dopaminergic manipulations and its effects on neurogenesis and motor function in a transgenic mouse model of Huntington's disease

Neurobiology of Disease 66 (2014) 19–27

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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

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(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

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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.

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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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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,

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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).

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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

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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

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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.

References Andrews, T.C., Weeks, R.A., Turjanski, N., Gunn, R.N., Watkins, L.H., Sahakian, B., Hodges, J.R., Rosser, A.E., Wood, N.W., Brooks, D.J., 1999. Huntington's disease progression. PET and clinical observations. Brain 122 (Pt 12), 2353–2363. Backhouse, B., Barochovsky, O., Malik, C., Patel, A.J., Lewis, P.D., 1982. Effects of haloperidol on cell proliferation in the early postnatal brain. Neuropathol. Appl. Neurobiol. 8, 109–116. Bäckman, L., Farde, L., 2001. Dopamine and cognitive functioning: brain imaging findings in Huntington's disease and normal aging. Scand. J. Psychol. 42, 287–296. Benn, C.L., Fox, H., Bates, G.P., 2008. Optimisation of region-specific reference gene selection and relative gene expression analysis methods for pre-clinical trials of Huntington's disease. Mol. Neurodegener. 3, 17. Benn, C.L., Luthi-Carter, R., Kuhn, A., Sadri-Vakili, G., Blankson, K.L., Dalai, S.C., Goldstein, D.R., Spires, T.L., Pritchard, J., Olson, J.M., Van Dellen, A., Hannan, A.J., Cha, J.H., 2010. Environmental enrichment reduces neuronal intranuclear inclusion load but has no effect on messenger RNA expression in a mouse model of Huntington disease. J. Neuropathol. Exp. Neurol. 69, 817–827. Blackwell, A.D., Paterson, N.S., Barker, R.A., Robbins, T.W., Sahakian, B.J., 2008. The effects of modafinil on mood and cognition in Huntington's disease. Psychopharmacology (Berl) 199, 29–36. Borta, A., Höglinger, G.U., 2007. Dopamine and adult neurogenesis. J. Neurochem. 100, 587–595. Brooks, S.P., Janghra, N., Workman, V.L., Bayram-Weston, Z., Jones, L., Dunnett, S.B., 2012. Longitudinal analysis of the behavioural phenotype in R6/1 (C57BL/6J) Huntington's disease transgenic mice. Brain Res. Bull. 88, 94–103. Cha, J.-H.J., Kosinski, C.M., Kerner, J.A., Alsdorf, S.A., Mangiarini, L., Davies, S.W., Penney, J.B., Bates, G.P., Young, A.B., 1998. Altered brain neurotransmitter receptors in transgenic mice expressing a portion of an abnormal human Huntington disease gene. Proc. Natl. Acad. Sci. 95, 6480–6485. Charvin, D., Vanhoutte, P., Pagès, C., Borelli, E., Caboche, J., 2005. Unraveling a role for dopamine in Huntington's disease: the dual role of reactive oxygen species and D2 receptor stimulation. Proc. Natl. Acad. Sci. U. S. A. 102, 12218–12223. Clelland, C.D., Choi, M., Romberg, C., Clemenson, G.D., Fragniere, A., Tyers, P., Jessberger, S., Saksida, L.M., Barker, R.A., Gage, F.H., Bussey, T.J., 2009. A functional role for adult hippocampal neurogenesis in spatial pattern separation. Science 325, 210–213. Crook, Z.R., Housman, D.E., 2012. Dysregulation of dopamine receptor D2 as a sensitive measure for Huntington disease pathology in model mice. Proc. Natl. Acad. Sci. U. S. A. 109, 7487–7492. Cummings, D.M., Milnerwood, A.J., Dallerac, G.M., Waights, V., Brown, J.Y., Vatsavayai, S.C., Hirst, M.C., Murphy, K.P., 2006. Aberrant cortical synaptic plasticity and dopaminergic dysfunction in a mouse model of Huntington's disease. Hum. Mol. Genet. 15, 2856–2868. Cyr, M., Beaulieu, J.-M., Laakso, A., Sotnikova, T.D., Yao, W.-D., Bohn, L.M., Gainetdinov, R.R., Caron, M.G., 2003. Sustained elevation of extracellular dopamine causes motor dysfunction and selective degeneration of striatal GABAergic neurons. Proc. Natl. Acad. Sci. 100, 11035–11040. Dallerac, G.M., Vatsavayai, S.C., Cummings, D.M., Milnerwood, A.J., Peddie, C.J., Evans, K.A., Walters, S.W., Rezaie, P., Hirst, M.C., Murphy, K.P., 2011. Impaired long-term potentiation in the prefrontal cortex of Huntington's disease mouse models: rescue by D1 dopamine receptor activation. Neurodegener. Dis. 8, 230–239. Dawirs, R.R., Hildebrandt, K., Teuchert-Noodt, G., 1998. Adult treatment with haloperidol increases dentate granule cell proliferation in the gerbil hippocampus. J. Neural Transm. 105, 317–327. Duan, W., Peng, Q., Masuda, N., Ford, E., Tryggestad, E., Ladenheim, B., Zhao, M., Cadet, J.L., Wong, J., Ross, C.A., 2008. Sertraline slows disease progression and increases neurogenesis in N171-82Q mouse model of Huntington's disease. Neurobiol. Dis. 30, 312–322. Garrett, M.C., Soares-Da-Silva, P., 1992. Increased cerebrospinal fluid dopamine and 3,4dihydroxyphenylacetic acid levels in Huntington's disease: evidence for an overactive dopaminergic brain transmission. J. Neurochem. 58, 101–106. Gasbarri, A., Sulli, A., Packard, M.G., 1997. The dopaminergic mesencephalic projections to the hippocampal formation in the rat. Prog. Neuropsychopharmacol. Biol. Psychiatry 21, 1–22. Gil, J.M.A.C., Mohapel, P., Araújo, I.M., Popovic, N., Li, J.-Y., Brundin, P., Petersén, Å., 2005. Reduced hippocampal neurogenesis in R6/2 transgenic Huntington's disease mice. Neurobiol. Dis. 20, 744–751. Ginovart, N., Lundin, A., Farde, L., Halldin, C., Backman, L., Swahn, C.G., Pauli, S., Sedvall, G., 1997. PET study of the pre- and post-synaptic dopaminergic markers for the neurodegenerative process in Huntington's disease. Brain 120 (Pt 3), 503–514. Glass, M., Van Dellen, A., Blakemore, C., Hannan, A.J., Faull, R.L., 2004. Delayed onset of Huntington's disease in mice in an enriched environment correlates with delayed loss of cannabinoid CB1 receptors. Neuroscience 123, 207–212. Grote, H.E., Hannan, A.J., 2007. Regulators of adult neurogenesis in the healthy and diseased brain. Clin. Exp. Pharmacol. Physiol. 34, 533–545. Grote, H.E., Bull, N.D., Howard, M.L., Van Dellen, A., Blakemore, C., Bartlett, P.F., Hannan, A.J., 2005. Cognitive disorders and neurogenesis deficits in Huntington's disease mice are rescued by fluoxetine. Eur. J. Neurosci. 22, 2081–2088. Hagihara, H., Toyama, K., Yamasaki, N., Miyakawa, T., 2009. Dissection of hippocampal dentate gyrus from adult mouse. J. Vis. Exp. 17, 1543. Hickey, M.A., Reynolds, G.P., Morton, A.J., 2002. The role of dopamine in motor symptoms in the R6/2 transgenic mouse model of Huntington's disease. J. Neurochem. 81, 46–59. Hoglinger, G.U., Rizk, P., Muriel, M.P., Duyckaerts, C., Oertel, W.H., Caille, I., Hirsch, E.C., 2004. Dopamine depletion impairs precursor cell proliferation in Parkinson disease. Nat. Neurosci. 7, 726–735.

M.L. Choi et al. / Neurobiology of Disease 66 (2014) 19–27 Huntington Study Group, 2006. Tetrabenazine as antichorea therapy in Huntington disease: a randomized controlled trial. Neurology 66, 366–372. Jahanshahi, A., Vlamings, R., Kaya, A.H., Lim, L.W., Janssen, M.L., Tan, S., Visser-Vandewalle, V., Steinbusch, H.W., Temel, Y., 2010. Hyperdopaminergic status in experimental Huntington disease. J. Neuropathol. Exp. Neurol. 69, 910–917. Jakel, R.J., Maragos, W.F., 2000. Neuronal cell death in Huntington's disease: a potential role for dopamine. Trends Neurosci. 23, 239–245. Jones, B.J., Roberts, D.J., 1968. The quantitative measurement of motor inco-ordination in naive mice using an accelerating rotarod. J. Pharm. Pharmacol. 20, 302–304. Kim, J.S., Lee, J.S., Im, K.C., Kim, S.J., Kim, S., Lee, D.S., Moon, D.H., 2007. Performance measurement of the microPET Focus 120 scanner. J. Nucl. Med. 48, 1527–1535. Kim, H.A., Jiang, L., Madsen, H., Parish, C.L., Massalas, J., Smardencas, A., O'Leary, C., Gantois, I., O'Tuathaigh, C., Waddington, J.L., Ehrlich, M.E., Lawrence, A.J., Drago, J., 2014. Resolving pathobiological mechanisms relating to Huntington disease: gait, balance, and involuntary movements in mice with targeted ablation of striatal D1 dopamine receptor cells. Neurobiol. Dis. 62, 323–337. Kippin, T.E., Kapur, S., Van Der Kooy, D., 2005. Dopamine specifically inhibits forebrain neural stem cell proliferation, suggesting a novel effect of antipsychotic drugs. J. Neurosci. 25, 5815–5823. Lalchandani, R.R., Van Der Goes, M.S., Partridge, J.G., Vicini, S., 2013. Dopamine D2 receptors regulate collateral inhibition between striatal medium spiny neurons. J. Neurosci. 33, 14075–14086. Lazic, S.E., Grote, H., Armstrong, R.J.E., Blakemore, C., Hannan, A.J., Van Dellen, A., Barker, R.A., 2004. Decreased hippocampal cell proliferation in R6/1 Huntington's mice. NeuroReport 15, 811–813. Lazic, S.E., Grote, H.E., Blakemore, C., Hannan, A.J., Van Dellen, A., Phillips, W., Barker, R.A., 2006. Neurogenesis in the R6/1 transgenic mouse model of Huntington's disease: effects of environmental enrichment. Eur. J. Neurosci. 23, 1829–1838. Leranth, C., Hajszan, T., 2007. Extrinsic afferent systems to the dentate gyrus. Prog. Brain Res. 163, 63–84. Logan, J., Fowler, J.S., Volkow, N.D., Wang, G.J., Ding, Y.S., Alexoff, D.L., 1996. Distribution volume ratios without blood sampling from graphical analysis of PET data. J. Cereb. Blood Flow Metab. 16, 834–840. Malberg, J.E., Eisch, A.J., Nestler, E.J., Duman, R.S., 2000. Chronic antidepressant treatment increases neurogenesis in adult rat hippocampus. J. Neurosci. 20, 9104–9110. Mangiarini, L., Sathasivam, K., Seller, M., Cozens, B., Harper, A., Hetherington, C., Lawton, M., Trottier, Y., Lehrach, H., Davies, S.W., Bates, G.P., 1996. Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell 87, 493–506. Mason, S.L., Barker, R.A., 2009. Emerging drug therapies in Huntington's disease. Expert Opin. Emerg. Drugs 14, 273–297. McLaughlin, B.A., Nelson, D., Erecińska, M., Chesselet, M.F., 1998. Toxicity of dopamine to striatal neurons in vitro and potentiation of cell death by a mitochondrial inhibitor. J. Neurochem. 70, 2406–2415. Ming, G.-L., Song, H., 2011. Adult neurogenesis in the mammalian brain: significant answers and significant questions. Neuron 70, 687–702. Mu, Y., Zhao, C., Gage, F.H., 2011. Dopaminergic modulation of cortical inputs during maturation of adult-born dentate granule cells. J. Neurosci. 31, 4113–4123. O'Keeffe, G.C., Tyers, P., Aarsland, D., Dalley, J.W., Barker, R.A., Caldwell, M.A., 2009. Dopamine-induced proliferation of adult neural precursor cells in the mammalian subventricular zone is mediated through EGF. Proc. Natl. Acad. Sci. 106, 8754–8759. Ortiz, A.N., Kurth, B.J., Osterhaus, G.L., Johnson, M.A., 2011. Impaired dopamine release and uptake in R6/1 Huntington's disease model mice. Neurosci. Lett. 492, 11–14. Pang, T.Y., Du, X., Zajac, M.S., Howard, M.L., Hannan, A.J., 2009. Altered serotonin receptor expression is associated with depression-related behavior in the R6/1 transgenic mouse model of Huntington's disease. Hum. Mol. Genet. 18, 753–766. Paoletti, P., Vila, I., Rife, M., Lizcano, J.M., Alberch, J., Gines, S., 2008. Dopaminergic and glutamatergic signaling crosstalk in Huntington's disease neurodegeneration: the role of p25/cyclin-dependent kinase 5. J. Neurosci. 28, 10090–10101. Pavese, N., Andrews, T.C., Brooks, D.J., Ho, A.K., Rosser, A.E., Barker, R.A., Robbins, T.W., Sahakian, B.J., Dunnett, S.B., Piccini, P., 2003. Progressive striatal and cortical dopamine receptor dysfunction in Huntington’s disease: a PET study. Brain 126, 1127–1135.

27

Pavese, N., Politis, M., Tai, Y.F., Barker, R.A., Tabrizi, S.J., Mason, S.L., Brooks, D.J., Piccini, P., 2010. Cortical dopamine dysfunction in symptomatic and premanifest Huntington's disease gene carriers. Neurobiol. Dis. 37, 356–361. Paxinos, G., Franklin, K.B.J., 2001. The mouse brain in stereotaxic coordinates, second ed. Academic Press, San Diego (Book). Petersén, Å., Larsen, K.E., Behr, G.G., Romero, N., Przedborski, S., Brundin, P., Sulzer, D., 2001. Expanded CAG repeats in exon 1 of the Huntington's disease gene stimulate dopamine-mediated striatal neuron autophagy and degeneration. Hum. Mol. Genet. 10, 1243–1254. Petersen, A., Puschban, Z., Lotharius, J., Nicniocaill, B., Wiekop, P., O'Connor, W.T., Brundin, P., 2002. Evidence for dysfunction of the nigrostriatal pathway in the R6/1 line of transgenic Huntington's disease mice. Neurobiol. Dis. 11, 134–146. Phillips, W., Morton, A.J., Barker, R.A., 2005. Abnormalities of neurogenesis in the R6/2 mouse model of Huntington's disease are attributable to the in vivo microenvironment. J. Neurosci. 25, 11564–11576. Priller, J., Ecker, D., Landwehrmeyer, B., Craufurd, D., 2008. A Europe-wide assessment of current medication choices in Huntington's disease. Mov. Disord. 23, 1788. Ramos, E.M., Latourelle, J.C., Gillis, T., Mysore, J.S., Squitieri, F., Di Pardo, A., Di Donato, S., Gellera, C., Hayden, M.R., Morrison, P.J., Nance, M., Ross, C.A., Margolis, R.L., GomezTortosa, E., Ayuso, C., Suchowersky, O., Trent, R.J., McCusker, E., Novelletto, A., Frontali, M., Jones, R., Ashizawa, T., Frank, S., Saint-Hilaire, M.H., Hersch, S.M., Rosas, H.D., Lucente, D., Harrison, M.B., Zanko, A., Abramson, R.K., Marder, K., Gusella, J.F., Lee, J.M., Alonso, I., Sequeiros, J., Myers, R.H., Macdonald, M.E., 2013. Candidate gluta matergic and dopaminergic pathway gene variants do not influence Huntington's disease motor onset. Neurogenetics 14, 173–179. Renoir, T., Pang, T.Y., Zajac, M.S., Chan, G., Du, X., Leang, L., Chevarin, C., Lanfumey, L., Hannan, A.J., 2012. Treatment of depressive-like behaviour in Huntington's disease mice by chronic sertraline and exercise. Br. J. Pharmacol. 165, 1375–1389. Reynolds, D.S., Carter, R.J., Morton, A.J., 1998. Dopamine modulates the susceptibility of striatal neurons to 3-nitropropionic acid in the rat model of Huntington's disease. J. Neurosci. 18, 10116–10127. Ross, C.A., Tabrizi, S.J., 2011. Huntington's disease: from molecular pathogenesis to clinical treatment. Lancet Neurol. 10, 83–98. Rossato, J.I., Bevilaqua, L.R., Izquierdo, I., Medina, J.H., Cammarota, M., 2009. Dopamine controls persistence of long-term memory storage. Science 325, 1017–1020. Sahay, A., Scobie, K.N., Hill, A.S., O'Carroll, C.M., Kheirbek, M.A., Burghardt, N.S., Fenton, A.A., Dranovsky, A., Hen, R., 2011. Increasing adult hippocampal neurogenesis is sufficient to improve pattern separation. Nature 472, 466–470. Stack, E.C., Dedeoglu, A., Smith, K.M., Cormier, K., Kubilus, J.K., Bogdanov, M., Matson, W.R., Yang, L., Jenkins, B.G., Luthi-Carter, R., Kowall, N.W., Hersch, S.M., Beal, M.F., Ferrante, R.J., 2007. Neuroprotective effects of synaptic modulation in Huntington's disease R6/2 mice. J. Neurosci. 27, 12908–12915. Stephans, S., Yamamoto, B., 1996. Methamphetamines pretreatment and the vulnerability of the striatum to methamphetamine neurotoxicity. Neuroscience 72, 593–600. Tang, T.-S., Chen, X., Liu, J., Bezprozvanny, I., 2007. Dopaminergic signaling and striatal neurodegeneration in Huntington's disease. J. Neurosci. 27, 7899–7910. Turjanski, N., Weeks, R., Dolan, R., Harding, A.E., Brooks, D.J., 1995. Striatal D1 and D2 receptor binding in patients with Huntington's disease and other choreas. A PET study. Brain 118 (Pt 3), 689–696. Van Oostrom, J.C., Dekker, M., Willemsen, A.T., De Jong, B.M., Roos, R.A., Leenders, K.L., 2009. Changes in striatal dopamine D2 receptor binding in pre-clinical Huntington's disease. Eur. J. Neurol. 16, 226–231. Von Bohlen und Halbach, O., 2011. Immunohistological markers for proliferative events, gliogenesis, and neurogenesis within the adult hippocampus. Cell Tissue Res. 345, 1–19. Wakade, C.G., Mahadik, S.P., Waller, J.L., Chiu, F.C., 2002. Atypical neuroleptics stimulate neurogenesis in adult rat brain. J. Neurosci. Res. 69, 72–79. Walker, T.L., Turnbull, G.W., MacKay, E.W., Hannan, A.J., Bartlett, P.F., 2011. The latent stem cell population is retained in the hippocampus of transgenic Huntington's disease mice but not wild-type mice. PLoS ONE 6, e18153. Wise, R.A., 2004. Dopamine, learning and motivation. Nat. Rev. Neurosci. 5, 483–494. Yang, P., Arnold, S.A., Habas, A., Hetman, M., Hagg, T., 2008. Ciliary neurotrophic factor mediates dopamine D2 receptor-induced CNS neurogenesis in adult mice. J. Neurosci. 28, 2231–2241.