[1] 18w Early decrease of type 1 cannabinoid receptor binding and phosphodiesterase 10A activity in vivo in R6/2 Huntington mice

[1] 345w Several lines of evidence imply early alterations in endocannabinoid and phosphodiesterase 10A (PDE10A) signaling in Huntington disease (HD). Using [ 18 F]MK-9470 and [ 18 F]JNJ42259152 small-animal positron emission tomography (PET), we investigated for the first time cerebral changes in type 1 cannabinoid (CB1) receptor binding and PDE10A levels in vivo in presymptomatic, early symptomatic, and late symptomatic HD (R6/2) mice, in relation to glucose metabolism ([ 18 F]FDG PET), brain morphology (magnetic resonance imaging) and motor function. Ten R6/2 and 16 wild-type (WT) mice were investigated at 3 different time points between the age of 4 and 13 weeks. Parametric CB1 receptor and PDE10A images were anatomically standardized to Paxinos space and analyzed voxelwise. Volumetric microMRI imaging was performed to assess HD pathology. In R6/2 mice, CB1 receptor binding was decreased in comparison with WT in a cluster comprising the bilateral caudate-putamen, globus pallidus, and thalamic nucleus at week 5 (À8.1% AE 2.6%, p ¼ 1.7 Â 10 À5 ). Longitudinal follow-up showed further progressive decline compared with controls in a cluster comprising the bilateral hippocampus, caudate-putamen, globus pallidus, superior colliculus, thalamic nucleus, and cerebellum (late vs. presymptomatic age: À13.7% AE 3.1% for R6/2 and þ1.5% AE 4.0% for WT, p ¼ 1.9 Â 10 À5 ). In R6/2 mice, PDE10A binding potential also decreased over time to reach significance at early and late symptomatic HD (late vs. presymptomatic age: À79.1% AE 1.9% for R6/2 and þ2.1% AE 2.7% for WT, p ¼ 1.5 Â 10 À4 ). The observed changes in CB1 receptor and PDE10A binding were correlated to anomalies exhibited by R6/2 animals in motor function, whereas no correlation was found with magnetic resonance imaging-based striatal volume. Our findings point to early regional dysfunctions in endocannabinoid and PDE10A signaling, involving the caudate-putamen and lateral globus pallidus, which may play a role in the progression of the disease in R6/2 animals. PET quantification of in vivo CB1 and/or PDE10A binding may thus be useful early biomarkers for HD. Our results also provide evidence of subtle motor deficits at earlier stages than previously described.

[1] 144w Huntington disease (HD) is an autosomal dominant inherited neurodegenerative disease, characterized by motor dysfunctions, behavioral changes, and cognitive decline. The causative mutation is an expanded CAG repeat in exon 1 of the gene encoding the protein huntingtin (htt) (The Huntington's Disease Collaborative Research Group, 1993). Adult onset is usually observed with trinucleotide repeat blocks between 40 and 50 units, whereas more than 60 repeat results in more severe and much less frequently observed juvenile, and even infantile, forms. The most striking pathophysiological feature of HD affected brains is the progressive atrophy of the caudate nucleus and the putamen, accompanied by a secondary enlargement of the lateral ventricles, and cortical degeneration in some patients (Vonsattel et al., 1985). Despite progress in elucidating the molecular pathology of HD, therapeutic benefit for patients in terms of effective pharmacotherapy with either symptomatic or protective effects, has been scarce.

[2] 269w The type 1 cannabinoid (CB1) receptor and phosphodiesterase 10A (PDE10A) enzyme play a role in changed neurotransmission in HD (Hebb et al., 2004;Katona and Freund, 2008). CB1 receptors presynaptically modulate the release of other neurotransmitters (Goutopoulos and Makriyannis, 2002) and are found at high densities on GABA-ergic striatal projection neurons (Richfield and Herkenham, 1994). PDE10A hydrolyzes the important second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate and is characterized by a restricted distribution, predominantly in medium spiny neurons of the striatum (Seeger et al., 2003). In HD, loss of CB1 receptor binding and/or signaling from the basal ganglia nuclei is one of the earliest neurochemical alterations observed in humans (Allen et al., 2009;Glass et al., 2000;Richfield and Herkenham, 1994) and experimental models (Blazquez et al., 2011;Centonze et al., 2005;Denovan-Wright and Robertson, 2000;Dowie et al., 2009;Lastres-Becker et al., 2002, 2004), preceding the development of cerebral metabolic deficits (Antonini et al., 1996) and striatal volume losses (Glass et al., 2000). Also, decreased levels of PDE10A expression occur before the onset of motor-related HD symptoms in transgenic HD mice (Hebb et al., 2004). In addition, genetic ablation of CB1 receptors showed to accelerate the onset of HD-like symptoms in transgenic R6/2 mice (Blazquez et al., 2011), whereas PDE10A inhibition ameliorated striatal and cortical pathology in experimental HD (Giampa et al., 2010;Giralt et al., 2013;Kleiman et al., 2011). Furthermore, pharmacologic studies demonstrated crosstalk of CB1 and PDE10A with striatal dopaminergic signaling (Chiang et al., 2013;Nishi et al., 2008). In symptomatic HD patients, we found a profound cortical and subcortical loss of CB1 receptor availability in vivo (Van Laere et al., 2010).

[3] 115w So far, only in vitro and ex vivo data exist on the CB1 receptor binding and PDE10A activity in genetic HD models. Ex vivo studies of CB1 receptor changes in transgenic mouse models of HD have focused on the basal ganglia, hippocampus, and motor cortex, but have not assessed other brain regions (Dowie et al., 2009(Dowie et al., , 2010 ) ). Also it remains unknown whether CB1 receptor and PDE10A levels are changed in vivo, and to what spatial and temporal extent. Thanks to the development of selective CB1 ([ 18 F]MK-9470; Burns et al., 2007) and PDE10A radioligands ([ 18 F]JNJ42259152; Celen et al., 2013), in vivo imaging of these proteins became feasible.

[4] 78w Our primary objective was thus to investigate CB1 receptor and PDE10A changes in vivo throughout the disease process of R6/2 transgenic HD mice. As the secondary objective, motor function, brain glucose metabolism, and morphology, which are shown to be altered (early) in patients and animal models of HD (Antonini et al., 1996;Carter et al., 1999;Cowin et al., 2011;Rattray et al., 2013), were investigated in the same animals and correlated to the regional CB1 receptor binding and PDE10A levels.

[1] 242w Functional imaging of brain CB1 receptor binding, PDE10A enzyme activity and glucose metabolism was performed in R6/2 and WT animals at the age of 4e5, 7e8, and 11e12 weeks to study the presymptomatic, early symptomatic, and late symptomatic phases, respectively (for time-line overview, see Fig. 1). Before small-animal positron emission tomography (PET) imaging, mice were anesthetized using 2% isoflurane in 2.0 L/min oxygen. Tail veins were catheterized for injection of the radioligands ([ 18 F]MK-9470 9.3 AE 1.6 MBq, [ 18 F]JNJ42259152 7.4 AE 2.3 MBq, and [ 18 F]FDG 11.7 AE 1.4 MBq; mass dose per body weight: 0.5 AE 0.2 nmol/kg). The radioligands were injected in a total volume of approximately 300 mL. [ 18 F]MK-9470 activity measurements were obtained after overnight fasting during a 20-minute interval, starting 1 hour postinjection, as previously validated in Casteels et al. (2012), although [ 18 F]FDG acquisitions were performed dynamically for 90 minutes (Casteels et al., 2013). [ 18 F]JNJ42259152 acquisitions were done dynamically for 60 minutes without overnight fasting (Celen et al., 2013). All small-animal PET data for one time point were acquired within a time span of 1 week. Small-animal PET imaging was performed using a FOCUS 220 tomograph (Siemens/ Concorde Microsystems, Knoxville, TN, USA), which has a transaxial resolution of 1.35 mm full-width at half-maximum. Data were reconstructed in a 256 Â 256 Â 95 matrix with a pixel width of 0.316 mm and a slice thickness of 0.796 mm.

[2] 118w To assess atrophy of the caudate-putamen (Rattray et al., 2013;Sawiak et al., 2009), anatomic magnetic resonance imaging (MRI) was performed in the same week as PET on a 9.4 Tesla Bruker Biospec Scanner (Bruker Biospin, Ettlingen, Germany) using a dedicated mouse brain surface coil (Rapid Biomedical, Rimpar, Germany). Three-dimensional high resolution images of the entire mouse brain were obtained in 20 minutes using a conventional 3D turboRARE sequence with following parameters: TR ¼ 1300 ms, TE ¼ 14.2 ms, rare factor ¼ 16, matrix 192 Â 256 Â 128, isotropic voxel size of 80 mm. Mice were anesthetized with 1.5%e2% isoflurane, the head fixed in a stereotaxic frame and body temperature maintained at 37 C AE 1 C.

[3] 130w Absolute [ 18 F]MK-9470 binding values were not significantly different in the brain of R6/2 mice and WT littermates at the age of 5, 7, and 11 weeks. Also, absolute [ 18 F]MK-9470 values of C57BL/6 mice were consistent in magnitude with those previously reported in Wistar (Casteels et al., 2010a(Casteels et al., , 2010b) ) and Sprague-Dawley rats (Casteels et al., 2011). Mean images of absolute [ 18 F]MK-9470 binding in the mouse brain of 11-week-old R6/2 mice and controls are shown in Fig. 2A. As it can be seen from this figure, the pattern of [ 18 F]MK-9470 binding observed in the mouse control brain is similar to that previously reported ex vivo (Herkenham et al., 1990), that is, high uptake in the cortex, cerebellum, hippocampus, and caudate-putamen.

[4] 155w In R6/2 mice, regional relative [ 18 F]MK-9470 binding was decreased in comparison to WT in the bilateral caudate-putamen, globus pallidus, and thalamic nucleus at week 5 (À8.1% AE 2.6%, À8.0% AE 3.3%, and À7.0% AE 2.2%, respectively, all p 2.0 Â 10 À5 ). Longitudinal follow-up showed further progressive decline between genotypes. Voxel-based analysis showed decreased binding in R6/2 mice versus WT between week 5 and 7 in a cluster comprising the bilateral hippocampus, caudate-putamen, globus pallidus, superior colliculus, thalamic nucleus, and cerebellum (Fig. 3A). The mean decrease at the Paxinos coordinate peak maximum was À11.6% AE 2.0% versus þ4.4% AE 4.4% (p ¼ 1.1 Â 10 À6 ) for R6/2 mice and WT, respectively. This decrease remained present at late symptomatic ages, predominantly in the left hemisphere (À13.7% AE 3.1% for R6/2 vs. þ1.5% AE 4.0% for WT between week 11 and week 5; p ¼ 1.9 Â 10 À5 ; Fig. 3B).

[5] 162w Representative images of mean PDE10A BP ND values in the caudate-putamen of 12-week-old R6/2 mice and controls for [ 18 F] JNJ42259152 are shown in Fig. 2B. Voxel-based analysis demonstrated decreased BP ND values in the bilateral caudate-putamen of R6/2 mice in comparison with controls over time that reached significance at 12 weeks of age (Fig. 4A). The decrease at the Paxinos coordinate peak maximum was À67.9% AE 0.1% for the left caudateputamen (p ¼ 1.5  10 À4 ) and À79.1% AE 1.9% for the right caudateputamen (p ¼ 2.9  10 À4 ) in R6/2, whereas BP ND values of WT animals changed nonsignificantly with À10.0% AE 3.7% and þ2.1% AE 2.7%, respectively, between week 5 and week 12. Predefined VOI analysis in Paxinos space confirmed SPM findings of week 12, but also showed significantly decreased PDE10A binding at week 8 (F [genotype  time]2,8 ¼ 15.8, p < 0.05 weeks 8 and p < 0.001 weeks 12, Fig. 4B).

[6] 27w For [ 18 F]FDG, no significant changes were detected between R6/ 2 and WT animals over time (p height < 0.005; k E > 200; Fig. 2C).

[7] 48w Above small-animal PET findings were also observed without a genotype  time-effect in caudate-putamen volume, although caudate-putamen volume of R6/2 mice was lower at week 13 in comparison with WT animals, that is, 24.8 AE 1.0 mm 3 versus 26.1 AE 0.9 mm 3 (2-way ANOVA, Not Significant).

[8] 36w Detailed cluster peak locations and p-values of all SPM findings at the SPM analysis threshold, that is, p height 0.005 uncorrected, k E > 200, and p cluster < 0.05 corrected, are shown in Table 1.

[9] 75w Throughout the study, control mice maintained longer times on the rotarod at all rotation speeds. In R6/2 mice, there was a progressive decline in performance over time at all 5 speeds (Fig. 5). Significant deficits were present for each velocity at 13 weeks of age (2-way ANOVA, p < 0.05). At the highest velocity (32 rpm) performance of R6/2 mice decreased by 62.0% compared with WT (F [genotype  time]2,54 ¼ 7.3, p < 0.01).

[10] 118w On the Catwalk, transgenic mice showed comparable results with controls at 6 and 9 weeks of age. At 13 weeks of age, however, R6/2 mice showed a lower swing speed (range: À31.1%, À47.9%, F [genotype]1,36 ¼ 21.4, p < 0.001) and shorter stride lengths (range: À25.8%, À34.8%, F [genotype]1,36 ¼ 42.1, p < 0.001), whereas the duration of paw contact, that is, stand (range: þ47.5%, þ89.3%; F [ge- notype]1,36 ¼ 24.8, p < 0.001) and print area (range: þ37.2%, þ204.1%; F [genotype]1,36 ¼ 11.9, p < 0.01) were increased as compared with WTs for all paws (Fig. 6A and B). Absolute values of body mass did not significantly differ between genotypes at week 13 (p ¼ 0.1, NS).

[11] 72w In addition, the number of errors made using the front paws on an irregular ladder design was higher at each time point for R6/2 mice (e.g., þ51.1% at week 6, F [genotype]1,54 ¼ 7.9, p < 0.01), but it did not progress over time. A similar genotype-effect was observed when the error score was used as quantitative outcome (e.g., À12.3% at week 6, F [genotype]1,54 ¼ 8.8; p < 0.01, Fig. 6C).

[12] 87w Voxel-based correlation analysis showed a positive correlation between the latency to fall off the rotarod and relative [ 18 F]MK-9470 binding in a cluster covering the bilateral hippocampus and primary sensory cortex (Spearman r ¼ 0.81, p < 0.0004, Fig. 7A and B and Table 1). VOI-based PDE10A BP ND values of the right caudateputamen inversely correlated to the print area and duration of paw contact of the left forepaw while crossing the Catwalk (Fig. 7C and D; both Spearman r ¼ À0.86; p < 0.02 corrected).

[13] 104w No correlations of relative [ 18 F]MK-9470 binding and PDE10A BP ND values of the caudate-putamen with MRI-based striatal volume were observed, nor between relative [ 18 F]MK-9470 binding and PDE10A BP ND values itself. Caudate-putamen values of absolute [ 18 F]MK-9470 binding and PDE10A BP ND determined by the individual MR-based segmentation highly correlated to the ones obtained using a predefined VOI map oriented in Paxinos space, pointing to a limited influence of striatal volume changes on our quantitative outcomes (Fig. 7E and F; [ 18 F]MK-9470: Spearman r ¼ 0.94, p < 0.0001 and PDE10A: Spearman r ¼ 0.97, p < 0.0001).

[1] 59w In this study, we have for the first time characterized CB1 receptor and PDE10A alterations in vivo in R6/2 transgenic mice of HD using [ 18 F]MK-9470 and [ 18 F]JNJ42259152 small-animal PET. We showed that in early and late stages of the symptomatic phase, both CB1 receptor binding and levels of PDE10A are decreased in basal ganglia regions.

[2] 280w Specifically for [ 18 F]MK-9470, at all studied ages, CB1 receptor binding was reduced in the caudate-putamen and globus pallidus of transgenic mice, where cell bodies and axon terminals of CB1 receptor-positive medium spiny neurons are located, respectively. Reduced binding of [ 18 F]MK-9470 to the CB1 receptor was also seen in the hippocampus, thalamic nucleus, superior colliculus, and cerebellum at symptomatic ages. Reduction in CB1 receptor binding of basal ganglia regions is in line with ex vivo observations in R6/2 mice. Horne et al. (2013) found a 16% and 26% reduction in the striatal and pallidal CB1 receptor binding of 12-week-old R6/2 mice using immunohistochemistry. They did not, in contrast to this study, observe any change in protein binding at earlier time points. It has been reported that CB1 receptor downregulation in these regions primarily occurred in medium-sized spiny neurons belonging to the indirect pathway (Chiodi et al., 2012;Horne et al., 2013), a neuronal subpopulation also particularly vulnerable in HD patients (Reiner et al., 1988). CB1 receptor downregulation also occurred in neuropeptide Y/neuronal nitric oxide synthaseexpressing interneurons of the striatum (Chiodi et al., 2012;Horne et al., 2013). In line with Horne et al. (2013), we also observed that the loss of striatal CB1 receptor binding is less pronounced compared with the loss of previously published CB1 receptor messenger RNA (mRNA) data in that region (Denovan-Wright and Robertson, 2000;Luthi-Carter et al., 2000;McCaw et al., 2004). These previous studies showed that striatal mRNA levels of the CB1 receptor decrease in R6/2 mice compared with wild-type animals from 3 weeks of age to a minimum level of approximately 30% by 4 weeks of age, and remained constant thereafter (McCaw et al., 2004).

[3] 455w We also found reduced binding of [ 18 F]MK-9470 in other brain regions, especially the hippocampus, cerebellum, and thalamic nucleus, a fact also found in HD patients given the ubiquitous expression of mutant htt that converts the disease multifactorial with symptoms (cognitive decline) and deterioration in cortical and subcortical (e.g., hippocampus) structures (Ross and Tabrizi, 2011). Similar observations have been done in transgenic mice, for example, symptomatic R6/2 mice are characterized by loss of CB1 receptor mRNA in a subset of hippocampal neurons (Denovan-Wright and Robertson, 2000) and by slightly reduced levels of the endogenous ligands, AEA, and 2-AG (Bisogno et al., 2008). Also, disturbed functioning of Purkinje cells in which CB1 receptors are located at the synapses (Suarez et al., 2008), have been described in this model before cell loss (Dougherty et al., 2012). Cerebellar dysfunction is not surprising, knowing that R6/2 mice more closely replicate juvenile HD (Sawiak et al., 2009) and several reports have proved the occurrence of cerebellum-related symptoms (e.g., Fig. 5. Balance and motor coordination on the rotarod R6/2 and WT mice were subjected to 5 different speeds on a fixed speed rotarod, receiving 2 trials per speed. The mean AE SD of the duration of balance or the latency to fall (maximum trial length ¼ 60 seconds) for the 2 trials at the lowest (16 rpm) and highest speed (32 rpm) is shown. R6/2 mice exhibited a decline in latency to fall from the rotarod with age, reaching statistical significance at 13 weeks of age; 2-way ANOVA; * p < 0.05; *** p < 0.001. Abbreviations: ANOVA, analysis of variance; SD, standard deviation; WT, wild type. ataxia) in these most severe HD patients (Nicolas et al., 2011;Ruocco et al., 2006). In addition, from an anatomic point of view, thalamic nuclei are connected to the striatum and participate in the control of voluntary movement. However, no preclinical data on CB1 receptor changes or endocannabinoid changes have been reported yet in this region, although we observed a profound subcortical decrease in CB1 receptor binding in symptomatic HD patients in vivo (Van Laere et al., 2010). Similar to CB1, PDE10A density was also found to decline in R6/2 mice. In 8-and 12-week-old animals, PDE10A binding quantified with [ 18 F]JNJ42259152 PET was reduced bilaterally in the striatum. These in vivo findings are in line with few ex vivo results obtained in the same model (Hebb et al., 2004). For example, Hebb et al. (2004) observed declining PDE10A mRNA levels in R6/2 mice that initiates at the age of 4 weeks and that reaches a stable value at 9 weeks of age. The lower mRNA levels resulted in PDE10A protein levels of approximately 70% lower at 9 weeks compared with 3-week-old R6/2 mice.

[4] 280w The mechanism by which HD promotes an early loss of CB1 receptors is suggested to be caused by interactions between mutant htt and nuclear transcription factors, similar to other key elements whose transcriptional regulation is affected by mutant htt (e.g., brain-derived neurotrophic factor). Blazquez et al. (2011) showed in striatal cells that mutant htt, because of loss of function, downregulates CB1 receptors through the inhibition of gene promoter activity via repressor element 1 silencing transcription factor and sensitizes cells to excitotoxic damage. The decrease in PDE10A, on the other hand, could conceivably be an indirect and compensating mechanism secondary to the loss of cAMP. Gines et al. (2003) showed impairment of cAMP signaling and gene transcription of its target molecule, cAMP response element binding protein (CREB), through arresting CREB-binding protein due to a gain-of-function of mutant htt. Reduced cAMP signaling has been hypothesized to contribute to neuronal loss (Klevytska et al., 2010), whereas PDE10A inhibition increased CREB-mediated signaling (Giralt et al., 2013) and thus plays a neuroprotective role. This CREB theory could also explain why PDE10A inhibition is beneficial (Giampa et al., 2010;Kleiman et al., 2011) and improves spatial and recognition memory in the R6/1 mouse model for HD (Giralt et al., 2013) despite the fact that PDE10A expression decreases during disease progression. However, a change in the cellular localization of PDE10A (Leuti et al., 2013) or direct interference of mutant htt with PDE10A transcription factors (Hu et al., 2004) cannot be excluded. The fact that alterations in CB1 receptor occurred slightly earlier than alterations of PDE10A levels fits with the different independent underlying mechanisms, that is, a direct effect of mhtt versus a compensatory (indirect) effect of cAMP, respectively.

[5] 349w In our work, we also demonstrated that changes in relative [ 18 F] MK-9470 binding of the bilateral hippocampus positively correlated to the rotarod performance, suggesting that animals with the lowest binding were related to a worse functional outcome. In agreement with this finding, especially abnormalities in T2 relaxivity of hippocampus and not in striatum volume have previously shown to be associated with rotarod performance in male and female R6/2 mice (Rattray et al., 2013). We also found that PDE10A BP ND values of the right caudate-putamen correlated to the print area and duration of paw contact of the left forepaw. Although there is no evidence of asymmetry in the pathology of HD, our data do suggest that PDE10A of the right caudate-putamen is more associated with motor deficits, in particular with the control of the forepaw rather than of the hindpaw. Values of PDE10A BP ND and [ 18 F]MK-9470 in the caudate-putamen itself did not correlate to each other, nor did we observe differences in [ 18 F]FDG uptake at symptomatic ages, similar to tgHD rats (Casteels et al., 2011). Nevertheless, our findings of an early in vivo loss of CB1 receptors and PDE10A enzyme, and their correlations with behavior, although modest, suggest that both [ 18 F]MK-9470 and [ 18 F] JNJ42259152 may be useful as early biomarkers of HD. Also, their decrease with age, although more narrow for CB1 receptors, suggests usefulness for testing potential therapeutic approaches aimed at delaying the onset and slowing down the disease progression. In line with this, inhibition of PDE10A showed to ameliorate striatal pathology and to delay disease onset in R6 mice (Giampa et al., 2010;Giralt et al., 2013). Also, CB1 receptor deletion showed to aggravate symptoms and the neuropathology of HD, whereas the pharmacologic administration of the cannabinoid D9tetrahydrocannabinol exerted a therapeutic effect and ameliorated above parameters (Blazquez et al., 2011). The more narrow age effect of CB1 receptors as compared with PDE10A could to some extent also be because of their abundant expression throughout the entire brain combined with the partial volume effect of smallanimal PET imaging.

[6] 385w With respect to behavioral testing, it appeared that the horizontal ladder test is the most sensitive in discriminating R6/2 mice from WT littermates, although their performance over time did not progress differently, indicating that dysfunctions and/or degeneration of the medium-sized spiny neurons minimally influenced our outcome. At each time point investigated, the numbers of errors made and the error score using the front paws on an irregular ladder design was higher and lower, respectively, in R6/2 mice. This early discriminatory power is not surprising, knowing that the horizontal ladder test has to be proven sufficiently challenging to reveal subtle impairments in forelimb and hindlimb use and unmask impairments that require forebrain control (Metz and Whishaw, 2002a). Especially, using the irregular rung condition, mice are not capable of anticipating the rung location and learning a specific gait pattern. The horizontal ladder test has also proven sensitive to normal aging as older rats showed more impaired rung walking than younger ones (Metz and Whishaw, 2002a), similar to what we observed in the present work. In addition, we succeeded in monitoring the progressive decline in motor performance that is characteristic of R6/2 mice using an incremental fixed speed rotarod protocol, as previously published (Carter et al., 1999;Samadi et al., 2013). Optimal rotation speeds for evaluating motor performance using this protocol were in the 20e32 rpm range, with the highest sensitivity at the highest speed. Using 32 rpm, impairments in performance became already apparent at 9 weeks, while only by the age of 13 weeks for all other speeds. However, we probably would have reached a higher sensitivity by the use of an accelerating rotarod. Previous data making use of continuously increasing speeds have reported on mild impairments in performances as early as 6e7 weeks of age (Blazquez et al., 2011;Luesse et al., 2001;Petr et al., 2013;Rattray et al., 2013). Using the Catwalk, deficiencies in gait were also detectable by 13 weeks of age; instead of walking in a straight line with evenly spaced and accurately positioned footprints, R6/2 mice showed reduced stride lengths and hypoactive behavior by decreased swing speed, increased print area, and longer duration of paw contact. Notably, these abnormalities were similar to gait disturbances described in this model using the footprint test (Carter et al., 1999) and open field activity test (Samadi et al., 2013).

[7] 157w A major hallmark of HD is loss of projection neurons in the neostriatum, with relative sparing of interneuron subpopulations (Vonsattel et al., 1985). Initial studies in R6/2 mice demonstrated striatal atrophy (Reiner et al., 1988;Richfield et al., 1995), but later ones also reported on neuronal loss, which is first detectable at 11 weeks (Samadi et al., 2013). Since PDE10A is expressed in medium spiny neurons (Coskran et al., 2006), which are the most affected ones in HD, structural changes could potentially confound our findings. However, correcting for the striatal volume using MRIbased segmentation yielded similar results of decreased PDE10A binding over time in R6/2 animals and indicated a clear correlation between the data acquired by a predefined VOI map oriented in Paxinos space and individual MR-based segmentation in native space (r ¼ 0.97), pointing to limited influence. Comparable findings were seen in the present work for absolute [ 18 F]MK-9470 binding in the caudate-putamen (r ¼ 0.94).

[1] 121w In vivo cerebral mapping of presymptomatic, early symptomatic, and late symptomatic mice transgenic for HD using [ 18 F]MK-9470 and [ 18 F]JNJ42259152 small-animal PET points to early regional dysfunctions in endocannabinoid and PDE10A signaling, incorporating the caudate-putamen. In vivo CB1 receptor and PDE10A enzyme measurements using [ 18 F]MK-9470 and [ 18 F]JNJ42259152 may thus be early biomarkers for HD. Translational studies in premanifest human carriers of the HD mutation using the same imaging techniques are needed to further demonstrate the clinical significance of this finding Our results also provide evidence of 1) in vivo CB1 receptor changes in the hippocampus, thalamic nucleus, and cerebellum at symptomatic ages and of 2) subtle motor deficits at earlier stages than previously described.

[2] 74w as well as the Leuven PET radiopharmacy team for tracer preparations. Financial support of the Fund for Scientific Research, Flanders, Belgium (FWO/G.0972.13), the KU Leuven In Vivo Molecular Imaging (IMIR) Consortium (KUL PF/10/017), and the European Commission (FP7, INMiND, grant agreement no. 278850) is gratefully acknowledged. Cindy Casteels and Kathleen Vunckx are supported by a postdoctoral mandate of the Research Foundation Flanders. Koen Van Laere is senior clinical investigator of the Research Foundation Flanders.

[1] 150w Heterozygous male and female mice transgenic for exon 1 of the human HD gene with a greatly expanded CAG repeat (R6/2 mice, C57BL/6 background) (Mangiarini et al., 1996) and wild-type (WT) littermates were obtained from Jackson Laboratories (Bar Harbour, Maine, USA) colony, developed at the Complutense University of Madrid, Spain. In total, 7 transgenic HD (R6/2) female, 3 R6/2 male, and 10 female and 6 male WT mice were bred. All mice were housed per 3 or 4, with the same gender and genotype. Housing conditions were standard (12-hour light and/or dark cycle) and animals had ad libitum access to food and water. Mice were allowed several days of habituation and were used between 4 and 13 weeks of age, a range that corresponds to the presymptomatic phase extending to the advanced stage of HD (Carter et al., 1999). Animal use and care followed the European Communities Council Directives (86/609/EEC).

[2] 9w Brain CB1 receptor imaging was done using the radioligand

[3] 159w which is characterized by high specificity and high affinity for the CB1 receptor (rat Ki 0.7 nM) (Casteels et al., 2012). The precursor for [ 18 F]MK-9470 was obtained from Merck Research Laboratories (West Point, NY, USA). Radiolabeling was performed onsite by alkylation of the precursor with 2-[ 18 F]fluoroethylbromide, as previously described in Burns et al. (2007). Brain PDE10A imaging was done using the radioligand [ 18 F]JNJ42259152 (rat IC 50 1.6 nM) (Celen et al., 2013). Its precursor was obtained from Janssen Research and Development (Beerse, Belgium). Radiolabeling was done by alkylation of the precursor with [ 18 F]fluoroethylbromide (Andres et al., 2011). In addition, glucose metabolism was quantified using [ 18 F]FDG. [ 18 F]FDG was prepared using a Cyclone 18/9 cyclotron and a routine [ 18 F]FDG synthesis module (IBA, Louvainla-Neuve, Belgium). All tracers were produced with a radiochemical purity of >95% and a specific activity range of 95e534 GBq/ mmol at end of synthesis.

[4] 60w All mice were tested within the light phase of a 12-hour light and/or dark cycle for motor function using the rotarod, Catwalk and horizontal ladder test, as described in the following. Motor function was tested at the age of 6, 9, and 13 weeks, that is, within 1e2 weeks from the small-animal PET experiments (for time-line overview, see Fig. 1).

[5] 166w To assess motor coordination and balance in R6/2 and WT animals, mice were trained to remain on a fixed speed rotarod, as previously reported in Carter et al. (1999). All mice underwent a 3day training program on a 3-cm diameter rotarod (Ugo Basil, Biological Research Apparatus, Varese, Italy). During the training period, each mouse was placed on the rotating cylinder at a constant speed (24 rotations per minute; rpm) for a maximum of 60 seconds, and the latency to fall of the rotarod within this time period was recorded. Mice received 4 trials per day, for 3 consecutive days, by which time a steady baseline level of performance was attained. The test took place on the fourth day and consisted of 2 trials at 5 different velocities, ranging from 16 rpm to 32 rpm. The latency to fall of the rotarod at each speed level was recorded. Data from the 2 trials on the test day were averaged per speed level and used in statistical analysis.

[6] 209w To assess skilled walking, limb placement and limb coordination, we performed the horizontal ladder test. The horizontal ladder rung walking test apparatus consisted of side walls made of clear Plexiglas and metal rungs (2 mm diameter) which could be inserted to create a floor with a minimum distance of 0.4 cm between rungs. The side walls were 50 cm long and 10 cm high measured from the height of the rungs. The entire apparatus was placed on 2 standard rat housing cages, 15 cm above the ground. The width of the alley was adjusted to the size of the animal, so that it was about 0.5 cm wider than an animal to prevent the animal from turning around. The difficulty of the task was modified by varying the position of the metal rungs. Animals were tested in 2 ladder conditions, a regular pattern and an irregular random pattern. In the regular pattern, rungs were arranged at a distance of 0.4 cm apart. In the irregular pattern, the distance of the rungs varied, ranging from 0.4 to 3.2 cm. A camera (GZ-MG335, HD Everio, JVC) was positioned at a slight ventral angle, so that both sides of the body and paw positions could be recorded simultaneously from a ventral view.

[7] 171w Before testing, animals were trained to cross the ladder from the beginning to the end of the ladder. All animals crossed the ladder in the same direction. No reinforcement was given to motivate the animals to cross the ladder. All animals were trained on the regular rung pattern for 5 times per session, for 2 consecutive days. The next day, animals were tested 3 times on the regular rung pattern and 3 times on the irregular rung pattern. Only the test session was filmed. All video recordings were analyzed frame-by-frame. Each step during a pass along the ladder was scored, however, the first 2 initiation steps and the last 2 final steps were omitted when an animal paused. Each correct and each incorrect step was scored using a 1e6 scoring scale as in Metz and Whishaw, (2002b). The number of errors in each session and the error score that was calculated from the total number of errors and the number of steps for each limb were subsequently used as quantitative outcomes.

[8] 113w Reported values in this manuscript are given as the mean AE standard deviation. Conventional statistics were carried out using Graphpad Prism 5.1 (Graphpad Software, La Jolla, CA, USA). Measurements of rotarod, horizontal ladder test, and caudate-putamen volume were analyzed using 2-way analysis of variance (ANOVA) tests with time and genotype as between-subject factors. Bonferroni post hoc test was applied for multiple comparisons. Catwalk data and body weight were analyzed using nonparametric Mann-Whitney U tests as time variations in size, weight, and skeletal morphometry have been shown to influence standard gait parameters (Wooley et al., 2009). The Spearman correlation analysis was used for all correlative tests. Significance was accepted at the 95% probability level.

[1] 225w For quantification purposes, PET scans were reconstructed using an iterative maximum a posteriori probability algorithm with ordered subsets (MAP; 18 iterations, 9 subsets, fixed resolution: 1.5 mm) (Qi et al., 1998). Standardized uptake value (SUV) images of [ 18 F]MK-9470 binding based on standard uptake values (SUV ¼ activity concentration (MBq/mL) Â body mass (g)/injected dose [MBq]) were generated as measure of absolute [ 18 F]MK-9470 binding. SUV was previously validated to give good estimates of total receptor volume of distribution as determined by full kinetic modeling in rats (Casteels et al., 2012). We investigated relative [ 18 F]MK-9470 binding as well to exclude the higher physiologic interindividual variability of absolute [ 18 F]MK-9470 determinations in the brain. Relative [ 18 F]MK-9470 binding have the advantage of being much more sensitive, allowing changes of 5%e10% to be measured using SPM (Van Laere et al., 2002). Relative [ 18 F]MK-9470 binding was expressed as SUV normalized on whole-brain SUV. For PDE10A, voxelwise parametric binding potential (BP ND ) images were reconstructed using a Logan reference tissue model using the cerebellum as the reference region in PMODv.3.1 (PMOD Inc, Zurich, Switzerland), as previously validated in Celen et al. (2013). Glucose metabolism was determined by normalizing [ 18 F]FDG data from the 45e60 minutes time interval to the whole-brain uptake in that time frame (Poisnel et al., 2012).

[2] 72w To obtain maximal use of information without a priori knowledge, PET data were analyzed voxelwise using SPM8 (Statistical Parametric Mapping, Wellcome Department of Cognitive Neurology, London, UK). For spatial normalization, individual PET data were manually normalized using affine transformations to tracer-specific custom-made mouse brain PET templates in Paxinos stereotactic space (Casteels et al., 2013). This methodology allows reporting results in coordinates directly corresponding to the Paxinos coordinate system for the mouse brain.

[3] 181w For SPM analysis, data were analyzed in a flexible factorial design using subjects (R6/2 vs. WT) Â conditions (4e5 weeks vs. 7e8 weeks vs. 11e12 weeks). T-maps were interrogated at a peak voxel threshold (height/amplitude) of p height ¼ 0.005 (uncorrected) and extent threshold k E > 200 voxels. Only significant clusters with p cluster < 0.05 (corrected for multiple comparisons) were retained, in combination with sufficient localizing power (p height < 0.005, uncorrected for multiple comparisons) (Casteels et al., 2011;Luyten et al., 2012). These p-values were set before SPM analysis using a Monte Carlo simulation (AlphaSim, AFNI, http://afni.nimh.nih.gov/afni) to ensure that familywise error was kept below 5%. For analysis of relative CB1 receptor binding and relative glucose metabolism, proportional scaling to the mean voxel value was used and an analysis threshold of 0.8 of the mean image intensity was applied. To exclude the influence of gender, analyses were done with this variable as covariate. In addition, a voxel-based correlation analysis between relative CB1 receptor binding/PDE10A BP ND /[ 18 F]FDG uptake and the behavioral outcomes for motor function was performed.

[4] 214w To assess the volumetric loss of HD in vivo, caudate-putamen volume was delineated automatically from the 3D turboRARE MR images (Rattray et al., 2013;Sawiak et al., 2009). For this purpose, all MR images were corrected for RF inhomogeneity (Likar et al., 2001) and spatially aligned by affine registration to a custom-made MRI template in Paxinos space (Casteels et al., 2013) using maximization of mutual information criteria (Maes et al., 1997). To model the regional atrophy in HD animals, affine transformations were supplemented by B-spline based deformable registration (Loeckx et al., 2003) using a spline degree of 2 and isotropic mesh size of 0.64 mm. Based on the transformation determined by this spatial normalization step, predefined delineations of caudate-putamen on the MR template were propagated to individual MR images and used as measure of caudateputamen volume. Any cerebrospinal fluid contrast falsely included in the segmented caudate-putamen volume was systematically removed in all images using low-level intensity thresholding. Individual labels of caudate-putamen volume were also propagated to individual PET data upon rigid manual PET-MRI registrations. The PET quantification using these individual labels of caudate-putamen volume was subsequently compared with the PET quantification by aid of a predefined VOI map in Paxinos space (Casteels et al., 2013), to assess impact of morphological changes on caudate-putamen quantitative outcomes.

[5] 90w The Catwalk (Noldus, Wageningen, the Netherlands) consists of a horizontal glass plate and video-capturing equipment placed below. It allows semi-automated quantification of a large number of locomotor parameters during walkway crossing. Based on the position, pressure, and surface area of each footfall multiple parameters are calculated such as stride length, base of support, interlimb coordination, and swing/stance phases (Vandeputte et al., 2010). For correct locomotor analysis, 3 uninterrupted runs were used with a minimum of 3 step sequence patterns. Body mass as confounding variable was measured at all test days.