Cannabinoid receptor 1 (CB₁ receptor) controls several neuronal functions, including neurotransmitter release, synaptic plasticity, gene expression and neuronal viability. Downregulation of CB₁ expression in the basal ganglia of patients with Huntington’s disease (HD) and animal models represents one of the earliest molecular events induced by mutant huntingtin (mHtt). This early disruption of neuronal CB₁ signaling is thought to contribute to HD symptoms and neurodegeneration. Here we determined whether CB₁ downregulation measured in patients with HD and mouse models was ubiquitous or restricted to specific striatal neuronal subpopulations. Using unbiased semi-quantitative immunohistochemistry, we confirmed previous studies showing that CB₁expression is downregulated in medium spiny neurons of the indirect pathway, and found that CB₁ is also downregulated in neuropeptide Y (NPY)/neuronal nitric oxide synthase (nNOS)-expressing interneurons while remaining unchanged in parvalbumin- and calretinin-expressing interneurons. CB₁downregulation in striatal NPY/nNOS-expressing interneurons occurs in R6/2 mice, Hdh^Q150/Q150 mice and the caudate nucleus of patients with HD. In R6/2 mice, CB₁ downregulation in NPY/nNOS-expressing interneurons correlates with diffuse expression of mHtt in the soma. This downregulation also occludes the ability of cannabinoid agonists to activate the pro-survival signaling molecule cAMP response element-binding protein in NPY/nNOS-expressing interneurons. Loss of CB₁ signaling in NPY/nNOS-expressing interneurons could contribute to the impairment of basal ganglia functions linked to HD.

Antibodies and drugs

The following antibodies were used in this study: CB₁ (L15 pAb guinea pig 1 : 2000; Berghuis et al., 2007); NPY [pAb rabbit 1 : 4000; Immunostar (Hudson, WI, USA) cat# 22940; mAb mouse 1 : 2000; Grouzmann et al., 1992]; leucine-enkephalin [pAb rabbit 1 : 500; Millipore (Billerica, MA, USA) cat# AB5024]; Substance P (pAb rabbit 1 : 4000; Millipore cat# AB1566); parvalbumin (pAb rabbit 1 : 500; Immunostar cat# 24428); phosphorylated cAMP response element-binding protein (pCREB) [(p-Ser133) pAb rabbit 1 : 800; Cell Signaling Technology (Danvers, MA, USA) cat# 9198]; mHtt (mAb mouse clone mEM48 1 : 300; Millipore cat# MAB5374); calretinin (pAb rabbit 1 : 1000; Millipore cat# AB5054); neuronal nitric oxide synthase (nNOS; CH₃-terminal, pAb 1 : 1000 rabbit; Immunostar cat# 24287); goat anti-rabbit Alexa-488, 555 and 647 [1 : 500; Invitrogen (Grand Island, NY, USA) cat# A11034, A21429, A21245]; goat anti-guinea pig Alexa-488 (1 : 500; Invitrogen cat# A11073); and goat anti-mouse Alexa-555 and 647 (1 : 500; Invitrogen cat# A21422, A21236). WIN 55,212-2 was purchased from Tocris Biosciences (Minneapolis, MN, USA).

R6/2 mice colony

Mice were housed in a specific pathogen-free facility in accordance with the National Institutes of Health, and the Institutional Animal Care and Use Committee at the University of Washington approved all experiments. Enrichment and ad libitum access to food and water were provided, and a 12-h light : dark cycle was maintained in the facility. R6/2 and wild-type littermates were given a wet food mash in addition to dry pellets beginning at 9 weeks old. Both female and male heterozygous and wild-type littermates were used in this study. The colony was maintained by breeding 6–8-week-old R6/2 males with two CBA/C57Bl/6 females (Jackson Labs, Bar Harbor, ME, USA). The average CAG repeat length was 114.1 ± 0.3 (n = 10), and was determined by polymerase chain reaction (PCR) from tail snips by Laragen (Culver City, CA, USA). CB₁receptor knockout colony was maintained by breading homozygote males with homozygote females from 6 to 8 weeks old (Marsicano et al, 2002). Experimenters were blind to the gender, genotype and age of all mice during tissue processing, image collection and digital image analysis.

Quantitative PCR (qPCR)

The cortex and striatum of R6/2 and littermate wild-type mice (4, 6, 8 and 12 weeks old) were dissected and rapidly stored in RNAlaterv® (Invitrogen). qPCR assays were performed using LightCycler 480 system [Roche Applied Science (Branchburg, NJ, USA)]. Probes for CB₁ receptor were from Roche Applied Science (Universal Probe Library: Set #47) and hypoxanthine phosphoribosyltransferase 1 (hprt1) from Applied Biosystems (Carlsbad, CA, USA) (VIC-tgcaaatacgaggagtcctgttgatgttgc-TAMRA). Primer sequences were:CNR1 forward 5′-cgttcaaggagaacgaggac-3′ and reverse 5′-tgaagcactccatgtc-cataa-3′; HPRT1 forward 5′-cctaagatgagcgcaagttgaa-3′ and reverse 5′-ccacaggactagaacacctgctaa-3′. Amplifications were run using a Stratagene Mx3000P QPCR system, and consisted of 30-min incubation at 45 °C, followed by 10 min at 95 °C, and 40 cycles of 1 min at 95 °C and 30 s at 60 °C.

Human tissue sections

The University of Washington Institutional Review Board approved this study. Appropriate measures for the protection of patient privacy were used. Paraffin-embedded human caudate-putamen tissue samples from HD and healthy patients were supplied by the University of Washington Alzheimer’s Disease Research Center [ADRC; grant # P50 AG05136 (Seattle, WA, USA)]. Five patients with HD and age-matched non-HD patients were studied by semi-quantitative immunohistochemistry [sq-IHC; genotype, age, gender and Vonsattel grade were (Vonsattel et al, 1985): non-HD 74 female, non-HD 77 female, non-HD 78 male, non-HD 67 male, non-HD 52 male, HD 55 male (3), HD 20 female (3), HD 55 male (3), HD 43 male (2–3), HD 74 male (3)]. The patients with HD died from complications due to HD, while the non-HD patients died from non-CNS diseases and had normal brains.

Immunohistochemistry

Mice were killed and perfused with paraformaldehyde [PFA; 4% in phosphate-buffered saline (PBS)], post-fixed for 24 h, and their brains cryoprotected in 15% sucrose (24 h) followed by 30% sucrose (48 h). Coronal sections that included the corticostriatal, globus pallidus or substantia nigra regions (30 µm) were prepared using a microtome, and then stored in PBS at 4 °C until processing. Sections from R6/2 mice and wild-type littermates were processed/ stained in parallel as follows – free-floating sections were rinsed 3× with PBS and incubated for 90 min at room temperature (RT) in PBS supplemented with donkey serum (5%) and Triton X-100 (1%). Primary antibody combinations were prepared as a master stock in PBS supplemented with donkey serum (2.5%) and Triton X-100 (0.5%), and applied to sections for 78 h at 4 °C with gentle agitation. Sections were then rinsed 8× with PBS supplemented with Tween-20 (0.05%, at RT) and incubated with secondary antibodies diluted in PBS supplemented with donkey serum (2.5%) and Triton X-100 (0.5%) for 1 h at RT with gentle agitation, followed by seven rinses with PBS and one rinse with deionized water. Sections were mounted onto slides and allowed to dry for ~ 18 h, after which cover slips were mounted with Vectashield and sealed with nail polish. For CREB phosphorylation studies, 12-week-old R6/2, CB₁ receptor knockout and wild-type littermates were injected with either vehicle (cremophore : ethanol : saline, 1 : 1 : 18) or WIN 55,212-2 (10 mg/kg, i.p.), returned to their home cage for 30 min, and then killed, perfused with 4% PFA, their brains dissected and stored in 10% neutral buffered formalin for paraffin embedding. Human and pCREB paraffin-embedded slices (5 and 4 µm, respectively) were deparaffinized with 3×5 min xylene washes followed by rehydrating washes in ethanol and antigen retrieval in citrate buffer (10 mm). Following addition of a hydrophobic barrier using an ImmunoPen™ (Millipore), sections were stained with the same protocol as the free-floating sections.

Microscopy

Dual-labeled images were collected on a Zeiss Axio Observer Z1 equipped with a Pan-Apochromatic 10×/0.3 numerical aperture (NA) air objective (to count somatic population) or a 63×/1.4 NA oil objective, ORCA-ER 1394 CCD camera [Hamamatsu (Hamamatsu City, Japan)] and AxioVision software [version 4.7.1, Zeiss (Jena, Germany)]. Illumination was set at 70% attenuation to limit photo bleaching and deconvolved using an Apotome (Optical Sectioning Average: medium; Noise: 3). Triple-labeled images were acquired using a Leica SP1 Confocal Laser Scanning microscope with a 40×/1.25 NA oil objective coupled to an argon laser (excites Alexa488), a DPSS laser (excites Alexa555) and a helium/neon laser (excites Alexa647) at the Keck Microscopy Facility of the University of Washington. Four scans were averaged using a 4× digital zoom with a medium scan rate and pinhole set at 102.9 µm. Sections that underwent parallel IHC staining were imaged using the same exposure settings. Five images of the dorsolateral striatum and layer V/VI of the sensorimotor cortex were taken per slice, and were processed for publication in parallel using Photoshop.

Semi-quantitative image analysis and statistics

Dual- and triple-labeled images were analysed and quantified using ImageJ (National Institutes of Health). Each channel was split into an individual image, and the mean intensity and standard deviation of each fluorophore in each image was measured. Background signal was removed by creating regions of interest (ROIs) for each fluorophore and setting the zero threshold value to the mean + standard deviation (Table 1). This threshold was used to analyse the 1/3 brightest pixels in the Poisson distribution of pixel intensity as the mean intensity is composed of a large majority of background intensities, as revealed by parallel staining of CB₁R^−/− tissue. While this threshold excludes ‘weakly’ stained synapses, the remaining pixels that are analysed and quantified are comprised of positively stained pixels. ROIs 1–2 were used to collect the average intensity of each fluorophore, and selectively quantify axons and cell bodies that have positive staining. ROI 3 was created by making binary masks of each ROI and using the ‘AND’ function to create new ROIs that only contain regions with overlapping positive pixels from the original ROIs (Table 1). The ‘Analysed Particles’ function was then used to extrapolate the mean and standard deviation for each channel in each ROI. To quantify pCREB and mHtt levels, ROIs were hand-drawn around positively labeled somas as determined by thresholding the interneuron marker to a mean + standard deviation (interneuron soma ROI). Positive pCREB or mHtt in interneurons was determined by thresholding total pCREB or mHtt staining to a mean + standard deviation (pCREB/mHtt ROI) and combining it with the interneuron soma ROI using the ‘AND’ function in each image (pCREB/mHtt NPY ROI). The ‘Analysed Particles’ function was then used to extrapolate the mean and standard deviation for each channel in each ROI. Neurons whose percent area of pCREB staining to NPY soma staining was < 10% or > 50% [(# of pCREB NPY ROI-positive pixels/# of NPY soma ROI-positive pixels) *100%] were excluded, so only interneurons that contained pCREB translocated to the nucleus were quantified [based on the average percent area of positive pCREB pixels in NPY interneurons with visible nuclei treated with either vehicle (24.9 ± 18.1%) or WIN 55,212–2 (42.2 ± 1.8%); these values specifically reported as standard deviations]. Mask statistics were then used to calculate the mean and standard deviation for each channel in each mask. All values were imported into Excel [Microsoft^® Office 2008 Version 12.2.7 (Redmond, WA, USA)], and means were normalized to wild-type expression for comparison. All values are expressed as mean ± standard error of the mean.

Table 1

ROIs created for sq-IHC

Statistical analysis

Statistical analysis and graphs were generated using graphpad prism (version 4; San Diego, CA, USA). Comparisons of mean intensities between genotype at specific ages were analysed by Student’s t-test. Comparisons between interneuron subtypes within R6/2 mice or pCREB levels between genotypes were analysed by one-way anova with a Tukey’s post hoc analysis, while a two-wayanova was used to analyse qPRC data. Data were considered significant if P < 0.05.

CB₁ receptor expression is decreased in R6/2 mouse striatum

It is known that CB₁ receptor expression is decreased in the basal ganglia of various HD mouse models (Fernandez-Ruiz, 2009). In agreement with several studies, we found that CNR1 mRNA expression decreased in R6/2 mice as a function of disease progression (Denovan-Wright & Robertson, 2000; Luthi-Carter et al, 2000; McCaw et al, 2004; Blazquez et al, 2011; Chiodi et al, 2011). Specifically, using qPCR we found that CNR1 mRNA is decreased by 25.6 ± 5.6% in the striatum of R6/2 at 4 weeks old, and that this downregulation reached its lowest level at 6 weeks old (57.7 ± 4.8% compared with wild-type: df = 1, F = 72.495, P < 0.05; Fig. 1A). CNR1 mRNA in the cortex of R6/2 mice decreased by 20.3 ± 1.6% at 4 weeks old, and by 43.6 ± 2.5% at 12 weeks old (R6/2 compared with wild-type: df = 1, F = 18.839, P < 0.05; Fig. 1A).

Fig. 1

Expression of total CB₁ receptor in the sensorimotor cortex and basal ganglia of R6/2 mice. (A) qPCR time course of CNR1 mRNA in the cortex and striatum of R6/2 mice normalized to percentage of levels measured in wild-type littermates (n = 3–5 …

To determine if the expression of CB₁ receptor protein was concomitantly decreased to CNR1 mRNA, a sq-IHC analysis of corticostriatal slices from R6/2 mice was performed. Control experiments confirmed that CB₁ receptor protein expression displays a stereotypical axonal pattering in the striatum of wild-type mice [(Uchigashima et al, 2007; Fig. 1B], and that this immunostaining is absent in CB₁^−/− mice (Fig. 1C, insert). Remarkably, CB₁ receptor protein staining in R6/2 striatum appeared more punctate (Fig. 1C). When quantifying CB₁ receptor expression by sq-IHC and defining ROIs for CB₁ receptor-positive axons (see Materials and methods for details; Fig. 2A and B), we found that CB₁receptor loss in the striatum of R6/2 mice was delayed and less pronounced compared with the loss of CNR1 mRNA, reaching a 16.2 ± 3.9% decrease in CB₁receptor protein at 12 weeks old as compared with wild-type littermates (CB₁receptor expression normalized to age-matched wild-type littermates: 4 weeks, P= 0.77; 8 weeks, P = 0.52; 12 weeks, P < 0.001; Fig. 1D). CB₁ receptor protein staining in layers V/VI of the sensorimotor cortex remained unchanged throughout the disease progression (CB₁ receptor expression normalized to age-matched wild-type littermates: 4 weeks, P = 0.28; 8 weeks, P = 0.11; 12 weeks, P= 0.60; Fig. 1E).

Fig. 2

sq-IHC analysis of CB₁ receptor at neuronal subtypes and synapses in the striatum of 12-week-old R6/2 mice. (A and B) Representative dual-labeled images of CB₁ receptor (Lin et al., 2001) and neuropeptide Y (NPY; Blazquez et al., 2011) interneurons in …

Approximately 95% of the striatal neurons are GABAergic MSNs, which project to the substantia nigra reticulata in the direct pathway and the globus pallidus in the indirect pathway. Because CNR1 mRNA in R6/2 striatum is significantly decreased and the majority of CB₁ receptors traffic to presynaptic axon terminals following translation (Matyas et al., 2006), we measured whether CB₁receptor expression in the substantia nigra reticulata and the globus pallidus of R6/2 mice was also decreased. We found that at 12 weeks old, CB₁ receptor protein levels in R6/2 mice were decreased in the globus pallidus by 26.4 ± 10.9% compared with wild-type, while remaining unchanged in the substantia nigra reticulata [CB₁ receptor expression normalized to age-matched wild-type littermates (globus pallidus): 4 weeks, P < 0.01; 8 weeks, P < 0.001; 12 weeks, P< 0.05; (substantia nigra reticulate): 4 weeks, P = 0.22; 8 weeks, P = 0.55; 12 weeks, P = 0.48; Fig. 1F]. Interestingly, the combined fold decrease in CB₁receptor protein measured in both the globus pallidus and the striatum at 12 weeks (13.9 + 26.4% = 40.3%) is within the range of the decrease in CNR1mRNA measured in the striatum (46.3%) at this age. This result suggests that the loss of CNR1 mRNA in the striatum is not a ubiquitous decrease in CNR1mRNA among all neuronal subpopulations, but is rather a selective decrease in specific neuronal subpopulations. Together, these results suggest that CB₁receptor downregulation occurs in MSNs belonging to the indirect pathway and in a yet unidentified neuronal subpopulation within the striatum.

Downregulation of CB₁ receptor protein in the striatum is selective to both NPY/nNOS+ interneurons and indirect pathway MSNs

Studies reporting the electrophysiological measurements in the striatum of R6/2 mice demonstrate that overall GABAergic transmission is increased at disease end-stage (12 weeks old), and CB₁ receptor-mediated inhibition of GABA release is lost in 12-week-old R6/2 mice (Cepeda et al., 2004; Chiodi et al., 2011). Here we tested if CB₁ receptor expression is changed in all subpopulations of striatal GABAergic neurons known to express CB₁ receptors: MSNs, parvalbumin interneurons, calretinin interneurons and NPY interneurons (Fusco et al., 2004;Narushima et al., 2006; Uchigashima et al, 2007). Cholinergic interneurons in the striatum of wild-type mice lack CB₁ receptors and thus were not analysed (Hohmann & Herkenham, 2000). To quantify CB₁ receptor expression in these neuronal subpopulations, we performed sq-IHC of corticostriatal slices from R6/2 mice and their wild-type littermates (double-labeled with antibodies directed against CB₁ receptors and specific neuronal markers). For MSN, we stained for the direct (substance P) and indirect (leucine-enkephalin) pathways, thus labeling the collaterals that project within the striatum. To identify interneurons, we co-labeled for parvalbumin, calretinin or NPY (Fig. 2A and B). In agreement with the decrease of the CB₁ receptor found in the globus pallidus, a 14.2 ± 6.2% decrease in indirect pathway MSN axon collaterals was observed (CB₁ receptor in leucine-enkephalin normalized to wild-type littermate: P = 0.025; Fig. 2C). CB₁ receptor was also decreased by 20.3 ± 3.5% at NPY interneurons, while remaining unchanged at parvalbumin and calretinin interneurons, as well as in the axonal collaterals of direct pathway MSN within the striatum of R6/2 mice [CB₁ receptor in neuronal subtypes normalized to wild-type littermate (NPY): P < 0.001; (substance P): P = 0.54; (parvalbumin):P = 0.68; (calretinin): P = 0.87; Fig. 2C].

A recent study has demonstrated that NPY interneurons in the striatum consist of two distinct populations of GABAergic interneurons (Ibanez-Sandoval et al, 2011). Approximately 90% of NPY interneurons are low-threshold spiking (LTS) interneurons that express NOS and somatostatin (NPY/nNOS+); the remaining 10% of NPY interneurons lack somatostatin and NOS (NPY/nNOS—) and display a distinct electrophysiological profile, suggesting they perform a unique role in the striatal microcircuitry (Ibanez-Sandoval et al, 2011). To test whether CB₁ receptor expression was decreased in these subclasses of NPY interneurons in 12-week-old R6/2 mice, corticostriatal slices were triple-labeled for CB₁receptor, NPY and nNOS. sq-IHC analysis on NPY/nNOS— interneurons showed no significant decrease in CB₁ receptor expression (CB₁ receptor normalized to wild-type littermates: P = 0.21; Fig 2D). These results indicate that the decrease of CB₁ receptors in NPY interneurons is selective to the main NPY interneuron subtype, the LTS NPY/nNOS+ interneurons.

Downregulation of CB₁ protein in NPY interneurons of Hdh^Q150/Q150 knock-in mice and human patients with HD

Decreased striatal CNR1 mRNA also occurs in the full-length HD mouse model (Lin et al, 2001; Woodman et al, 2007). Here we sought to determine if the expression of CB₁ receptor protein was also decreased in NPY interneurons in two full-length HD models (Hdh^Q150/Q150 and BACHD; Woodman et al., 2007;Gray et al., 2008). Coronal corticostriatal sections of Hdh^Q150/Q150 mice (16–17 months old) and BACHD (9 months old) were co-labeled for CB₁ receptor and NPY. Similar to what we detected in R6/2 mice, CB₁ receptor expression in the striatum of Hdh^Q150/Q150 mice was decreased by 15.8 ± 6.5% in the striatum as a whole (CB₁ receptor in Hdh^Q150/Q150 normalized to wild-type littermates: P = 0.019; data not shown) and by 13.5 ± 6.5% at NPY interneurons (CB₁ receptor in NPY interneurons of Hdh^Q150/Q150 -normalized wild-type littermates: P = 0.042; Fig. 3A). In contrast, CB₁ receptor expression remained unchanged both in the striatum as a whole (CB₁ receptor in BACHD normalized to wild-type littermates: P = 0.12; data not shown) and at NPY interneurons in BACHD mice compared with their wild-type littermates (CB₁ receptor in NPY interneurons of BACHD-normalized wild-type littermates: P = 0.23; Fig 3A). These results suggest that not all HD mice models reproduce the downregulation of CB₁receptor known to occur in patients with HD.

Fig. 3

CB₁ receptor expression at neuropeptide Y (NPY) interneurons in full-length HD mouse models and human patients with HD. (A) sq-IHC analysis of striatal sections from wild-type and Hdh^Q150/Q150 or BACHD mice co-labeled for CB₁ receptor, and neuropeptide …

A recent PET study demonstrated that downregulation of CB₁ receptor in the striatum of human presymptomatic patients with HD can be detected (Van Laere et al., 2010), extending autoradiography studies that had detected a global loss of CB₁ receptor in the caudate and putamen of patients with HD as early as grade 0 (neuropathological grading scale criteria of Vonsattel and colleagues; Vonsattel et al., 1985; Glass et al., 2000). This loss has been attributed to the loss of CB₁ receptor from MSN, first from indirect pathway MSNs in early stages of the disease and then spreading to direct pathway MSNs in later stages (Richfield & Herkenham, 1994; Glass et al., 2000). To determine if CB₁ receptors at NPY interneurons are also downregulated in human patients with HD, sq-IHC was performed on the caudate and putamen tissue sections from patients with HD. We found a 15.2 ± 3.6% decrease of CB₁ receptors in NPY interneurons located in the caudate nucleus of patients with HD compared with non-HD patients [CB₁ receptor in NPY interneuron of patients with HD normalized to non-HD patients (caudate): P < 0.001; (putamen): P = 0.8; Fig. 3B]. These results suggest that the decrease of CB₁ receptors at NPY neurons is a common phenomenon occurring in both human patients with HD and HD mouse models.

NPY interneurons display diffuse mHtt load in R6/2 mice

mHtt forms aggregates within the nucleus and micro-aggregates within the cytoplasma of neuropila that have been implicated in disrupting specific cellular mechanisms, including gene transcription and axonal protein transport, both of which control CB₁ receptor expression and distribution within neurons (Li et al, 1999; Lee et al, 2004). To investigate if mHtt formed nuclear aggregates in NPY interneurons, corticostriatal slices were double-labeled for mHtt and the three GABAergic interneuron subtypes. We found that large mHtt aggregates were prominently located in parvalbumin interneurons in the striatum of 12-week-old R6/2 (Fig. 4A). While it is thought that mHtt aggregates in a cell-autonomous manner, calretinin interneurons express very low levels of mHtt and do not display the large aggregates observed in parvalbumin interneurons (Fig. 4B). Some NPY interneurons displayed large aggregates similar to parvalbumin interneurons, but the majority of mHtt expression within NPY interneurons appeared as diffuse staining suggesting the presence of micro-aggregates (Fig. 4C). Interestingly, small mHtt aggregates were observed in some NPY neurites (Fig. 4C1). Semi-quantitative analysis confirmed that parvalbumin interneurons contain 130% higher levels of mHtt compared with calretinin interneurons and 105% higher levels compared with NPY interneurons [mHtt in interneuron subtypes normalized to mHtt in calretinin interneurons: F = 15.73,P < 0.001 (parvalbumin vs. calretinin, and parvalbumin vs. NPY); Fig. 4D]. These results indicate that the presence of micro-aggregate mHtt correlates with the decrease in CB₁ receptor expression in NPY interneurons.

Fig. 4

Mutant huntingtin (mHtt) expression levels in GABAergic interneurons in the striatum of 12-week-old R6/2 mice. R6/2 striatal tissue sections dual-labeled for mHtt (green, left column; scale bar: 50 µm) and parvalbumin (Parv; A), calretinin (B) …

A recent study suggested that monomeric or micro-aggregates represent the toxic form of mHtt that could participate in neurodegeneration (Miller et al, 2011). To determine if the number of NPY cells is reduced in R6/2 mice, NPY-positive somas were outlined and counted in both R6/2 mice and human patients with HD. We found that the overall number of NPY interneurons within the striatum of R6/2 mice was not lost (11.7 ± 5.8 NPY interneurons/ 500 000 µm² in wild-type vs. 10.1 ± 5.4 NPY interneurons/ 500 000 µm² in R6/2; P = 0.07; Fig. 5A). However, closer inspection of the two subtypes of NPY interneurons revealed that the number of NPY/nNOS– interneurons is significantly reduced in the dorsolateral striatum of 12-weeks-old R6/2 mice (1.6 ± 0.3 NPY interneurons/500 000 um² in wild-type vs. 0.4 ±0.1 NPY interneurons/500 000 µm² R6/2; P = 0.012; data not shown). In human patients with HD, NPY interneurons were spared in the caudate (where CB₁receptor levels are decreased at NPY interneurons; 1.7 ± 0.29 NPY interneurons/500 000 µm² in patients with HD vs. 1.6 ± 0.20 NPY interneurons/500 000 µm² in non-HD patients), but were significantly higher in the putamen [where CB₁ receptors are spared; 3.5 ± 0.40 NPY interneurons/500 000 µm² in patients with HD vs. 1.8 ± 0.25 NPY interneurons/500 000 µm² in non-HD patients; F = 9.357, P < 0.001 (non-HD putamen vs. HD putamen, HD putamen vs. HD caudate, HD putamen vs. non-HD caudate); Fig. 5B]. These results confirm previous studies that also found an increase in the number of NPY interneurons in the caudate and putamen of patients with HD (Dawbarn et al., 1985).

Fig. 5

Population of neuropeptide Y (NPY) interneurons in the striatum of 12-week-old R6/2 mice and the caudate-putamen of patients with HD. (A) Number of NPY-positive soma in the dorsolateral striatum of 12-week-old R6/2 compared with wild-type littermates …

Functional loss of CREB signaling downstream of CB ₁ receptors activation in striatal NPY interneurons in R6/2 mice

Activation of CREB through phosphorylation of serine 133 (pCREB) regulates cell survival pathways, and defects in this signaling pathway are thought to contribute to the neurodegeneration of MSN in the dorsolateral striatum (Mantamadiotis et al., 2002). Acute activation of CB₁ receptors increases pCREB levels in the brain and, accordingly, CB₁ receptor downregulation could lead to a decrease in pCREB levels at specific neuronal subtypes (Rubino et al., 2006; Isokawa, 2009). To test if decreased CB₁ receptor expression in R6/ 2 mice affected CREB signaling, we treated 12-week-old R6/2 mice and their wild-type littermates with the CB₁ receptor agonist WIN 55,212–2 and measured changes in pCREB levels in NPY interneurons located within the dorsolateral striatum by sq-IHC. While CB₁ receptor activation in wild-type mice caused a significant 12.4 ± 2.5% increase in the amount of pCREB in striatal NPY interneurons of wild-type mice [pCREB in NPY of R6/2 normalized to pCREB in NPY of wild-type littermate: F = 5.677, P < 0.001 (wild-type WIN 55,212−2 vs. wild-type vehicle and knockout vehicle), P < 0.05 (wild-type WIN 55,212-2 vs. R6/2 vehicle and R6/2 WIN 55,212–2); Fig. 6A, B and E], this treatment did not increase pCREB in NPY interneurons of R6/2 or CB₁ knockout mice (F = 5.677, P > 0.05; Fig. 6C – E). These results suggest that CB₁ receptor downregulation results in a functional loss of cannabinoid-dependent pCREB signaling in striatal NPY interneurons of R6/2 mice.

Fig. 6

Phosphorylated cAMP response element-binding protein (pCREB) in striatal neuropeptide Y (NPY) interneurons following in vivo WIN 55,212-2 administration. (A–D) Striatal sections from 12-week-old R6/2 and wild-type littermates dual-labeled for …

The authors would like to thank Dr Ingo Willuhn, Dr Vicente Martinez, Dr George Yohrling and Dr Seung Kwak for helpful discussions, as well as Aimee Schantz and Kim Howard for assistance with human tissue studies. This work was funded by the CHDI.

CB₁ receptor

cannabinoid receptor 1

CREB

cAMP response element-binding protein

GABA

γ-aminobutyric acid

HD

Huntington’s disease

Htt

huntingtin protein

IPSC

inhibitory postsynaptic current

LTS

low-threshold spiking

mHtt

mutant huntingtin

MSN

medium spiny neuron

NA

numerical aperture

NMDA

N-methyl-d-aspartic acid

nNOS

neuronal nitric oxide synthase

NPY

neuropeptide Y

NR2B

NMDA receptor subunit 2B

PBS

phosphate-buffered saline

PCR

polymerase chain reaction

pCREB

phosphorylated CREB

PET

positron emission tomography

PFA

paraformaldehyde

qPCR

quantitative polymerase chain reaction

ROI

region of interest

RT

room temperature

sq-IHC

semi-quantitative immunohistochemistry

Conflicts of interest

The authors report no conflict of interest.

• Andre VM, Cepeda C, Venegas A, Gomez Y, Levine MS. Altered cortical glutamate receptor function in the R6/2 model of Huntington’s disease.J. Neurophysiol. 2006;95:2108–2119. [PubMed]
• Arrasate M, Mitra S, Schweitzer ES, Segal MR, Finkbeiner S. Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death. Nature. 2004;431:805–810. [PubMed]
• Berghuis P, Rajnicek AM, Morozov YM, Ross RA, Mulder J, Urban GM, Monory K, Marsicano G, Matteoli M, Canty A, Irving AJ, Katona I, Yanagawa Y, Rakic P, Lutz B, Mackie K, Hark-any T. Hardwiring the brain: endocannabinoids shape neuronal connectivity. Science.2007;316:1212–1216. [PubMed]
• Blazquez C, Chiarlone A, Sagredo O, Aguado T, Pazos R, Resel E, Palazuelos J, Julien B, Salazar M, Borner C, Benito C, Carrasco C, Diez-Zaera M, Paoletti P, Guzman M. Loss of striatal CB1 cannabinoid receptors is a key pathogenic factor in Huntington’s disease. Brain.2011;134:119–136. [PubMed]
• Bouaboula M, Poinot-Chazel C, Bourrie B, Canat X, Calandra B, Ri-naldi-Carmona M, Le Fur G, Casellas P. Activation of mitogen-activated protein kinases by stimulation of the central cannabinoid receptor CB1.Biochem. J. 1995;312(Pt 2):637–641. [PMC free article] [PubMed]
• Callahan JW, Abercrombie ED. In vivo dopamine efflux is decreased in striatum of both fragment (R6/2) and full-length (YAC128) transgenic mouse models of Huntington’s disease. Front. Syst. Neurosci. 2011;5:61.[PMC free article] [PubMed]
• Cepeda C, Hurst RS, Calvert CR, Hernandez-Echeagaray E, Nguyen OK, Jocoy E, Christian LJ, Ariano MA, Levine MS. Transient and progressive electrophysiological alterations in the corticostriatal pathway in a mouse model of Huntington’s disease. J. Neurosci.2003;23:961–969. [PubMed]
• Cepeda C, Starling AJ, Wu N, Nguyen OK, Uzgil B, Soda T, Andre VM, Ariano MA, Levine MS. Increased GABAergic function in mouse models of Huntington’s disease: reversal by BDNF. J. Neurosci. Res.2004;78:855–867. [PubMed]
• Chiodi V, Uchigashima M, Beggiato S, Ferrante A, Armida M, Mar-tire A, Potenza RL, Ferraro L, Tanganelli S, Watanabe M, Domenici MR, Popoli P. Unbalance of CB1 receptors expressed in GABAergic and glutamatergic neurons in a transgenic mouse model of Huntington’s disease. Neurobiol. Dis. 2011;45:983–991. [PubMed]
• Cummings DM, Cepeda C, Levine MS. Alterations in striatal synaptic transmission are consistent across genetic mouse models of Huntington’s disease. ASN Neuro. 2010;2:e00036. [PMC free article][PubMed]
• Dawbarn D, De Quidt ME, Emson PC. Survival of basal ganglia neuropeptide Y-somatostatin neurones in Huntington’s disease. Brain Res. 1985;340:251–260. [PubMed]
• Dehorter N, Guigoni C, Lopez C, Hirsch J, Eusebio A, Ben-Ari Y, Hammond C. Dopamine-deprived striatal GABAergic interneurons burst and generate repetitive gigantic IPSCs in medium spiny neurons.J. Neurosci. 2009;29:7776–7787. [PubMed]
• Denovan-Wright EM, Robertson HA. Cannabinoid receptor messenger RNA levels decrease in a subset of neurons of the lateral striatum, cortex and hippocampus of transgenic Huntington’s disease mice. Neuro-science. 2000;98:705–713. [PubMed]
• Derkinderen P, Valjent E, Toutant M, Corvol JC, Enslen H, Ledent C, Trzaskos J, Caboche J, Girault JA. Regulation of extracellular signal-regulated kinase by cannabinoids in hippocampus. J. Neurosci.2003;23:2371–2382. [PubMed]
• DiFiglia M, Sapp E, Chase KO, Davies SW, Bates GP, Vonsattel JP, Aronin N. Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. Science. 1997;277:1990–1993. [PubMed]
• Dowie MJ, Bradshaw HB, Howard ML, Nicholson LF, Faull RL, Hannan AJ, Glass M. Altered CB1 receptor and endocannabinoid levels precede motor symptom onset in a transgenic mouse model of Huntington’s disease. Neuroscience. 2009;163:456–465. [PubMed]
• Fernandez-Ruiz J. The endocannabinoid system as a target for the treatment of motor dysfunction. Br. J. Pharmacol. 2009;156:1029–1040.[PMC free article] [PubMed]
• Finkbeiner S. CREB couples neurotrophin signals to survival messages.Neuron. 2000;25:11–14. [PubMed]
• Fusco FR, Martorana A, Giampa C, De March Z, Farini D, D’Angelo V, Sancesario G, Bernardi G. Immunolocalization of CB1 receptor in rat striatal neurons: a confocal microscopy study. Synapse. 2004;53:159–167. [PubMed]
• Gerdeman G, Lovinger DM. CB1 cannabinoid receptor inhibits synaptic release of glutamate in rat dorsolateral striatum. J. Neurophysiol.2001;85:468–471. [PubMed]
• Gittis AH, Nelson AB, Thwin MT, Palop JJ, Kreitzer AC. Distinct roles of GABAergic interneurons in the regulation of striatal output pathways. J. Neurosci. 2010;30:2223–2234. [PMC free article] [PubMed]
• Giuffrida A, Parsons LH, Kerr TM, Rodriguez de Fonseca F, Navarro M, Piomelli D. Dopamine activation of endogenous cannabinoid signaling in dorsal striatum. Nat. Neurosci. 1999;2:358–363. [PubMed]
• Glass M, Faull RL, Dragunow M. Loss of cannabinoid receptors in the substantia nigra in Huntington’s disease. Neuroscience. 1993;56:523–527. [PubMed]
• Glass M, Dragunow M, Faull RL. The pattern of neurodegeneration in Huntington’s disease: a comparative study of cannabinoid, dopamine, adenosine and GABA(A) receptor alterations in the human basal ganglia in Huntington’s disease. Neuroscience. 2000;97:505–519.[PubMed]
• Graham ES, Ball N, Scotter EL, Narayan P, Dragunow M, Glass M. Induction of Krox-24 by endogenous cannabinoid type 1 receptors in Neuro2A cells is mediated by the MEK-ERK MAPK pathway and is suppressed by the phosphatidylinositol 3-kinase pathway. J. Biol. Chem.2006;281:29085–29095. [PubMed]
• Gray M, Shirasaki DI, Cepeda C, Andre VM, Wilburn B, Lu XH, Tao J, Yamazaki I, Li SH, Sun YE, Li XJ, Levine MS, Yang XW. Full-length human mutant huntingtin with a stable polyglutamine repeat can elicit progressive and selective neuropathogenesis in BACHD mice. J. Neurosci. 2008;28:6182–6195. [PMC free article] [PubMed]
• Group Hs.D.C.R. A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington’s disease chromosomes. The Huntington’s Disease Collaborative Research Group. Cell. 1993;72:971–983. [PubMed]
• Grouzmann E, Comoy E, Walker P, Burnier M, Bohuon C, Waeber B, Brunner H. Production and characterization of four anti-neuropeptide Y monoclonal antibodies. Hybridoma. 1992;11:409–424. [PubMed]
• Gunawardena S, Her LS, Brusch RG, Laymon RA, Niesman IR, Gordesky-Gold B, Sintasath L, Bonini NM, Goldstein LS. Disruption of axonal transport by loss of huntingtin or expression of pathogenic polyQ proteins in Drosophila. Neuron. 2003;40:25–40. [PubMed]
• Hardingham GE, Fukunaga Y, Bading H. Extrasynaptic NMDARs oppose synaptic NMDARs by triggering CREB shut-off and cell death pathways. Nat. Neurosci. 2002;5:405–414. [PubMed]
• Heng MY, Detloff PJ, Wang PL, Tsien JZ, Albin RL. In vivo evidence for NMDA receptor-mediated excitotoxicity in a murine genetic model of Huntington disease. J. Neurosci. 2009;29:3200–3205. [PubMed]
• Her LS, Goldstein LS. Enhanced sensitivity of striatal neurons to axonal transport defects induced by mutant huntingtin. J. Neurosci.2008;28:13662–13672. [PubMed]
• Herkenham M, Lynn AB, Johnson MR, Melvin LS, de Costa BR, Rice KC. Characterization and localization of cannabinoid receptors in rat brain: a quantitative in vitro autoradiographic study. J. Neurosci.1991;11:563–583. [PubMed]
• Hohmann AG, Herkenham M. Localization of cannabinoid CB (1) receptor mRNA in neuronal subpopulations of rat striatum: a double-label in situ hybridization study. Synapse. 2000;37:71–80. [PubMed]
• Hudson BD, Hebert TE, Kelly ME. Physical and functional interaction between CB1 cannabinoid receptors and beta2-adrenoceptors. Br. J. Pharmacol. 2010;160:627–642. [PMC free article] [PubMed]
• Ibanez-Sandoval O, Tecuapetla F, Unal B, Shah F, Koos T, Tepper JM. A novel functionally distinct subtype of striatal neuropeptide Y interneuron. J. Neurosci. 2011;31:16757–16769. [PMC free article][PubMed]
• Isokawa M. Time-dependent induction of CREB phosphorylation in the hippocampus by the endogenous cannabinoid. Neurosci. Lett.2009;457:53–57. [PMC free article] [PubMed]
• Johnson MA, Rajan V, Miller CE, Wightman RM. Dopamine release is severely compromised in the R6/2 mouse model of Huntington’s disease. J. Neurochem. 2006;97:737–746. [PubMed]
• Kofalvi A, Rodrigues RJ, Ledent C, Mackie K, Vizi ES, Cunha RA, Sperlagh B. Involvement of cannabinoid receptors in the regulation of neurotransmitter release in the rodent striatum: a combined immunochemical and pharmacological analysis. J. Neurosci.2005;25:2874–2884. [PubMed]
• Kreitzer AC. Physiology and pharmacology of striatal neurons. Annu. Rev. Neurosci. 2009;32:127–147. [PubMed]
• Kreitzer AC, Malenka RC. Dopamine modulation of state-dependent endocannabinoid release and long-term depression in the striatum. J. Neurosci. 2005;25:10537–10545. [PubMed]
• Lee WC, Yoshihara M, Littleton JT. Cytoplasmic aggregates trap polyglutamine-containing proteins and block axonal transport in a Drosophila model of Huntington’s disease. Proc. Natl. Acad. Sci. USA.2004;101:3224–3229. [PMC free article] [PubMed]
• Li H, Li SH, Cheng AL, Mangiarini L, Bates GP, Li XJ. Ultrastructural localization and progressive formation of neuropil aggregates in Huntington’s disease transgenic mice. Hum. Mol. Genet. 1999;8:1227–1236. [PubMed]
• Lin CH, Tallaksen-Greene S, Chien WM, Cearley JA, Jackson WS, Crouse AB, Ren S, Li XJ, Albin RL, Detloff PJ. Neurological abnormalities in a knock-in mouse model of Huntington’s disease. Hum. Mol. Genet. 2001;10:137–144. [PubMed]
• Luthi-Carter R, Strand A, Peters NL, Solano SM, Hollingsworth ZR, Menon AS, Frey AS, Spektor BS, Penney EB, Schilling G, Ross CA, Borchelt DR, Tapscott SJ, Young AB, Cha JH, Olson JM. Decreased expression of striatal signaling genes in a mouse model of Huntington’s disease. Hum. Mol. Genet. 2000;9:1259–1271. [PubMed]
• Mangiarini L, Sathasivam K, Seller M, Cozens B, Harper A, Hethe-rington C, Lawton M, Trottier Y, Lehrach H, Davies SW, Bates GP. Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell.1996;87:493–506. [PubMed]
• Mantamadiotis T, Lemberger T, Bleckmann SC, Kern H, Kretz O, Martin Villalba A, Tronche F, Kellendonk C, Gau D, Kapfhammer J, Otto C, Schmid W, Schutz G. Disruption of CREB function in brain leads to neurodegeneration. Nat. Genet. 2002;31:47–54. [PubMed]
• Marsicano G, Wotjak CT, Azad SC, Bisogno T, Rammes G, Cascio MG, Hermann H, Tang J, Hofmann C, Zieglgansberger W, Di Marzo V, Lutz B. The endogenous cannabinoid system controls extinction of aversive memories. Nature. 2002;418:530–534. [PubMed]
• Marsicano G, Goodenough S, Monory K, Hermann H, Eder M, Cannich A, Azad SC, Cascio MG, Gutierrez SO, van der Stelt M, Lopez-Rodriguez ML, Casanova E, Schutz G, Zieglgansberger W, Di Marzo V, Behl C, Lutz B. CB1 cannabinoid receptors and on-demand defense against excitotoxicity. Science. 2003;302:84–88. [PubMed]
• Massouh M, Wallman MJ, Pourcher E, Parent A. The fate of the large striatal interneurons expressing calretinin in Huntington’s disease.Neurosci. Res. 2008;62:216–224. [PubMed]
• Matyas F, Yanovsky Y, Mackie K, Kelsch W, Misgeld U, Freund TF. Subcellular localization of type 1 cannabinoid receptors in the rat basal ganglia. Neuroscience. 2006;137:337–361. [PubMed]
• McCaw EA, Hu H, Gomez GT, Hebb AL, Kelly ME, Denovan-Wright EM. Structure, expression and regulation of the cannabinoid receptor gene (CB1) in Huntington’s disease transgenic mice. Eur. J. Biochem.2004;271:4909–4920. [PubMed]
• Miller BR, Bezprozvanny I. Corticostriatal circuit dysfunction in Huntington’s disease: intersection of glutamate, dopamine and calcium.Future Neurol. 2010;5:735–756. [PMC free article] [PubMed]
• Miller J, Arrasate M, Brooks E, Libeu CP, Legleiter J, Hatters D, Curtis J, Cheung K, Krishnan P, Mitra S, Widjaja K, Shaby BA, Lotz GP, Newhouse Y, Mitchell EJ, Osmand A, Gray M, Thulasiramin V, Saudou F, Segal M, Yang XW, Masliah E, Thompson LM, Muchowski PJ, Weisgraber KH, Finkbeiner S. Identifying polyglutamine protein speciesin situ that best predict neurodegeneration. Nat. Chem. Biol.2011;7:925–934. [PMC free article] [PubMed]
• Milnerwood AJ, Gladding CM, Pouladi MA, Kaufman AM, Hines RM, Boyd JD, Ko RW, Vasuta OC, Graham RK, Hayden MR, Murphy TH, Raymond LA. Early increase in extrasynaptic NMDA receptor signaling and expression contributes to phenotype onset in Huntington’s disease mice. Neuron. 2010;65:178–190. [PubMed]
• Narushima M, Uchigashima M, Hashimoto K, Watanabe M, Kano M. Depolarization-induced suppression of inhibition mediated by endocannabinoids at synapses from fast-spiking interneurons to medium spiny neurons in the striatum. Eur. J. Neurosci.2006;24:2246–2252. [PubMed]
• Okamoto S, Pouladi MA, Talantova M, Yao D, Xia P, Ehrnhoefer DE, Zaidi R, Clemente A, Kaul M, Graham RK, Zhang D, Vincent Chen HS, Tong G, Hayden MR, Lipton SA. Balance between synaptic versus extrasynaptic NMDA receptor activity influences inclusions and neurotoxicity of mutant huntingtin. Nat. Med. 2009;15:1407–1413.[PMC free article] [PubMed]
• Palop JJ, Chin J, Mucke L. A network dysfunction perspective on neurodegenerative diseases. Nature. 2006;443:768–773. [PubMed]
• Richfield EK, Herkenham M. Selective vulnerability in Huntington’s disease: preferential loss of cannabinoid receptors in lateral globus pallidus. Ann. Neurol. 1994;36:577–584. [PubMed]
• Rosas HD, Koroshetz WJ, Chen YI, Skeuse C, Vangel M, Cudkowicz ME, Caplan K, Marek K, Seidman LJ, Makris N, Jenkins BG, Goldstein JM. Evidence for more widespread cerebral pathology in early HD: an MRI-based morphometric analysis. Neurology. 2003;60:1615–1620.[PubMed]
• Rubino T, Vigano D, Premoli F, Castiglioni C, Bianchessi S, Zippel R, Parolaro D. Changes in the expression of G protein-coupled receptor kinases and beta-arrestins in mouse brain during cannabinoid tolerance: a role for RAS-ERK cascade. Mol. Neurobiol. 2006;33:199–213. [PubMed]
• Silva AJ, Kogan JH, Frankland PW, Kida S. CREB and memory. Annu. Rev. Neurosci. 1998;21:127–148. [PubMed]
• Stella N, Piomelli D. Receptor-dependent formation of endogenous cannabinoids in cortical neurons. Eur. J. Pharmacol. 2001;425:189–196.[PubMed]
• Tepper JM, Tecuapetla F, Koos T, Ibanez-Sandoval O. Heterogeneity and diversity of striatal GABAergic interneurons. Front. Neuroanat.2010;4:150. [PMC free article] [PubMed]
• Uchigashima M, Narushima M, Fukaya M, Katona I, Kano M, Watanabe M. Subcellular arrangement of molecules for 2-arachidonoyl-glycerol-mediated retrograde signaling and its physiological contribution to synaptic modulation in the striatum. J. Neurosci.2007;27:3663–3676. [PubMed]
• Van Laere K, Casteels C, Dhollander I, Goffin K, Grachev I, Bormans G, Vandenberghe W. Widespread decrease of type 1 cannabinoid receptor availability in Huntington disease in vivo . J. Nucl. Med. 2010;51:1413–1417. [PubMed]
• Vonsattel JP, Myers RH, Stevens TJ, Ferrante RJ, Bird ED, Richardson EP., Jr Neuropathological classification of Huntington’s disease. J. Neuropathol. Exp. Neurol. 1985;44:559–577. [PubMed]
• Walker FO. Huntington’s disease. Lancet. 2007;369:218–228.[PubMed]
• Woodman B, Butler R, Landles C, Lupton MK, Tse J, Hockly E, Moffitt H, Sathasivam K, Bates GP. The Hdh(Q150/Q150) knock-in mouse model of HD and the R6/2 exon 1 model develop comparable and widespread molecular phenotypes. Brain Res. Bull. 2007;72:83–97.[PubMed]