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Journal of Alzheimer’s Disease 40 (2014) 701–712
DOI 10.3233/JAD-131910
IOS Press
701
Altered Expression of the CB1 Cannabinoid
Receptor in the Triple Transgenic Mouse
Model of Alzheimer’s Disease
Gaurav Bedsea,1, Adele Romanoa,1, Silvia Ciancia, Angelo M. Lavecchiaa, Pace Lorenzof,
Maurice R. Elphickb, Frank M. LaFerlac, Gianluigi Vendemialed, Caterina Grilloe, Fabio Altierie,
Tommaso Cassanof,∗and Silvana Gaetania
aDepartment of Physiology and Pharmacology, Sapienza University of Rome, Rome, Italy
bSchool of Biological and Chemical Sciences, Queen Mary University of London, Mile End Road, London, UK
cDepartment of Neurobiology and Behaviour, University of California, Irvine, CA, USA
dDepartment of Medical and Surgical Sciences, University of Foggia, Foggia, Italy
eDepartment of Biochemical Sciences, Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza University of Rome,
Rome, Italy
fDepartment of Clinical and Experimental Medicine, University of Foggia, Foggia, Italy
Handling Associate Editor: Patrizia Mecocci
Accepted 18 December 2013
Abstract. The endocannabinoid system has gained much attention as a new potential pharmacotherapeutic target in various
neurodegenerative diseases, including Alzheimer’s disease (AD). However, the association between CB1 alterations and the
development of AD neuropathology is unclear and often contradictory. In this study, brain CB1 mRNA and CB1 protein levels
were analyzed in 3×Tg-AD mice and compared to wild-type littermates at 2, 6 and 12 months of age, using in-situ hybridization
and immunohistochemistry, respectively. Semiquantitative analysis of CB1 expression focused on the prefrontal cortex (PFC),
prelimbic cortex, dorsal hippocampus (DH), basolateral amygdala complex (BLA), and ventral hippocampus (VH), all areas
with high CB1 densities that are strongly affected by neuropathology in 3×Tg-AD mice. At 2 months of age, there was no
change in CB1 mRNA and protein levels in 3×Tg-AD mice compared to Non-Tg mice in all brain areas analyzed. However,
at 6 and 12 months of age, CB1 mRNA levels were significantly higher in PFC, DH, and BLA, and lower in VH in 3×Tg-AD
mice compared to wild-type littermates. CB1 immunohistochemistry revealed that CB1 protein expression was unchanged in
3×Tg-AD at 2 and 6 months of age, while a significant decrease in CB1 receptor immunoreactivity was detected in the BLA and
DH of 12-month-old 3×Tg-AD mice, with no sign of alteration in other brain areas. The altered CB1 levels appear, rather, to
be age-and/or pathology-dependent, indicating an involvement of the endocannabinoid system in AD pathology and supporting
the ECS as a potential novel therapeutic target for treatment of AD.
Keywords: 3×Tg-AD mice, Alzheimer’s disease, basolateral amygdala complex, CB1 mRNA, CB1 receptor, endocannabinoid
system, hippocampus, prefrontal cortex
1These authors contributed equally to this manuscript.
∗Correspondence to: Tommaso Cassano, Department of Clinical
and Experimental Medicine, University of Foggia, Viale Luigi Pinto
1, Foggia 71100, Italy. Tel.: +39 0881 588042; Fax: +39 0881 188
0432; E-mail: tommaso.cassano@unifg.it.
INTRODUCTION
Alzheimer’s disease (AD) is a progressive, degener-
ative, and irreversible neurological disorder that causes
deterioration of memory, judgment, and reasoning
in the elderly. AD is characterized by accumulation
ISSN 1387-2877/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved
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702 G. Bedse et al. / Altered CB1 Receptor Expression in 3×Tg-AD
of extracellular insoluble plaques, intracellular neu-
rofibrillary tangles (NFTs) in the brain and selective
synaptic and neuronal loss. Extracellular plaques con-
sist of amyloid-(A) protein and NFTs are composed
of hyperphosphorylated tau protein [1]. Although A
plaques and NFTs pathology are prominent, other
pathological alterations in neurotransmitter systems
and concomitant changes in synthetic enzymes and
associated receptors are also an important feature of
AD. For example, cholinergic and glutamatergic neu-
rotransmitter systems are known to be affected by AD
[2].
The endocannabinoid system (ECS) has gained
much attention as a new potential pharmacothera-
peutic target in various neurodegenerative diseases
including AD. The CB1-type cannabinoid receptor
(CB1) is the most abundant G protein-coupled recep-
tor expressed in the central nervous system (CNS) and
through the activation of CB1 receptors in the CNS,
the ECS exerts important functions such as retrograde
inhibition of neurotransmitter release, control of neu-
ronal excitability, and regulation of various forms of
synaptic plasticity [3]. Aberrant patterns of brain CB1
receptor expression and densities have been observed
postmortem in patients suffering from AD and in ani-
mal models of AD. However, these observations are
sparse and often contradictory [4–8], so the relation-
ship between alterations in CB1 expression and the
development of AD neuropathology is still unclear.
Oddo and his colleagues developed a triple trans-
genic mouse model of AD (3×Tg-AD) harboring
three mutant human genes PS1M146 V, APPSwe, and
TauP301 L [9]. This model mimics critical aspects of AD
neuropathology observed in the human AD patients
[10, 11]: it 1) progressively develops both plaques and
tangles in AD relevant brain regions (mainly cortex,
hippocampus, and amygdala); 2) exhibits early deficits
in synaptic plasticity, including long-term potentiation;
and 3) shows selective loss of ␣7 neuronal nicotinic
acetylcholine receptors [9, 12], severe deficits in gluta-
matergic neurotransmission, and altered mitochondrial
functions in hippocampus and cortex [13].
The aim of the present study was to evaluate whether
brain CB1 expression is altered in 3×Tg-AD mice
in comparison with wild type littermates (Non-Tg).
Moreover, to investigate whether the temporal and
regional patterns of such possible alterations might
overlap with those of Aand tau pathology in this
AD model, brain CB1 expression was analyzed at dif-
ferent ages [9]. As a consequence, by studying the
temporal expression of CB1 in the wild-type litter-
mates, our study has also allowed us to analyze the
impact of aging on CB1 levels. Our analyses were con-
ducted on both CB1 mRNA and CB1 protein levels in
3×Tg-AD and wild-type mice at 2, 6 and 12 months of
age, by in situ hybridization and immunohistochem-
istry, respectively, followed by the semi-quantitative
analysis of the respective signals obtained in prefrontal
cortex (PFC), prelimbic cortex (PrL), dorsal hippocam-
pus (DH), basolateral amygdala complex (BLA), and
ventral hippocampus (VH), all areas strongly affected
by the neuropathology and characterized by high CB1
densities.
MATERIALS AND METHODS
Animals
Male 3×Tg-AD and Non-Tg mice aged 2-, 6-
and 12-months old were used in this study. The
3×Tg-AD mice harboring PS1M146 V, APPSwe, and
TauP301 L transgenes were genetically engineered by
LaFerla and colleagues at the Department of Neuro-
biology and Behavior, University of California, Irvine
[9]. Colonies of 3×Tg-AD mice and Non-Tg litter-
mates were established at the vivarium of the Puglia
and Basilicata Experimental Zooprophylactic Insti-
tute (Foggia, Italy). The 3×Tg-AD mice background
strain is C57BL6/129SvJ hybrid and genotypes were
confirmed from tail biopsy, according to the pro-
cedures described previously [9, 14]. The housing
conditions were controlled (temperature 22◦C, light
from 07 : 00–19 : 00, humidity 50%–60%), and fresh
food and water were freely available.
In situ hybridization
In situ hybridization was performed on coronal
sections of brains using a 35S-labeled RNA probe com-
plementary to rat CB1 mRNA. Riboprobes in antisense
and sense orientation were generated from linearized
vector constructs (520 bp, a kind gift of Dr. Jin Fu,
Xiamen University) by in vitro transcription using the
appropriate RNA polymerases [15].
Mice (n= 5 per group) were euthanized by decapi-
tation; their brains were rapidly removed, snap frozen
in 2-methylbutane (−50◦C), and stored at −80◦C.
Brain sections (20 m) were cut on a cryostat (−20◦C)
and thaw-mounted on RNAse-free positively charged
slides to be hybridized at 60◦C for 16 h in a buffer
containing [35S]cRNA (45,000 dpm ml−1), 10%
dextran sulfate, 50% formamide, 1×Denhardt’s solu-
tion, 100 gml
−1denatured salmon sperm DNA,
0.15 mg ml−1tRNA, and 40 mM dithiothreitol. After
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G. Bedse et al. / Altered CB1 Receptor Expression in 3×Tg-AD 703
hybridization, the sections were exposed to Kodak
Biomax film (Sigma-Aldrich) for 3 days. Autoradiog-
raphy films were first scanned (Epson perfection 3200
PHOTO) at high resolution (900 dpi). Optical densities
were converted to radioactivity measurements (Ci)
by densitometric analysis of 14C-microscale standards
that were used to create a calibration curve.
Immunohistochemistry
Mice (n= 3 per group) were intra-
cardioventricularly perfused with saline followed
by fixation solution (4% paraformaldehyde in 0.1
M phosphate buffer, PB, pH 7.4) at a flow rate of
36 ml min−1. Then brains were fixed for 48 h in 4%
paraformaldehyde. Free-floating coronal sections of
50 m thickness were obtained using a vibratome
slicing system (microM, Walldorf, Germany) and
stored at 4◦C in 0.02% sodium azide in phosphate
buffered (PB).
Immunohistochemistry was performed using both
peroxidase-based and fluorescence-based revealing
systems.
For peroxidase-based immunohistochemistry,
endogenous peroxidase activity was quenched for
30 min in 0.3% H2O2. The brain sections were blocked
with 10% normal goat serum/PBS with 0.3% Triton
X-100 and then incubated with CB1 2825.3 antiserum
(raised against C-terminal residues 461–473, 1 : 1500
dilution) overnight at 4◦C [16]. Evidence of the selec-
tivity of this antiserum in revealing CB1 expression in
the rat nervous system has been previously obtained
by pre-absorption tests with the CB1 C-terminal
peptide antigen and by western blotting, which reveals
a band in rat brain homogenates (∼53 kDa) consistent
with the expected molecular mass for CB1 [16, 17].
Furthermore, the selectivity of the antiserum for CB1
has been previously confirmed by analysis of brain
tissue and dorsal root ganglia from CB1-knockout
mice [17, 18]. After removing the primary antiserum
in excess, sections were incubated with secondary
antibody (Biotin-SP-conjugated fragment donkey
anti rabbit IgG) for 1 h at room temperature. After
washing excess of antibody, sections were treated
with avidin–biotin–peroxidase complex (ABC, 1 : 200
dilution, Vector Laboratories) and then developed with
diaminobenzidine substrate using the avidin-biotin
horseradish peroxidase system (Vector Laboratories).
For fluorescence-based immunohistochemistry, free
floating coronal brain sections of 30 m thickness were
obtained using a cryostat (Microm HM550, Thermo
scientific) and stored at 4◦C in 0.02% sodium azide in
PB. The brain sections were treated with 90% of formic
acid for 7 min followed by PB washes. Then the brain
sections were blocked in a solution containing 5% nor-
mal goat serum and 0.3% Triton X-100 in PB and then
incubated with both CB1 2825.3 antiserum (1 : 1500
dilution) and Amonoclonal antibody (6E10, Cov-
ance, 1 : 1500 dilution) for 16 h at 4◦C. After removing
primary antibodies, sections were incubated with both
secondary antibodies Alexa Fluor 555 donkey anti-
rabbit IgG (1 : 250 dilution) and Alexa Fluor 488 goat
anti-mouse IgG (1 :250 dilution) for 1 h and 30 min at
room temperature. All washes after this step were car-
ried out in dark. After washing off excess antibodies,
sections were treated with Hoechst, Sigma (1 : 5000
dilution). After washing excess Hoechst with PB, brain
slices were mounted on slides. Furthermore, to con-
firm the background staining level, immunofluorescent
staining for CB1 was also carried out without the pri-
mary antibody.
Peroxidase-based immunolabeled sections were
viewed using a Nikon 80i Eclipse microscope equipped
with a DS-U1 digital camera, and NIS-elements BR
software (Nikon, Tokyo, Japan). Fluorescence-based
immunolabeled sections were observed under the con-
focal microscope Olympus FV-1000.
Semiquantitative analyses of the autoradiographic
signal of hybridized CB1 mRNA and of CB1 immunos-
taining were performed using freeware software from
the National Institutes of Health (Scion Image soft-
ware) and were expressed as optical densities.
Statistical analysis
The optical densities obtained by the semiquantita-
tive analyses were analyzed by two way analysis of
variance (ANOVA), with genotype and age as vari-
ables. Tukey’s honestly significant difference test was
used for multiple post hoc comparisons. The correla-
tion analysis between Aand CB1 protein levels was
performed on the respective optical densities measured
on double immunofluorescent slices and expressed as
percentage of those measured in Non-Tg mice, by
using the Pearson correlation test. Statistical signifi-
cance threshold was set at p< 0.05.
RESULTS
CB1 mRNA expression
Representative images of CB1 mRNA distribution
in the mouse brain is shown in Fig. 1A and quan-
titative analysis of CB1 mRNA expression in PFC,
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Fig. 1. CB1 mRNA distribution pattern in Non-Tg and 3xTg-AD mice. A) Representative micrographs of coronal sections from mouse brain
showing distribution of CB1 mRNA scanned from autoradiographic film exposed for 3 days. The dashed lines indicate the brain regions where
the optical density was measured. B–F) CB1 mRNA expression levels in Non-Tg (open bars) and 3xTg-AD mice (black bars) at 2, 6, and 12
months (2M, 6M, 12M, respectively) of age in PFC (B), PrL (C), DH (D), VH (E) and BLA (F). The data are expressed as means±SEM
*p< 0.05 versus Non-Tg and ◦p< 0.05 (n= 5 per group).
PrL, DH, VH, and BLA is shown in Fig. 1B–F.
The results from ANOVA revealed an overall effect
of genotype [F(genotype)1,122 = 31.992, p< 0.001], age
[F(age)2,122 = 16.177, p< 0.001], and genotype ×age
interaction [F(age x genotype)2,122 = 4.288, p< 0.05] on
CB1 mRNA expression in PFC (Fig. 1B). Post hoc
comparisons revealed that CB1 mRNA expression
was significantly higher in 3×Tg-AD mice compared
to Non-Tg mice at 6 months (+56%, p< 0.05) and
12 months (+15%, p< 0.05) of age. Different results
were obtained for PrL, where a significant over-
all effect of age was observed [F(age)2,113 = 18.212,
p< 0.001], with no significant overall effect of
genotype [F(genotype)1,113 = 1.161, n.s.] and genotype
by age interaction [F(age x genotype)2,113 = 0.871, n.s.]
(Fig. 1C). ANOVA analysis of CB1 mRNA expres-
sion in DH and VH demonstrated a significant
overall effect of age, genotype, and age by geno-
type interaction [DH: F(age)2,151 = 81.052, p< 0.001;
F(age x genotype)2,151 = 3.166, p< 0.05; F(genotype)1,151 =
19.079, p< 0.001; VH: F(age)2,182 = 10.431, p< 0.001;
F(age x genotype)2,182 = 6.987, p< 0.001; F(genotype)1,182 =
10.116, p< 0.01]. Interestingly, post hoc compar-
isons revealed a clear dissociation between the dorsal
and ventral hippocampus (Fig. 1D and E, respec-
tively). In particular, the former showed a significantly
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G. Bedse et al. / Altered CB1 Receptor Expression in 3×Tg-AD 705
higher expression of CB1 mRNA in the 3×Tg-AD
mice compared to Non-Tg mice both at 6 months
(+29%, p< 0.05) and 12 months (+33%, p< 0.05)
of age, while in the latter there was a significant
decrease in CB1 mRNA expression in the trans-
genic mice compared to the control group (−40%
and −35%, respectively at 6 and 12 months of
age; p< 0.05). Statistical analysis of CB1 mRNA
expression in the BLA revealed a significant overall
effect of age [F(age)2,160 = 14.888, p< 0.001], geno-
type [F(genotype)1,160 = 31.774, p< 0.001], and age
by genotype interaction [F(age x genotype)2,160 = 8.916,
p< 0.001] (Fig. 1F). Post hoc comparisons revealed
that CB1 mRNA expression was significantly higher in
3×Tg-AD mice compared to Non-Tg mice at 6 months
(+78%, p< 0.05) and 12 months (+49%, p< 0.05) of
age.
CB1 protein expression
Representative microphotographs of CB1 immunos-
taining are shown in Fig. 2A. Figure 2B–F shows
the semiquantitative analysis of CB1 protein expres-
sion in the PFC, PrL, DH, VH, and BLA. The
results from ANOVA revealed an overall effect
of genotype [F(genotype)1,314 = 12.687, p< 0.001] and
age [F(age)2,314 = 59.579, p< 0.001] in DH, with no
significant overall effect of genotype ×age interac-
tion [F(age x genotype)2,314 = 0.345, n.s.] on CB1 protein
expression (Fig. 2D). Post hoc comparisons revealed
that CB1 protein levels were significantly lower in
3×Tg-AD mice compared to Non-Tg mice at 12
months of age (−20%, p< 0.05) and that, in within-
genotype comparisons, both groups of mice at 12
months of age showed significantly lower CB1 protein
levels compared to 2- and 6-month old mice.
For the BLA, ANOVA showed an overall effect
of age [F(age)2,67 = 6.735, p< 0.01], with no signifi-
cant overall effect of genotype [F(genotype)1,67 = 2.736,
n.s.] and significant genotype ×age interaction
[F(age x genotype)2,67 = 3.279, p< 0.05] on CB1 protein
expression (Fig. 2F). Interestingly, at 12 months of
age 3×Tg-AD mice showed (i) lower CB1 protein
expression compared to age-matched Non-Tg mice
(−42%), and (ii) significantly lower CB1 protein levels
compared to 2-month-old (−48%, p< 0.05) and 6-
month-old (−47%, p< 0.05) transgenic mice. Finally,
no significant difference was found between genotypes
at 2, 6 and 12 months of age in PFC, PrL, and VH
(Fig. 2B, C, and E).
Lowered CB1 protein expression in DH and BLA
were further confirmed by immunofluorescent stain-
ing (Fig. 3A, B lower panel). At 12 months of age,
3×Tg-AD mice showed lower CB1 protein levels in
DH (−22%, p< 0.05) and BLA (−48%, p< 0.05) com-
pared to Non-Tg mice (Fig. 3C, D). Moreover, by
performing double immunofluorescence labelling for
CB1 and A(Fig. 3E, F), we could semiquantitatively
measure both protein levels and found an inverse corre-
lation between the decline of CB1 receptor expression
and the buildup of Apathology in both the DH
(Fig. 3G, DH: ρ= –0.7599, p< 0.0001) and the BLA
(Fig. 3H, BLA: ρ= –0.5052, p< 0.001, Pearson Corre-
lation test).
DISCUSSION
In this study, the general pattern of CB1 mRNA
expression and of CB1 protein distribution through-
out the mouse brain revealed similarity with previous
reports [16, 19, 20]. Furthermore, this study has
revealed for the first time that CB1 mRNA and CB1
protein expression in 3×Tg-AD mice is altered in brain
areas particularly involved in learning and memory
processes and where the impact of AD neuropathol-
ogy is more prominent. More specifically, a significant
increase of CB1 mRNA levels in PFC, DH, and BLA
and a reduction in VH were found in 3×Tg-AD mice
compared to Non-Tg mice at 6 and 12 months of age.
Such differences were found to be opposite for CB1
protein levels in the DH and BLA, where CB1 pro-
tein levels were lower in 12-month-old 3×Tg-AD mice
compared to their age-matched Non-Tg mice. No dif-
ferences between genotypes were found in the brains
of 2-month-old mice.
Furthermore, the comparisons within mice from the
same genotype at different ages revealed significant
effects of aging on both CB1 mRNA and CB1 pro-
tein levels in several brain regions. In particular, we
observed an age-dependent increase of CB1 mRNA
levels in most areas for both genotypes (except BLA
for Non-Tg mice and VH for 3×Tg-AD mice), while
a decrease of CB1 protein expression was detected in
two brain areas of aged mice (the DH for both geno-
types and the BLA for 3×Tg-AD mice) as compared to
2- and 6-months-old mice of the respective genotype.
In this study, the correlation between CB1 mRNA
and protein levels observed was not direct. This
observation is not surprising, as it was previously
demonstrated that in general mRNA levels do not
necessarily predict the respective protein levels [21].
Moreover, the discrepant results obtained here are
complex to interpret considering also that CB1
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706 G. Bedse et al. / Altered CB1 Receptor Expression in 3×Tg-AD
Fig. 2. CB1 protein distribution pattern in Non-Tg and 3xTg-AD mice. A) Representative microphotographs of brain coronal sections showing
CB1 immunostaining in the selected brain areas. The dashed lines indicate the brain regions where the optical density was measured. B–F) CB1
protein expression levels in Non-Tg (open bars) and 3xTg-AD mice (black bars) at 2, 6, and 12 months (2M, 6M, 12M, respectively) in PFC
(B), PrL (C), DH (D), VH (E), and BLA (F). The data are expressed as means ±SEM *p<0.05 versus Non-Tg and ◦p< 0.05 (n=3 per group).
receptors are expressed mostly on synaptic termi-
nals while CB1 mRNA is synthesized mostly in the
cell body. For example, CB1 receptors are abundantly
expressed on GABAergic interneurons of several brain
areas that also receive CB1 expressing nerve terminals
from other regions. In this case, the CB1 protein lev-
els may be more abundant than would be expected for
the respective CB1 mRNA level. Other sites may con-
tain only CB1 expressing terminals with no cell bodies
expressing CB1 mRNA and in these areas not neces-
sarily CB1 protein levels correspond to CB1 mRNA
levels. Alternatively or additionally discrepancy in
CB1 protein and mRNA levels might also be due to
modifications at translational and/or post-translational
levels, occurring at the three different ages considered.
Two months of age in our murine AD-model cor-
responds to a pre-pathologic phase characterized by
the absence of any Aand tau pathological expres-
sion [9]. The lack of differences in CB1 expression
between genotypes at this age suggests that 3×Tg-
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G. Bedse et al. / Altered CB1 Receptor Expression in 3×Tg-AD 707
Fig. 3. CB1 protein expression revealed by immunofluorescence staining in 12 month old 3xTg-AD mice. A, B) Representative microphotographs
of brain coronal sections showing nuclear staining with Hoechst (blue) and CB1 immunofluorescence staining (red) in the DH (A) and BLA
(B) without or with CB1 antiserum incubation step. C, D) CB1 protein expression levels measured in the DH (C) and in the BLA (D) of 12
month-old Non-Tg (open bars) and 3xTg-AD mice (black bars). E, F) Representative photographs for CB1 protein (red), Aprotein (green),
and nuclear staining with Hoechst (blue) in DH (E) and BLA (F). G, H) Scatterplot of Aprotein levels versus CB1 protein levels showing
an inverse correlationship (Pearson test) in both the DH (G, ρ= –0.7599, p< 0.0001) and the BLA (H, ρ= –0.5052, p< 0.001) The data are
expressed as means ±SEM *p< 0.05 versus Non-Tg (n= 3 per group).
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708 G. Bedse et al. / Altered CB1 Receptor Expression in 3×Tg-AD
Table 1
Summary of age related molecular and behavioral changes in 3×Tg-AD mice
Age of 3×Tg-AD mice CB1 receptor Molecular and behavioral observation References
2 months mRNA and protein unchanged –no Aand tau pathology [9]
–cognitively unimpaired
4 months – –intraneuronal Ain hippocampus and amygdala [9, 42, 43, 57]
–cognitively impaired
–activated central HPA axis
–normal corticosterone levels
–altered mRNA levels of corticoid receptors and CRH
6 months Increased mRNA in PFC, DH, BLA –extracellular Ain neocortex [9, 14]
Decreased in mRNA in VH –intraneuronal buildup in hippocampus, amygdala and
cortex
Protein unchanged –impaired LTP
–synaptic dysfunction
9 months – –increased corticosterone levels [42]
12 months Increased mRNA in PFC, PrL, DH, BLA [9, 14]
Decreased mRNA in VH extracellular Adeposits is evident in frontal cortex,
amygdala, DH and VH
Decreased protein in DH and BLA –Tau pathology evident in hippocampus
Protein unchanged in PFC, PrL and VH
18 months – – deficits in glutamate neurotransmission and
mitochondrial functions in prefrontal cortex and
hippocampus
[13, 48]
– emotionality and depressive like behavior
A, amyloid-; BLA, basolateral amygdala complex; CRH, corticotropin-releasing hormone; DH, dorsal hippocampus; HPA, hypothalamic-
pituitary-adrenal; LTP, long-term potentiation; PFC, prefrontal cortex; PrL, prelimbic cortex; VH, ventral hippocampus.
AD mice do not have inborn altered CB1 expression
in the brain regions analyzed. Therefore, we specu-
late that the altered pattern of CB1 expression found
at older ages in their brains can be interpreted as
age- and/or pathology-dependent. In accordance with
this hypothesis, an extensive set of age-related and
pathology-related alterations are described in our
murine model (see Table 1).
At 6 months of age, extracellular Adeposits first
become apparent in the frontal cortex of 3×Tg-AD
mice, while intracellular Aimmunoreactivity starts
to build-up in hippocampus, cortex, and amygdala [9,
22]. At 12 months of age, extracellular Adeposits
are readily evident in frontal cortex, amygdala, DH,
and VH; the immunoreactivity for hyperphosphory-
lated tau starts to be evident in CA1 neurons of
hippocampus, particularly at the somatodendritic level
of pyramidal neurons (progressing later to involve cor-
tical structures) [9, 14].
From our results, alterations of CB1 mRNA but not
protein levels appear at 6 months of age, when the
AD neuropathology seems to impact on CB1 expres-
sion first at transcriptional levels. Alterations in CB1
expression become more evident at 12 months of age,
when they involve also the protein levels in the BLA
and DH, remaining unaltered in the other areas.
Interestingly, the temporal pattern of the changes of
CB1 protein expression observed in our study seemed
to correlate with the temporal pattern of the devel-
opment of Apathology, at least in the two brain
areas analyzed, namely the DH and the BLA. Previ-
ous studies corroborate our finding that CB1 receptors
are unchanged in cortex [4, 8] but are lowered in DH
[5, 7] in AD. However, some reports showed that CB1
levels are altered in cortex [23, 24] and unaltered in
DH of AD patients [4, 6, 8]. These discrepancies might
be due to different disease models used in each study.
Until now, much emphasis has been given to the role of
the ECS in cortex and hippocampus in AD pathology,
while leaving the BLA poorly investigated, in spite of
its well-known role in learning, its involvement in AD
neuropathology and its quite high expression of CB1
receptors.
Age-related changes of CB1 mRNA expression in
the rodent brain have been already reported in the liter-
ature, although data are still sparse and in some cases
discrepant from our results. In particular, CB1 mRNA
was observed to increase steadily throughout neuronal
development of rats and mice until animals reach 2
months of age [25, 26]. Conversely, a decrease of CB1
mRNA has been described in hippocampus and BLA,
with no change in cortex, when rats are 24-months-old
[27]. These discrepancies with our results might be due
to different species used in these studies.
CB1 receptors play important roles in neuroprotec-
tion and the enhancement of endocannabinoid tone is
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G. Bedse et al. / Altered CB1 Receptor Expression in 3×Tg-AD 709
now considered an attractive therapeutic approach to
treat AD. It has been demonstrated, indeed, that the
enhancement of brain endocannabinoid tone is able
to reverse memory impairment and neurotoxic effects
triggered by soluble Ain murine models of AD [28].
The neuroprotective function of ECS is thought to
occur through a variety of mechanisms. For exam-
ple, through CB1 receptor activation anandamide was
recently shown to positively regulate Notch-1 pathway.
This pathway plays a key role in neurogenesis, long
term memory, and neuronal development, and thus
restores AD neurodegeneration and memory impair-
ments [29]. Moreover, ECS was also demonstrated
to be involved in clearing Afrom the blood-
brain barrier, as demonstrated in vitro by Bachmeier
et al. by incubation with cannabinoid receptor agonists
or inhibitors of endocannabinoid-degrading enzymes
[30]. Based on our results, we speculate that increas-
ing the endocannabinoid tone or hyperactivating CB1
receptors might produce such ameliorating effects by
counterbalancing the loss of CB1 receptors in selected
brain areas, such as the BLA and the DH.
In this latter area, we recently observed a dramatic
deficit of glutamate neurotransmission in aged 3×Tg-
AD mice. These lower levels of glutamate did not
appear to be due to synaptic loss, as synaptophysin,
a presynaptic vesicle marker of synaptic density, was
not altered [13, 31–34]. Within the hippocampus,
CB1 receptors are highly expressed by GABAer-
gic interneurons [35], where they negatively control
GABA release on excitatory glutamatergic neurons.
Therefore, it can be hypothesized that the reduced glu-
tamatergic neurotransmission in this area might result
from the reduced CB1 expression on GABA termi-
nals and the consequent excessive GABA-mediated
inhibition of glutamatergic neurons.
Recently, CB1 was found to be expressed in mito-
chondria, and a novel role for CB1 receptors in the
regulation of energy metabolism in the brain was
proposed [36]. Aged 3×Tg-AD mice show severe
mitochondrial impairment, as was previously shown by
our group and by others [13, 37], and the hippocampus
is the most severely affected area. This previous obser-
vation is in line with the current findings of reduced
CB1 levels in DH of aged mutant mice.
Apart from genetic factors, stress has also been sug-
gested as a risk factor in developing AD and severe
cognitive decline in AD patients. Hypothalamic-
pituitary-adrenal (HPA) axis dysregulation and ele-
vated cortisol levels have been described in a
substantial proportion of patients with AD [38–40].
Moreover, animal studies, including some performed
on 3×Tg-AD mice, suggest some sort of interac-
tion between corticosterone, dysregulation of the HPA
axis, and A/tau pathology in AD [41, 42], although
the mechanisms underlying this interaction remain
unknown. In particular, when corticosterone levels in
3×Tg-AD mice were evaluated, Green and colleagues
found that basal corticosterone levels were unchanged
until 9 months of age compared to aged-matched non-
transgenic mice. After 9 months of age, corticosterone
levels were significantly elevated in 3×Tg-AD mice
compared to age-matched non-transgenic mice [42].
Although corticosterone levels were normal at early
age, these mice showed activated HPA axis in 3–4-
month-old 3×Tg-AD. At this age increased mRNA
levels of mineralocorticoid receptor and glucocorti-
coids receptor were also observed in the hippocampus
and PVN with no change in the amygdala, while the
mRNA of corticotropin releasing hormone decreased
in the PVN and increased in both the central nucleus of
the amygdala and the bed nucleus of the stria terminalis
[43].
There is evidence that the ECS regulates the HPA
axis by negatively modulating its activation induced
by the exposure to stress [44–46]. Among other areas,
CB1 receptors expressed in DH and BLA seem to be
involved in negative feedback of glucocorticoids in
these brain regions [47]. As a consequence, CB1 recep-
tor blockade with the antagonist, SR141716, results in
activation of the HPA axis as measured by an increase
in plasma corticosterone levels in rodents [44]. Apart
from dysregulated HPA axis, increased emotionality
and depressive-like behavior are reported in these mice
[48]. We have observed depressive-like behavior in
these mice when subjected to a forced swimming
and tail suspension test (unpublished data). Moreover,
these mice are reported to show symptoms of anxiety
and fear associated with spatial memory deficits and a
deleterious effect of intraneuronal Aon amygdala-
dependent emotional responses has been proposed
[49]. A similar behavioral phenotype was observed
in CB1-knockout mice (CB1–/–), which show also
increased circulating levels of adrenocorticotropic hor-
mone [46], corticosterone [50, 51], anxiety-like and
fear responses [52–54] as well as depressive-like
behavior [55, 56]. Our results might suggest that
the decreased CB1 immunoreactivity found in DH
and BLA in 3×Tg-AD mice could play a role in
hippocampus-related memory deficits and amygdala-
related behavioral alterations.
Interestingly, a recent study by Stumm et al. showed
that the lack of CB1 receptors in CB1–/–mice
overexpressing APP23 can result in reduction of
AUTHOR COPY
710 G. Bedse et al. / Altered CB1 Receptor Expression in 3×Tg-AD
amyloid plaque load, reduced in situ inflammation, and
impaired learning and memory in aged mice [34]. We
propose that lowered CB1 receptor expression might
contribute to the cognitive impairments and dysregu-
lated HPA axis found in 3×Tg-AD mice.
Overall our results show that 3×Tg-AD do not have
inborn altered CB1 mRNA and protein expression,
as they did not show any alteration at 2 months of
age when their phenotype is still normal. The altered
CB1 mRNA/protein levels appear, rather, to be age-
and/or pathology-dependent, thus supporting the idea
of a critical role of the ECS in AD and its possible
impact as novel pharmacological target. It seems that
decreased CB1 receptor expression in DH and BLA
might be involved in depressive-like behavior observed
in this mouse model of AD. How AD pathology exactly
affects CB1 receptors and whether CB1 receptors and
AD pathology are directly or indirectly linked needs to
be further explored.
ACKNOWLEDGMENTS
This study was supported by PRIN (2009) (to GV).
The authors thank Dr. Antonio Petrella from the Puglia
and Basilicata Experimental Zooprophylactic Institute
(Foggia, Italy) for his invaluable veterinary assistance.
All experiments were performed in strict compliance
with the Italian National Laws (DL 116/92), the Euro-
pean Communities Council Directives (86/609/EEC).
All efforts were made to minimize the number of ani-
mals used in the study and their suffering.
Authors’ disclosures available online (http://www.j-
alz.com/disclosures/view.php?id=2066).
REFERENCES
[1] Selkoe DJ (2001) Alzheimer’s disease: Genes, proteins, and
therapy. Physiol Rev 81, 741-766.
[2] Whitehouse PJ, Price DL, Struble RG, Clark AW, Coyle JT,
Delon MR (1982) Alzheimer’s disease and senile dementia:
Loss of neurons in the basal forebrain. Science 215, 1237-
1239.
[3] Freund TF, Katona I, Piomelli D (2003) Role of endogenous
cannabinoids in synaptic signaling. Physiol Rev 83, 1017-
1066.
[4] Lee JH, Agacinski G, Williams JH, Wilcock GK, Esiri MM,
Francis PT, Wong PT, Chen CP, Lai MK (2010) Intact
cannabinoid CB1 receptors in the Alzheimer’s disease cortex.
Neurochem Int 57, 985-989.
[5] Westlake TM, Howlett AC, Bonner TI, Matsuda LA, Herken-
ham M (1994) Cannabinoid receptor binding and messenger
RNA expression in human brain: An in vitro receptor autora-
diography and in situ hybridization histochemistry study of
normal aged and Alzheimer’s brains. Neuroscience 63, 637-
652.
[6] Mulder J, Zilberter M, Pasquare SJ, Alpar A, Schulte G,
Ferreira SG, Kofalvi A, Martin-Moreno AM, Keimpema E,
Tanila H, Watanabe M, Mackie K, Hortobagyi T, de Ceballos
ML, Harkany T (2011) Molecular reorganization of endo-
cannabinoid signalling in Alzheimer’s disease. Brain 134,
1041-1060.
[7] Kalifa S, Polston EK, Allard JS, Manaye KF (2011)
Distribution patterns of cannabinoid CB1 receptors in the hip-
pocampus of APPswe/PS1DeltaE9 double transgenic mice.
Brain Res 1376, 94-100.
[8] Benito C, Nunez E, Tolon RM, Carrier EJ, Rabano A, Hillard
CJ, Romero J (2003) Cannabinoid CB2 receptors and fatty
acid amide hydrolase are selectively overexpressed in neuritic
plaque-associated glia in Alzheimer’s disease brains. J Neu-
rosci 23, 11136-11141.
[9] Oddo S, Caccamo A, Shepherd JD, Murphy MP, Golde TE,
Kayed R, Metherate R, Mattson MP, Akbari Y, LaFerla FM
(2003) Triple-transgenic model of Alzheimer’s disease with
plaques and tangles: Intracellular Abeta and synaptic dysfunc-
tion. Neuron 39, 409-421.
[10] Mesulam MM (1999) Neuroplasticity failure in Alzheimer’s
disease: Bridging the gap between plaques and tangles. Neu-
ron 24, 521-529.
[11] Mesulam MM (2000) A plasticity-based theory of the patho-
genesis of Alzheimer’s disease. Ann N Y Acad Sci 924, 42-52.
[12] Oddo S, Caccamo A, Green KN, Liang K, Tran L, Chen
Y, Leslie FM, LaFerla FM (2005) Chronic nicotine admin-
istration exacerbates tau pathology in a transgenic model of
Alzheimer’s disease. Proc Natl Acad Sci U S A 102, 3046-
3051.
[13] Cassano T, Serviddio G, Gaetani S, Romano A, Dipasquale
P, Cianci S, Bellanti F, Laconca L, Romano AD, Padalino
I, LaFerla FM, Nicoletti F, Cuomo V, Vendemiale G (2012)
Glutamatergic alterations and mitochondrial impairment in
a murine model of Alzheimer disease. Neurobiol Aging 33,
1121.e1121-1112.
[14] Oddo S, Caccamo A, Kitazawa M, Tseng BP, LaFerla FM
(2003) Amyloid deposition precedes tangle formation in a
triple transgenic model of Alzheimer’s disease. Neurobiol
Aging 24, 1063-1070.
[15] van Rijn CM, Gaetani S, Santolini I, Badura A, Gabova A,
Fu J, Watanabe M, Cuomo V, van Luijtelaar G, Nicoletti F,
Ngomba RT (2010) WAG/Rij rats show a reduced expression
of CB(1) receptors in thalamic nuclei and respond to the CB(1)
receptor agonist, R(+)WIN55,212-2, with a reduced incidence
of spike-wave discharges. Epilepsia 51, 1511-1521.
[16] Egertova M, Elphick MR (2000) Localisation of cannabinoid
receptors in the rat brain using antibodies to the intracellular
C-terminal tail of CB. J Comp Neurol 422, 159-171.
[17] Bridges D, Rice AS, Egertova M, Elphick MR, Winter J,
Michael GJ (2003) Localisation of cannabinoid receptor 1
in rat dorsal root ganglion using in situ hybridisation and
immunohistochemistry. Neuroscience 119, 803-812.
[18] Monory K, Massa F, Egertova M, Eder M, Blaudzun H, West-
enbroek R, Kelsch W, Jacob W, Marsch R, Ekker M, Long
J, Rubenstein JL, Goebbels S, Nave KA, During M, Klug-
mann M, Wolfel B, Dodt HU, Zieglgansberger W, Wotjak
CT, Mackie K, Elphick MR, Marsicano G, Lutz B (2006) The
endocannabinoid system controls key epileptogenic circuits
in the hippocampus. Neuron 51, 455-466.
[19] Marsicano G, Lutz B (1999) Expression of the cannabinoid
receptor CB1 in distinct neuronal subpopulations in the adult
mouse forebrain. Eur J Neurosci 11, 4213-4225.
[20] Egertova M, Cravatt BF, Elphick MR (2003) Comparative
analysis of fatty acid amide hydrolase and cb(1) cannabinoid
AUTHOR COPY
G. Bedse et al. / Altered CB1 Receptor Expression in 3×Tg-AD 711
receptor expression in the mouse brain: Evidence of a
widespread role for fatty acid amide hydrolase in regulation
of endocannabinoid signaling. Neuroscience 119, 481-496.
[21] Pascal LE, True LD, Campbell DS, Deutsch EW, Risk M,
Coleman IM, Eichner LJ, Nelson PS, Liu AY (2008) Corre-
lation of mRNA and protein levels: Cell type-specific gene
expression of cluster designation antigens in the prostate.
BMC Genomics 9, 246.
[22] Kitazawa M, Oddo S, Yamasaki TR, Green KN, LaFerla FM
(2005) Lipopolysaccharide-induced inflammation exacer-
bates tau pathology by a cyclin-dependent kinase 5-mediated
pathway in a transgenic model of Alzheimer’s disease. J Neu-
rosci 25, 8843-8853.
[23] Ramirez BG, Blazquez C, Gomez del Pulgar T, Guzman
M, de Ceballos ML (2005) Prevention of Alzheimer’s dis-
ease pathology by cannabinoids: Neuroprotection mediated
by blockade of microglial activation. J Neurosci 25, 1904-
1913.
[24] Solas M, Francis PT, Franco R, Ramirez MJ (2013)
CB2 receptor and amyloid pathology in frontal cortex of
Alzheimer’s disease patients. Neurobiol Aging 34, 805-808.
[25] Psychoyos D, Vinod KY, Cao J, Xie S, Hyson RL, Wlodar-
czyk B, He W, Cooper TB, Hungund BL, Finnell RH (2012)
Cannabinoid receptor 1 signaling in embryo neurodevelop-
ment. Birth Defects Res B Dev Reprod Toxicol 95, 137-150.
[26] Romero J, Garcia-Palomero E, Berrendero F, Garcia-Gil L,
Hernandez ML, Ramos JA, Fernandez-Ruiz JJ (1997) Atyp-
ical location of cannabinoid receptors in white matter areas
during rat brain development. Synapse 26, 317-323.
[27] Berrendero F, Romero J, Garcia-Gil L, Suarez I, De la Cruz P,
Ramos JA, Fernandez-Ruiz JJ (1998) Changes in cannabinoid
receptor binding and mRNA levels in several brain regions of
aged rats. Biochim Biophys Acta 1407, 205-214.
[28] van der Stelt M, Mazzola C, Esposito G, Matias I, Petrosino
S, De Filippis D, Micale V, Steardo L, Drago F, Iuvone T,
Di Marzo V (2006) Endocannabinoids and beta-amyloid-
induced neurotoxicity in vivo: Effect of pharmacological
elevation of endocannabinoid levels. Cell Mol Life Sci 63,
1410-1424.
[29] Tanveer R, Gowran A, Noonan J, Keating SE, Bowie AG,
Campbell VA (2012) The endocannabinoid, anandamide, aug-
ments Notch-1 signaling in cultured cortical neurons exposed
to amyloid-beta and in the cortex of aged rats. J Biol Chem
287, 34709-34721.
[30] Bachmeier C, Beaulieu-Abdelahad D, Mullan M, Paris D
(2013) Role of the cannabinoid system in the transit of beta-
amyloid across the blood-brain barrier. Mol Cell Neurosci 56,
255-262.
[31] Arsenault D, Dal-Pan A, Tremblay C, Bennett DA, Guitton
MJ, De Koninck Y, Tonegawa S, Calon F (2013) PAK inacti-
vation impairs social recognition in 3xTg-AD Mice without
increasing brain deposition of tau and Abeta. J Neurosci 33,
10729-10740.
[32] Noristani HN, Meadows RS, Olabarria M, Verkhratsky A,
Rodriguez JJ (2011) Increased hippocampal CA1 density of
serotonergic terminals in a triple transgenic mouse model of
Alzheimer’s disease: An ultrastructural study. Cell Death Dis
2, e210.
[33] Rodriguez-Ortiz CJ, Hoshino H, Cheng D, Liu-Yescevitz
L, Blurton-Jones M, Wolozin B, LaFerla FM, Kitazawa
M (2013) Neuronal-specific overexpression of a mutant
valosin-containing protein associated with IBMPFD pro-
motes aberrant ubiquitin and TDP-43 accumulation and
cognitive dysfunction in transgenic mice. Am J Pathol 183,
504-515.
[34] Stumm C, Hiebel C, Hanstein R, Purrio M, Nagel H, Conrad
A, Lutz B, Behl C, Clement AB (2013) Cannabinoid receptor
1 deficiency in a mouse model of Alzheimer’s disease leads
to enhanced cognitive impairment despite of a reduction in
amyloid deposition. Neurobiol Aging 34, 2574-2584.
[35] Pertwee RG (2008) The diverse CB1 and CB2 receptor
pharmacology of three plant cannabinoids: Delta9-
tetrahydrocannabinol, cannabidiol and delta9-
tetrahydrocannabivarin. Br J Pharmacol 153, 199-215.
[36] Benard G, Massa F, Puente N, Lourenco J, Bellocchio L,
Soria-Gomez E, Matias I, Delamarre A, Metna-Laurent M,
Cannich A, Hebert-Chatelain E, Mulle C, Ortega-Gutierrez
S, Martin-Fontecha M, Klugmann M, Guggenhuber S, Lutz
B, Gertsch J, Chaouloff F, Lopez-Rodriguez ML, Grandes
P, Rossignol R, Marsicano G (2012) Mitochondrial CB(1)
receptors regulate neuronal energy metabolism. Nat Neurosci
15, 558-564.
[37] Yao J, Irwin RW, Zhao L, Nilsen J, Hamilton RT,
Brinton RD (2009) Mitochondrial bioenergetic deficit pre-
cedes Alzheimer’s pathology in female mouse model of
Alzheimer’s disease. Proc Natl Acad SciUSA106, 14670-
14675.
[38] Davis KL, Davis BM, Greenwald BS, Mohs RC, Mathe
AA, Johns CA, Horvath TB (1986) Cortisol and Alzheimer’s
disease, I: Basal studies. Am J Psychiatry 143, 300-
305.
[39] Masugi F, Ogihara T, Sakaguchi K, Otsuka A, Tsuchiya Y,
Morimoto S, Kumahara Y, Saeki S, Nishide M (1989) High
plasma levels of cortisol in patients with senile dementia of
the Alzheimer’s type. Methods Find Exp Clin Pharmacol 11,
707-710.
[40] Swanwick GR, Kirby M, Bruce I, Buggy F, Coen RF, Coak-
ley D, Lawlor BA (1998) Hypothalamic-pituitary-adrenal
axis dysfunction in Alzheimer’s disease: Lack of associa-
tion between longitudinal and cross-sectional findings. Am
J Psychiatry 155, 286-289.
[41] Elliott EM, Mattson MP, Vanderklish P, Lynch G, Chang
I, Sapolsky RM (1993) Corticosterone exacerbates kainate-
induced alterations in hippocampal tau immunoreactivity and
spectrin proteolysis in vivo.J Neurochem 61, 57-67.
[42] Green KN, Billings LM, Roozendaal B, McGaugh JL, LaFerla
FM (2006) Glucocorticoids increase amyloid-beta and tau
pathology in a mouse model of Alzheimer’s disease. J Neu-
rosci 26, 9047-9056.
[43] Hebda-Bauer EK, Simmons TA, Sugg A, Ural E, Stewart
JA, Beals JL, Wei Q, Watson SJ, Akil H (2013) 3xTg-AD
mice exhibit an activated central stress axis during early-stage
pathology. J Alzheimers Dis 33, 407-422.
[44] Patel S, Roelke CT, Rademacher DJ, Cullinan WE, Hillard
CJ (2004) Endocannabinoid signaling negatively modulates
stress-induced activation of the hypothalamic-pituitary-
adrenal axis. Endocrinology 145, 5431-5438.
[45] Hill MN, McLaughlin RJ, Bingham B, Shrestha L, Lee TT,
Gray JM, Hillard CJ, Gorzalka BB, Viau V (2010) Endoge-
nous cannabinoid signaling is essential for stress adaptation.
Proc Natl Acad SciUSA107, 9406-9411.
[46] Barna I, Zelena D, Arszovszki AC, Ledent C (2004) The role
of endogenous cannabinoids in the hypothalamo-pituitary-
adrenal axis regulation: In vivo and in vitro studies in CB1
receptor knockout mice. Life Sci 75, 2959-2970.
[47] De Kloet ER, Vreugdenhil E, Oitzl MS, Joels M (1998) Brain
corticosteroid receptor balance in health and disease. Endocr
Rev 19, 269-301.
[48] Gimenez-Llort L, Blazquez G, Canete T, Johansson B,
Oddo S, Tobena A, LaFerla FM, Fernandez-Teruel A (2007)
AUTHOR COPY
712 G. Bedse et al. / Altered CB1 Receptor Expression in 3×Tg-AD
Modeling behavioral and neuronal symptoms of Alzheimer’s
disease in mice: A role for intraneuronal amyloid. Neurosci
Biobehav Rev 31, 125-147.
[49] Espana J, Gimenez-Llort L, Valero J, Minano A, Rabano A,
Rodriguez-Alvarez J, LaFerla FM, Saura CA (2010) Intraneu-
ronal beta-amyloid accumulation in the amygdala enhances
fear and anxiety in Alzheimer’s disease transgenic mice. Biol
Psychiatry 67, 513-521.
[50] Cota D, Steiner MA, Marsicano G, Cervino C, Herman JP,
Grubler Y, Stalla J, Pasquali R, Lutz B, Stalla GK, Pagotto
U (2007) Requirement of cannabinoid receptor type 1 for
the basal modulation of hypothalamic-pituitary-adrenal axis
function. Endocrinology 148, 1574-1581.
[51] Steiner MA, Marsicano G, Wotjak CT, Lutz B (2008)
Conditional cannabinoid receptor type 1 mutants reveal
neuron subpopulation-specific effects on behavioral and neu-
roendocrine stress responses. Psychoneuroendocrinology 33,
1165-1170.
[52] Haller J, Bakos N, Szirmay M, Ledent C, Freund TF (2002)
The effects of genetic and pharmacological blockade of the
CB1 cannabinoid receptor on anxiety. Eur J Neurosci 16,
1395-1398.
[53] Maccarrone M, Valverde O, Barbaccia ML, Castane A,
Maldonado R, Ledent C, Parmentier M, Finazzi-Agro A
(2002) Age-related changes of anandamide metabolism in
CB1 cannabinoid receptor knockout mice: Correlation with
behaviour. Eur J Neurosci 15, 1178-1186.
[54] Martin M, Ledent C, Parmentier M, Maldonado R, Valverde
O (2002) Involvement of CB1 cannabinoid receptors in emo-
tional behaviour. Psychopharmacology (Berl) 159, 379-387.
[55] Aso E, Ozaita A, Serra MA, Maldonado R (2011) Genes dif-
ferentially expressed in CB1 knockout mice: Involvement in
the depressive-like phenotype. Eur Neuropsychopharmacol
21, 11-22.
[56] Valverde O, Torrens M (2012) CB1 receptor-deficient mice
as a model for depression. Neuroscience 204, 193-206.
[57] Billings LM, Oddo S, Green KN, McGaugh JL, LaFerla
FM (2005) Intraneuronal Abeta causes the onset of early
Alzheimer’s disease-related cognitive deficits in transgenic
mice. Neuron 45, 675-688.
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