activity of muscarinic, galanin and cannabinoid … · 1 2 activity of muscarinic, galanin and...
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Please cite this article in press as: Manuel I et al. Activity of muscarinic, galanin and cannabinoid receptors in the prodromal and advanced stages in the
triple transgenic mice model of Alzheimer’s disease. Neuroscience (2016), http://dx.doi.org/10.1016/j.neuroscience.2016.05.012
NSC 17098 No. of Pages 10
21 May 2016
Neuroscience xxx (2016) xxx–xxx
ACTIVITY OF MUSCARINIC, GALANIN AND CANNABINOID RECEPTORSIN THE PRODROMAL AND ADVANCED STAGES IN THE TRIPLETRANSGENIC MICE MODEL OF ALZHEIMER’S DISEASE
16
IVAN MANUEL, a LAURA LOMBARDERO, a
FRANK M. LAFERLA, c LYDIA GIMENEZ-LLORT b ANDRAFAEL RODRIGUEZ-PUERTAS a*
aDepartment of Pharmacology, Faculty of Medicine, University of
the Basque Country (UPV/EHU), B� Sarriena s/n, 48940 Leioa, Spain
b Institut de Neurosciencies & Department of Psychiatry and Forensic
Medicine, Faculty of Medicine, Universitat Autonoma de Barcelona,
Avgda Can Domenech s/n, 08193 Bellaterra, Spain
cDepartment of Neurobiology & Behavior, University of
California Irvine, Irvine, CA, USA
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Abstract—Neurochemical alterations in Alzheimer’s disease
(AD) include cholinergic neuronal loss in the nucleus basa-
lis of Meynert (nbM) and a decrease in densities of the M2
muscarinic receptor subtype in areas related to learning
and memory. Neuromodulators present in the cholinergic
pathways, such as neuropeptides and neurolipids, control
these cognitive processes and have become targets of
research in order to understand and treat the pathophysio-
logical and clinical stages of the disease. This is the case
of the endocannabinoid and galaninergic systems, which
have been found to be up-regulated in AD, and could there-
fore have a neuroprotective role. In the present study, the
functional coupling of G/o protein-coupled receptors to
GalR1, and the CB1 receptor subtype for endocannabinoids
were analyzed in the 3xTg-AD mice model of AD. In addition,
the activity mediated by Gi/o protein-coupled M2/4 muscarinic
receptor subtypes was also analyzed in brain areas involved
in anxiety and cognition. Thus, male mice were studied at 4
and 15 months of age (prodromal and advanced stages,
respectively) and compared to age-matched
non-transgenic (NTg) mice (adult and old, respectively). In
4-month-old 3xTg-AD mice, the [35S]GTPcS binding
stimulated by galanin was significantly increased in the
hypothalamus, but a decrease of functional M2/4 receptors
was observed in the posterior amygdala. The CB1
cannabinoid receptor activity was up-regulated in the ante-
rior thalamus at that age. In 15-month-old 3xTg-AD mice,
muscarinic receptor activity was found to be increased in
motor cortex, while CB1 activity was decreased in nbM. No
changes were found in GalR1-mediated activity at this age.
Our results provide further evidence of the relevance of lim-
bic areas in the prodromal stage of AD, the profile of which
is characterized by anxiety. The up-regulation of galaniner-
gic and endocannabinoid systems support the hypothesis
http://dx.doi.org/10.1016/j.neuroscience.2016.05.0120306-4522/� 2016 IBRO. Published by Elsevier Ltd. All rights reserved.
*Corresponding author. Address: Department of Pharmacology,Faculty of Medicine, University of the Basque Country, E-48940Leioa, Vizcaya, Spain. Tel: +34-94-6012739; fax: +34-94-6013220.
E-mail address: [email protected] (R. Rodrıguez-Puertas).Abbreviations: bA, b-amyloid; AD, Alzheimer’s disease; EGTA,ethylene glycol tetraacetic acid; NTg, non-transgenic.
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of their neuroprotective roles, and these are established
prior to the onset of clear clinical cognitive symptoms of
the disease. � 2016 IBRO. Published by Elsevier Ltd. All
rights reserved.
Key words: cholinergic, neuropeptides, neurolipids, Alzhei-
mer, G protein, autoradiography.
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INTRODUCTION
Alterations in cholinergic neurotransmission seem to be
one of the most characteristic hallmarks of Alzheimer’s
disease (AD), as was firstly described, due to the
impairment of cholinergic neurons in the basal nucleus
of Meynert (nbM) (Whitehouse et al., 1982). Moreover,
a decrease in the density of M2 muscarinic receptors
has been described in areas related to memory and learn-
ing, such as the hippocampus and entorhinal cortex
(Rodrıguez-Puertas et al., 1997a). However, neurochem-
ical alterations also include the impairment of noradrener-
gic (Marcyniuk et al., 1986) and serotonergic (Palmer
et al., 1987) systems. Other neuromodulators, such as
neuropeptides and neurolipids, present in cholinergic
pathways, have become targets of research in order to
understand, prevent or treat the disease. This is the case
of the galaninergic and endocannabinoid systems, which
have been found to be up-regulated in AD and could have
a neuroprotective role (Manuel et al., 2014; Rodrıguez-
Puertas et al., 1997b). In fact, it has been described that
cholinergic cells of nbM are hyperinnervated by galanin
(Chan-Palay, 1988). Besides, an increase of 125I-galanin
binding was observed in AD brains in the hippocampus
and entorhinal cortex, the same areas in which M2 recep-
tor density was reduced (Rodrıguez-Puertas et al.,
1997b). In nbM and amygdala an increase in galanin125I-binding has also been observed (Mufson et al.,
2000; Perez et al., 2002). In the case of cannabinoid neu-
rotransmission, there is a reduction in CB1 receptors in
advanced Braak stages of the disease in different layers
of the hippocampus (Westlake et al., 1994). Furthermore,
there is an increase in both the activity and density of CB1
receptors in early and moderate stages (Manuel et al.,
2014). Nevertheless, cannabinoids are involved in
neuroprotective functions against excitotoxic damage
(Marsicano et al., 2003). In the same way, cannabinoids
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are also involved in the suppression of neuroinflammatory
processes in AD (Ehrhart et al., 2005).
Interestingly, pathophysiological processes underlying
AD, seem to occur almost a decade prior to obvious
clinical symptoms. The study of this long preclinical
phase is now considered to be of crucial importance,
together with classical research focused on the study of
more advanced stages of the disease (Sperling et al.,
2013). The identification of early pathophysiological alter-
ations is necessary in order to lead to an effective therapy
prior to the onset of the first clinical symptoms of AD. In
the prodromal stage of the disease, not only biological
markers specific for AD neuropathology and oxidative
stress, but also non-specific markers of neuronal degen-
eration and inflammation are explored as targets for early
drug therapies for AD (Bailey, 2007). As clearly stated by
Welsh-Bohmer (2008), in the very early stages, the neu-
ropsychological symptoms may not be apparent at all
(latent phase), but as the pathology worsens, the first
symptoms emerge (prodromal stage) followed by a full
manifestation of the clinical disease (dementia stage).
This growing concern for the initial stages of AD has lead
to basic research on the validity of animal models to
reproduce the wide spectrum of alterations found in AD
patients, ranging from the pathological processes to the
cognitive deficits and alterations in behavior (Gimenez-
Llort et al., 2007). Due to the genetic background of trans-
genic mice models of familial AD mutations, they may
prove to be a useful tool for the study of the pathophysio-
logical processes involved in AD, from the asymptomatic
stages to the prodromal and the advanced stages of the
disease. This is the case of the triple transgenic mice
model of AD (3xTg-AD) harboring bAPPSwe, PS1M146V
and tauP301L human transgenes, which mimics the devel-
opment of the main histopathological features of the dis-
ease (Oddo et al., 2003). These mice progressively
develop the neuropathological markers of AD in a tempo-
ral and regional-specific profile similar to that found in the
brain of AD patients. At 4 months of age, 3xTg-AD mice
develop intraneuronal b-amyloid (bA) deposits and this
age could be compared to the prodromal stages of the
disease. Indeed, we have consistently reported the pres-
ence of an anxious-like profile, together with some of the
first cognitive deficits (Billings et al., 2005; Gimenez-Llort
et al., 2006, 2007; Canete et al., 2015). The presence of
intracellular bA is initially detected in the hippocampal CA1
region, but at 6 months of age, it is also visible in the cor-
tex (Billings et al., 2005) and in the basolateral amygdala
(Espana et al., 2010), and is clearly related to cognitive
deficits and behavioral alterations. At 12 months of age
the appearance of extraneuronal bA is described and is
followed, at 15 months of age, by concomitant hyperphos-
phorylated tau protein in the hippocampus (Oddo et al.,
2003). At the neurochemical level, the basal forebrain
cholinergic system starts to be affected in 3xTg-AD mice,
early in the intracellular bA stage (Perez et al., 2011).
Histopathological alterations in the primary motor cortex
also appear soon after 3 months of age (Mastrangelo
and Bowers, 2008).
Within this context, the present work aims to describe
the activity of muscarinic, galanin and cannabinoid
Please cite this article in press as: Manuel I et al. Activity of muscarinic, galanin
triple transgenic mice model of Alzheimer’s disease. Neuroscience (2016), http
receptors in 4- and 15-month-old 3xTg-AD mice. These
ages were chosen as those which mimic the above-
mentioned stages in the progress of the disease, that is,
the prodromal stage and the advanced stages,
respectively. Age-matched non-transgenic mice (NTg)
were used as controls. Therefore, the objective of the
study is to validate the neurochemical alterations in this
animal model at different stages, but also to contribute
to the understanding of the neuroanatomical substrates
which are involved in the prodromal and advanced
stages of the disease.
EXPERIMENTAL PROCEDURES
Animals
The group of Dr. Frank LaFerla (Dept. of Neurobiology
and Behavior, University of California Irvine, USA)
created 3xTg-AD transgenic mice as described by Oddo
et al. (2003). Brain samples used in this study were from
4- and 15-month-old homozygous male 3xTg-AD mice
and age-matched NTg mice (n= 6 in each group) of
the colonies established at Universitat Autonoma de Bar-
celona, Spain. All animals were born and bred in the ani-
mal housing facilities in the Medical Psychology Unit and
maintained under standard laboratory conditions in a 12-h
light/dark cycle, 22 ± 2 �C, 50–60% humidity, with food
and water ad libitum.
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[35S]GTPcS binding assay
Brains were quickly removed and sectioned down the
midline into the right and left hemispheres. Then tissues
were frozen on dry ice, and kept at �80 �C. The brains
were cut in a Microm cryostat (HM 550, Thermo) to
obtain 20 lm sections that were mounted onto gelatin-
coated slides and these were stored at �20 �C until used.
The tissue sections were air-dried for 15 min and then
immersed in a Tris–HCl buffer 50 mM (pH 7.4) with
100 mM NaCl, 3 mM MgCl2 and 0.2 mM EGTA, and
preincubated in the previous mentioned Tris–HCl buffer
50 mM supplemented with 2 mM GDP and 1 mM
DL-dithiothreitol (DTT) and adenosine deaminase
(3 mU/ml). Later, sections were incubated for 2 h at
30 �C in the same buffer containing 0.04 nM [35S]GTPcS(1250 Ci/mmol; Perkin Elmer, Boston, MA, USA). The
agonist-stimulated binding was measured under the
same conditions in the presence of the specific GPCR
agonist: carbachol (100 lM) for M2/4 muscarinic
receptors, galanin (1 lM) for GalR1 receptors and WIN
55212,2 (100 lM) for CB1 receptors The receptor
subtype specificity was demonstrated by inhibiting the
[35S]GTPcS binding by co-incubation with atropine
(10 lM), M15 (1 lM) and SR141716 (10 lM), as
respective antagonists, together with carbachol, galanin
and WIN 55212,2. The chosen concentrations of
agonists and antagonists were similar to those used in
previous studies, and the criteria was based on the
potencies of the different compounds to produce a
receptor-mediated effect. Therefore, the concentrations
are more similar to physiological studies (EC50) than to
receptor binding analysis (affinity, Kd). Note that
and cannabinoid receptors in the prodromal and advanced stages in the
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I. Manuel et al. / Neuroscience xxx (2016) xxx–xxx 3
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antagonist concentrations were usually lower than those
of the agonists, since they bind to the G-protein
uncoupled conformation of the receptors that is favored
during the preincubation washing out of the endogenous
ligands and by the exogenous addition of GDP to the
incubation buffer. Non-specific binding was determined
in the presence of 10 lM of non-labeled GTPcS.Sections were washed twice in Tris–HCl buffer 50 mM
(pH 7.4), once in distilled water, and air-dried. Sections
were exposed to Kodak Biomax MR films with 14C
standards.
Quantitative image analysis of film autoradiograms
Films were scanned and quantified by transforming the
optical densities into nCi/g tissue equivalent (nCi/g t.e.)
using the 14C standards by means of NIH-IMAGE
analysis system (Bethesda, MA, USA). The percentages
of stimulation were calculated from the basal and
agonist stimulation of [35S]GTPcS. The slide
background and non-specific densities were subtracted.
Data were expressed as mean value ± SEM.
Differences between regions of genotypes were
analyzed by Student’s t test.
Thionine staining
The thionine staining was performed to facilitate the
identification of the different areas of the rat brain.
Tissues mounted onto gelatine-coated slides were
hydrated after thawing. The hydration was performed by
immersing tissues for 5 min in ethanol solutions in
descending order (100%, 96%, 70% and 50%). Then
sections were submerged in thionine solution for 5 min.
Later, tissues were washed with deionized water and
placed in ethanol solutions in ascending order to
dehydrate the tissue.
RESULTS
The results from the [35S]GTPcS autoradiography assays
were obtained as autoradiograms of the slices for each
mouse and for every experimental condition. A
representative example from one mouse is represented
in Fig. 1, where the basal activity is measured in the
autoradiogram obtained in the absence of agonist (A),
and the stimulation evoked by the muscarinic agonist
carbachol is observed in a consecutive section (B). The
muscarinic activity is antagonized by atropine (C), and
the non-specific activity is obtained from the incubation
in the presence of 10 lM GTPcS (D).
Cholinergic, endocannabinoid and galaninergicsystems at 4 months of age
Firstly, the basal [35S]GTPcS binding measured in 4-
month-old 3xTg-AD and NTg mice was quantified. The
presence of the three mutations did not modify the
basal activity measured in the absence of any
compound in any of the analyzed brain areas. Note that
the basal binding indicates the activity mediated by the
whole population of the different GPCR subtypes for any
neurotransmitter system coupled to Gi/o proteins in the
Please cite this article in press as: Manuel I et al. Activity of muscarinic, galanin
triple transgenic mice model of Alzheimer’s disease. Neuroscience (2016), http
conditions of the present assay. The analysis of the
[35S]GTPcS binding mainly quantifies the activity
mediated by Gi/o proteins. The reason for this seems to
be that there is both a higher ratio of Glo/i proteins
compared to that of other families, and higher rates of
GTP exchange, resulting in a much stronger signal
which precludes the stimulation of the other types of G
proteins (Harrison and Traynor, 2003).
On the contrary, the cholinergic muscarinic receptor
activity induced by carbachol measured as Gi/o proteins
bound to [35S]GTPcS, was found to be decreased in
specific brain areas of 3xTg-AD mice. One of these
regions was the posterior portion of the amygdala in
which the percentage of carbachol-induced stimulation
was 60 ± 14% in NTg and 25 ± 8% (p< 0.05) in
3xTg-AD mice. In the nbM a reduction in the activity of
these receptors was also observed (NTg 118 ± 25%;
3xTg-AD 76 ± 17%), but this change was not
statistically significant. However, in the hippocampus, an
increase in the [35S]GTPcS binding induced by
carbachol was found in dentate gyrus (NTg 33 ± 16%;
3xTg-AD 67 ± 27%) (Fig. 2, Table 1).
The functional coupling of GalR1 to Gi/o proteins
induced by galanin was only modified in the
hypothalamic area in which there was an increase in the
[35S]GTPcS binding in the transgenic mice (NTg 14
± 15%; 3xTg-AD 47 ± 12%, p< 0.05). There was a
decrease in the nbM (NTg 35 ± 13%; 3xTg-AD 24
± 9%), although it was not of statistical significance.
[35S]GTPcS binding stimulated by the cannabinoid
agonist WIN55,212-2 was only different from that of,
NTg in the ventrolateral part of the anteroventral
thalamic nucleus (anterior thalamus) (NTg 66 ± 21%;
3xTg-AD 136 ± 15%; p< 0.05) (Fig. 2, Table 2).
In summary, in 4-month-old 3xTg-AD mice no
changes were observed in the basal GPCR-mediated
activity. MR activity was lower than that observed in
age-matched NTg mice in the basal forebrain, but
higher in hippocampus. GalR1 activity was up-regulated
in hypothalamus, and CB1 activity in the anterior
thalamus in comparison with controls.
Cholinergic, endocannabinoid and galaninergicsystems at 15 months of age
To examine the functionality of the GPCR in 3xTg-AD
mice in advanced neuropathological stages and age-
matched NTg mice, we also analyzed the basal activity.
At 15 months of age, the 3xTg-AD mice presented an
increase in the basal [35S]GTPcS binding compared to
NTg mice in different brain areas, such as the anterior
portion of the amygdala (NTg 639 ± 18 nCi/g t.e.; 3xTg-
AD 817 ± 31nCi/g t.e.; p< 0.01), the anteroventral
portion of the thalamus (NTg 578 ± 32 nCi/g t.e.; 3xTg-
AD 621 ± 18 nCi/g t.e.; p< 0.01), and the ventral
portion of this area (NTg 471 ± 29 nCi/g t.e.; 3xTg-AD
562 ± 15 nCi/g t.e.; p< 0.05). We also found an
increase in the basal [35S]GTPcS binding in the nbM
(NTg 607 ± 19 nCi/g t.e.; 3xTg-AD 752 ± 14 nCi/g t.e.;
p< 0.01). There was more activity in the striatum of
3xTg-AD mice than in that of NTg mice (NTg 665
± 24 nCi/g t.e.; 3xTg-AD 752 ± 16 nCi/g t.e.; p< 0.05);
and cannabinoid receptors in the prodromal and advanced stages in the
://dx.doi.org/10.1016/j.neuroscience.2016.05.012
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Fig. 1. Autoradiographic images of a representative NTg mouse of 4 months of age, showing [35S]GTPcS basal binding that was similar to that
observed in 3xTgAD mice (A), carbachol stimulation (100 lM) (B), carbachol stimulation antagonized with atropine (10 lM) (C) and non-specific
binding determined in the presence of 10 lM GTPcS (D).
4 I. Manuel et al. / Neuroscience xxx (2016) xxx–xxx
NSC 17098 No. of Pages 10
21 May 2016
and the same was observed in globus pallidus (NTg 672
± 18 nCi/g t.e.; 3xTg-AD 741 ± 20 nCi/g t.e.; p< 0.05)
(Fig. 3, Table 3).
In both NTg and 3xTg-AD mice the activity mediated
by the three different GPCR analyzed was lower at
15 months than at 4 months of age. Although it has
been described that there is a general decrease with
age in the GPCR activity measured by [35S]GTPcSbinding, the different groups of age were analyzed in
different experiments at different times, and the inter-
experimental variations could also have contributed to
those differences (Gonzalez-Maeso et al., 2002). Further-
more, 15-month-old 3xTg-AD mice showed an increase in
the activity of Gi/o-coupled cholinergic muscarinic recep-
tors induced by carbachol in discrete areas of the cortex
such as the motor cortex in the 3xTg-AD mice (NTg 15
± 4%; 3xTg-AD 34 ± 5%; p< 0.05) (Fig. 4). No signifi-
cant changes were observed in the activity induced by
galanin and in fact, the areas with the highest
stimulations, corresponding to hippocampus CA1 (NTg
17 ± 7%; 3xTg-AD 21 ± 8%) and dentate gyrus (NTg
16 ± 7%; 3xTg-AD 12 ± 6%), were also low. However,
there were changes in CB1-mediated activity in the
15-month-old 3xTg-AD mice, in which a reduction in the
nbM was found (NTg 116 ± 10%; 3xTg-AD 57 ± 7%;
p< 0.05) (Fig. 5).
In summary, the basal activity was increased only in
the 15-month-old 3xTg-AD mice, but the regulation of
the three different GPCR analyzed was only statistically
significant for the increase in motor cortex of M2/4 and
for the decrease of CB1 in nbM.
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DISCUSSION
The neurotransmission in AD is characterized by specific
and early alterations of the cholinergic system in the basal
forebrain pathways that innervate limbic structures such
Please cite this article in press as: Manuel I et al. Activity of muscarinic, galanin
triple transgenic mice model of Alzheimer’s disease. Neuroscience (2016), http
as the hippocampus and cortical areas. However, other
systems, which modulate cholinergic pathways, are
hyperactive, such as the galaninergic system (Chan-
Palay, 1988; Rodrıguez-Puertas et al., 1997b). A further
finely tuned modulation seems to be controlled by other
systems using neurolipids, e.g. the endocannabinoid sys-
tem that responds in the earliest stages of AD by increas-
ing its activity (Manuel et al., 2014). The present study
uses 3xTg-AD mice to analyze these three systems by
measuring GPCR activity at ages corresponding to the
prodromal and advanced stages of the disease. The
results obtained indicate that the 3xTg-AD mouse model
mimics the neurochemical alterations observed in AD
patients in relation to muscarinic, galanin and cannabinoid
receptors in both early and late stages of the disease.
Basal binding
The presence of the three genetic alterations, bAPPSwe,
PS1M146V and tauP301L transgenes in the 4-month-old
3xTg-AD mice, did not produce any alteration in the
basal activity of GPCR. Recently, different studies have
demonstrated that at least part of this basal activity
accounts for endogenous ligands for GPCR that remain
present during the assay as a consequence of their
lipidic structure. It is very difficult to remove the
endogenous lipid ligands or neurolipids during the
preincubation stage of the [35S]GTPcS binding assay,
which is carried out in an aqueous buffer in adequate
physiologic pH and temperature conditions (Aaltonen
et al., 2008, 2012; Gonzalez de San Roman et al.,
2015).However, the observed up-regulation in the basal
activity of 15-month-old 3xTg-AD mice could be a com-
pensatory response to more severe synaptic impairments
that arise with aging and that are mediated by GPCR sub-
types, probably related to neurolipid signaling. In this
sense, at 4 months of age the bA is only detected inside
and cannabinoid receptors in the prodromal and advanced stages in the
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Fig. 2. Autoradiographic images of sagittal tissue sections of [35S]GTPcS in 4-month-old NTg and 3xTg-AD mice showing stimulation by carbachol
(A and B), stimulation by galanin (C and D) and stimulation by WIN55,212-2 (E and F). Note that the carbachol stimulation in the posterior amygdala
is decreased in 3xTg-AD mice in comparison with NTg mice. On the contrary, stimulation by galanin is increased in the hypothalamus of 3xTg-AD
mice. The stimulation by WIN55,212-2, indicating mainly CB1-mediated cannabinoid receptor activity in the CNS (Llorente et al., 2014), was only
modified in the ventrolateral part of the anteroventral thalamic nucleus. Scale bar = 2.5 mm.
I. Manuel et al. / Neuroscience xxx (2016) xxx–xxx 5
NSC 17098 No. of Pages 10
21 May 2016
the neurons of 3xTg-AD mice (Oddo et al., 2003), and
there is also a deficit in long-term retention that correlates
with the accumulation of intraneuronal bA in hippocampus
and amygdala (Billings et al., 2005). Moreover, there is
little evidence of a decrease in the number of neurons in
3xTg-AD mice at 4 months, but a reduction in cells
has been observed at the medial septum/vertical limb of
the diagonal band of Broca in aged 3xTg-AD mice
(18–20 months) (Perez et al., 2011).
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Activity mediated by muscarinic receptors
The cholinergic alterations in 3xTg-AD mice include
degeneration of the cholinergic septo-hippocampal
pathway, reflected by a decrease in ChAT-positive
fibers in the hippocampus and a decrease in positive
neurons in the medial septum at 4 months of age (Girao
da Cruz et al., 2012). Regarding the observed decrease
of the MR response in amygdala, it is worth noting that
this nucleus has an important role in the processing of
anxiety and emotions. In 3xTg-AD mice, an increase of
Please cite this article in press as: Manuel I et al. Activity of muscarinic, galanin
triple transgenic mice model of Alzheimer’s disease. Neuroscience (2016), http
anxiety and fear-related behavior has been observed at
a similar age (Espana et al., 2010). The cholinergic con-
trol of cortical GABA activity involves M2 muscarinic
receptors on GABAergic terminals (Kawaguchi and
Kubota, 1997; Steriade and Descarries, 2006). The loss
of M2 receptors on GABA terminals in the amygdala,
could also account for the detected impairment of the
muscarinic receptor signaling.
The up-regulation of MR activity in the motor cortex
may be a response to the described increase in positive
ChAT-ir dystrophic neurites in the cortex in aged
animals (Perez et al., 2011), accompanied by the intra-
neuronal bA in the primary motor cortex (Mastrangelo
and Bowers, 2008). In other murine models of AD,
expressing the APP Swedish and the PS1DE9 mutations,
the ChAT activity was diminished at advanced ages
(Perez et al., 2007). The cholinergic regulation or alter-
ation in these transgenic models is only detected in aged
animals, as is also the case for the regulation of the MR
activity. Nevertheless, the TgCRND8 mice, which develop
demyelination and many Ab plaques, also show choliner-
and cannabinoid receptors in the prodromal and advanced stages in the
://dx.doi.org/10.1016/j.neuroscience.2016.05.012
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Table 1. [35S]GTPcS binding in different areas of 4-month-old 3xTg-AD and NTg mice induced by carbachol (100 lM) and galanin (1 lM) expressed as
the percentage of stimulation over the basal
Brain region Carbachol stimulation (%) Galanin stimulation (%)
NTg 3xTg-AD NTg 3xTg-AD
Amygdala
Anterior 101.5 ± 29.2 128.9 ± 25.9 19.6 ± 5.4 16.6 ± 15.1
Posterior 59.8 ± 14.1 25.4 ± 7.9* 39.9 ± 10.3 44.6 ± 5.5
Thalamic nuclei
Anteroventral 165.1 ± 21.8 208.7 ± 51.7 40.7 ± 30.3 70.8 ± 21.3
Thalamus 134.1 ± 21.3 146.5 ± 5.8 �3.1 ± 5.8 12.7 ± 12.5
Striatum 210.8 ± 24.2 193.0 ± 22.4 10.8 ± 7.9 5.7 ± 4.6
Hypothalamus 70.9 ± 11.1 74.5 ± 27.9 14.4 ± 15.1 46.6 ± 12.4*
Cortex
Cingular 118.5 ± 30.9 202.6 ± 99.5 22.4 ± 6.3 43.9 ± 30.1
Motor 111.7 ± 12.8 98.2 ± 32.6 �4.3 ± 4.2 �15.7 ± 5.5
Hippocampus
CA1 94.6 ± 14.8 92.7 ± 32.3 �5.4 ± 5.8 3.2 ± 7.9
Dentate gyrus 33.2 ± 16.2 66.9 ± 27.5 �17.6 ± 11.4 9.9 ± 12.2
Basal nucleus 118.0 ± 25.4 75.9 ± 16.6 35.0 ± 13.2 23.7 ± 8.7
Mean ± SEM of six animals by genotype.
Thalamus includes MDL (mediodorsal nucleus), PC (paracentral nucleus), VL (ventrolateral nucleus) and VPM (ventral posteromedial nucleus).* p< 0.05 (two tailed Student’s ‘‘t” test).
Table 2. [35S]GTPcS binding in different areas of 4-month-old 3xTg-AD
and NTg mice induced by WIN55,212-2 (100 lM) expressed as the
percentage of stimulation over the basal
Brain region WIN 55212-2 stimulation (%)
NTg 3xTg-AD
Amygdala
Anterior 124.8 ± 50.1 55.2 ± 17.0
Posterior 147.4 ± 14.3 187.5 ± 24.3
Thalamic nuclei
Anteroventral 64.8 ± 21.2 136.3 ± 15.5*
Thalamus 73.4 ± 24.0 115.9 ± 30.9
Striatum 254.2 ± 39.4 247.9 ± 18.9
Cortex
Cingular 364.9 ± 87.7 456.5 ± 58.2
Motor 381.4 ± 39.5 360.1 ± 25.5
Hippocampus
CA1 309.2 ± 43.4 304.9 ± 34.0
Dentate gyrus 276.2 ± 62.4 369.7 ± 64.6
Basal nucleus 203.5 ± 21.5 359.8 ± 70.2
Hypothalamus 93.3 ± 6.1 101.0 ± 18.7
Globus pallidus 507.8 ± 123.0 594.5 ± 88.8
Sustantia nigra 549.6 ± 224.7 737.6 ± 152.6
Mean ± SEM of six animals by genotype.
Thalamus includes MDL (mediodorsal nucleus), PC (paracentral nucleus), VL
(ventrolateral nucleus) and VPM (ventral posteromedial nucleus).* p< 0.05 (two tailed Student’s ‘‘t” test).
6 I. Manuel et al. / Neuroscience xxx (2016) xxx–xxx
NSC 17098 No. of Pages 10
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gic dysfunction with a reduction in ChAT in the nbM and
M2 reduction in the cortex (Bellucci et al., 2006). The
cholinergic activity seems to depend on the different
mutations expressed in each transgenic mouse model
of familial AD.
Please cite this article in press as: Manuel I et al. Activity of muscarinic, galanin
triple transgenic mice model of Alzheimer’s disease. Neuroscience (2016), http
Activity mediated by galaninergic receptors
The GalR1-mediated activity was measured using rat
galanin. The GalR1 subtype is the main galanin receptor
present in the CNS and is coupled to Gi/o family of G
proteins. Therefore, the recorded galanin stimulations of
[35S]GTPcS binding should be attributable to GalR1-
mediated activity receptors. Studies assessing different
behavior parameters of 4-month-old mice using the
open field test have described an anxious-like state in
these animals. The freezing latency was more
pronounced in 3xTg-AD mice than in NTg, and they also
showed a decrease in locomotion during the first
minute. Other fear-associated parameters such as
increased defecations and a delay in the onset of
grooming could also be an indication of the fact that the
anxious responses are exacerbated in these mice
(Gimenez-Llort et al., 2007). The increase in the activity
of GalR1 in limbic brain areas involved in the control of
anxiety, such as the hypothalamus, could be related to
the reported increase in anxious-like behavior.
Galanin seems to exert an important role in the
hypothalamus by regulating glutamate release in that
area (Kinney et al., 1998). Furthermore, under stress con-
ditions, an increase in the production of galanin in the
hypothalamus does exist (Palkovits, 2000), but both
exogenous galanin administration and agonists are able
to induce anxiolytic-like effects in rats (Bing et al., 1993;
Moller et al., 1999; Rajarao et al., 2007). A possible up-
regulation of GalR1 activity in 4-month-old 3xTg-AD mice
could be a physiological response to compensate for the
increased anxiety during their development that is not
necessary in more aged mice. In other mice models of
AD in which there is an overexpression of Ab, such as
PDAPP, APPSwe/PS1DE9 double transgenic or APP23,
an increase in galanin has also been described, but
and cannabinoid receptors in the prodromal and advanced stages in the
://dx.doi.org/10.1016/j.neuroscience.2016.05.012
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Fig. 3. Autoradiographic images of sagittal tissue sections showing basal [35S]GTPcS binding of 15-month-old NTg (A) and 3xTg-AD (B) mice. Note
that basal binding is higher in the anterior amygdala of 3xTg-AD mice in comparison with NTg mice. Scale bar = 2.5 mm.
Table 3. Basal [35S]GTPcS binding in different areas of 15-month-old
3xTg-AD and NTg mice
Brain region Basal binding (nCi/g t.e.)
NTg 3xTg-AD
Amygdala
Anterior 639 ± 18 817 ± 31**
Posterior 506 ± 19 711 ± 39
Thalamic nuclei
Anteroventral 578 ± 32 621 ± 18**
Thalamus 471 ± 29 562 ± 15*
Striatum 665 ± 24 752 ± 16*
Cortex
Cingular 677 ± 32 761 ± 22
Motor 542 ± 19 601 ± 38
Hippocampus
CA1 665 ± 38 714 ± 36
Dentate gyrus 436 ± 20 474 ± 24
Basal nucleus 607 ± 19 752 ± 14**
Locus coeruleus 342 ± 29 314 ± 22
Globus pallidus 672 ± 18 741 ± 20*
Sustantia nigra 635 ± 31 670 ± 31
Mean ± SEM of six animals by genotype.
Thalamus includes MDL (mediodorsal nucleus), PC (paracentral nucleus), VL
(ventrolateral nucleus) and VPM (ventral posteromedial nucleus).* p< 0.05.
** p< 0.01 (two tailed Student’s ‘‘t” test).
I. Manuel et al. / Neuroscience xxx (2016) xxx–xxx 7
NSC 17098 No. of Pages 10
21 May 2016
mainly in hippocampus and cortex (Diez et al., 2000;
Mufson et al., 2005).
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Activity mediated by cannabinoid receptors
The endocannabinoid system is implicated in
inflammatory responses and neurogenesis, but also in
memory deficits. Memory impairments induced by bApeptide administration are reverted by the CB1
antagonist, SR141716A or rimonabant (Mazzola et al.,
2003). Nevertheless, cannabinoid agonist administration
(HU-210) did not have any effects either in memory tests
or in amyloid progression in APP23/PS45 double trans-
genic mice (Chen et al., 2010). However, other authors
Please cite this article in press as: Manuel I et al. Activity of muscarinic, galanin
triple transgenic mice model of Alzheimer’s disease. Neuroscience (2016), http
using both CB1 and CB2 agonists described some protec-
tive effects in Tg APP mice (Martın-Moreno et al., 2012).
Moreover, the administration of the CB2 specific agonist,
JWH-133, could improve some inflammatory responses
present in AbetaPP/PS1 transgenic mice, but not the bAproduction or deposition in cortex and hippocampus
(Aso et al., 2013). Cannabinoids seem to act in excitotoxic
and inflammatory processes by increasing cannabinoid
receptor activity (Fowler et al., 2010). Thus, 4-month-old
3xTg-AD mice that only present intraneuronal bA, onlyhave an up-regulation of CB1-mediated activity in thala-
mus, while this is decreased in nbM when CNS damage
is evident at 15 months of age. The CB1-mediated activity
is increased in AD patients from the early stages of the
disease in hippocampal and cortical areas, suggesting
the existence of a possible neuroprotective response that
is not clearly observed in 3xTg-AD mice (Manuel et al.,
2014). Other early damaging factors or causes inherent
to the sporadic forms of AD, not present in the hereditary
forms of AD represented by 3xTg-AD mice, could be
responsible for initiating the early response of the endo-
cannabinoid system.
CONCLUSIONS
The 3xTg-AD mice represent an animal model of three
human transgenes and allow us to reproduce the
classical histopathological markers of AD, neuritic
plaques and neurofibrillary tangles. The model develops
the most representative biochemical modifications
associated with AD, i.e. the hyperphosphorylation of tau
and bA accumulation, which have been widely analyzed
in this mouse model at different ages. The behavior of
these mice has also been an important subject of study,
but only a few neurotransmission systems more
associated with memory impairment, such as the
cholinergic system, have been analyzed.
The activity of cholinergic muscarinic receptors is
analyzed in the present study together with the activity
mediated by a system of neuropeptides, the
galaninergic, and a system of neurolipids, the
endocannabinoid. Both systems modulate cholinergic
inputs from the basal forebrain to cortical and
and cannabinoid receptors in the prodromal and advanced stages in the
://dx.doi.org/10.1016/j.neuroscience.2016.05.012
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Fig. 5. Autoradiographic images of sagittal tissue sections of basal [35S]GTPcS binding of 15-month-old NTg (A) and 3xTg-AD (B) mice. Note that
[35S]GTPcS binding stimulated by WIN55,212-2 is not modified in 3xTg-AD mice in comparison with NTg mice. Scale bar = 2.5 mm.
Fig. 4. Autoradiographic images of sagittal tissue sections of basal [35S]GTPcS binding of 15-month-old NTg (A) and 3xTg-AD (B) mice. Note that
[35S]GTPcS binding stimulated by carbachol is increased in the motor cortex and dentate gyrus of the hippocampus of 3xTg-AD mice in comparison
with NTg mice. Scale bar = 2.5 mm.
8 I. Manuel et al. / Neuroscience xxx (2016) xxx–xxx
NSC 17098 No. of Pages 10
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subcortical areas involved in the control of learning and
memory processes, and both systems are up-regulated
in AD patients.
The present results are similar to those reported in
postmortem brain samples of AD patients in several
aspects in relation to the regulation of receptor
activities, but are not found in the same brain areas. MR
activity is decreased in 3xTg-AD mice at the age of
4 months, which mimics the early stages of AD, but only
in some limbic areas such as the amygdala, whereas a
general reduction of M2 MR densities has been found in
hippocampus and cortex of AD patients. Furthermore,
MR activity is increased in motor cortex in 15-month-old
transgenic mice. On the other hand, it is not
counteracted by a regulation of GalR1 and CB1 activity,
as seems to be the case in AD patients, in whom
hyperactivity of galanin and CB1 receptor has been
described mainly in hippocampal areas. The up-
regulation of both neuromodulators of the basal
cholinergic signaling has been interpreted as a
neuroprotective response.
In summary, the 3xTg-AD model has a mild
impairment of the cholinergic muscarinic activity and the
Please cite this article in press as: Manuel I et al. Activity of muscarinic, galanin
triple transgenic mice model of Alzheimer’s disease. Neuroscience (2016), http
regulation of neuromodulatory systems by
neuropeptides, such as galanin, and endocannabinoids
have a role that is likely to be related to the anxious
phenotype, which is characteristic of the prodromal
stages of AD, prior to the onset of clear clinical cognitive
symptoms of the disease.
UNCITED REFERENCE
Blazquez et al. (2014).
Acknowledgments—Supported by grants from the Basque
Government IT584-13 and Spanish Government, Ministry for
Health, I. S. C. III PI 10/01202- co-funded by European Research
Development. Thanks to the Animal facilities of the UPV/EHU at
the Biscay Campus, SGiker. We thank Adele Hopley and M. Ter-
esa Giralt for critical and careful reading of the manuscript. There
are no potential conflicts of interest.
REFERENCES
Aaltonen N, Palomaki VA, Lecklin A, Laitinen JT (2008)
Neuroanatomical mapping of juvenile rat brain regions with
and cannabinoid receptors in the prodromal and advanced stages in the
://dx.doi.org/10.1016/j.neuroscience.2016.05.012
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
567
568
569
570
571
572
573
574
575
576
577
578
579
580
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
596
597
598
599
600
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
619
620
621
622
623
624
625
626
627
628
629
630
631
632
633
634
635
636
637
638
639
640
641
642
643
644
645
646
647
648
649
650
651
652
653
654
655
656
657
658
659
660
661
662
663
664
665
666
667
668
669
I. Manuel et al. / Neuroscience xxx (2016) xxx–xxx 9
NSC 17098 No. of Pages 10
21 May 2016
prominent basal signal in [(35)S]GTPgammaS autoradiography. J
Chem Neuroanat 35(2):233–241.
Aaltonen N, Lehtonen M, Varonen K, Goterris GA, Laitinen JT (2012)
Lipid phosphate phosphatase inhibitors locally amplify
lysophosphatidic acid LPA1 receptor signalling in rat brain
cryosections without affecting global LPA degradation. BMC
Pharmacol 12:7.
Aso E, Juves S, Maldonado R, Ferrer I (2013) CB2 cannabinoid
receptor agonist ameliorates Alzheimer-like phenotype in AbPP/PS1 mice. J Alzheimers Dis 35(4):847–858.
Bailey P (2007) Biological markers of Alzheimer’s disease. Can J
Neurol Sci 34:72–76.
Bellucci A, Luccarini I, Scali C, Prosperi C, Giovannini MG, Pepeu G,
Casamenti F (2006) Cholinergic dysfunction, neuronal damage
and axonal loss in TgCRND8 mice. Neurobiol Dis 23(2):260–272.
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
(5):675–688.
Bing O, Moller C, Engel JA, Soderpalm B, Heilig M (1993) Anxiolytic-
like action of centrally administered galanin. Neurosci Lett 164(1–
2):17–20.
Blazquez G, Canete T, Tobena A, Gimenez-Llort L, Fernandez-
Teruel A (2014) Cognitive and emotional profiles of aged
Alzheimer’s disease (3xTg-AD) mice: effects of environmental
enrichment and sexual dimorphism. Behav Brain Res
268:185–201.
Canete T, Blazquez G, Tobena A, Gimenez-Llort L, Fernandez-
Teruel A (2015) Cognitive and emotional alterations in young
Alzheimer’s disease (3xTg-AD) mice: effects of neonatal handling
stimulation and sexual dimorphism. Behav Brain Res
281:156–171.
Chan-Palay V (1988) Galanin hyperinnervates surviving neurons of
the human basal nucleus of Meynert in dementias of Alzheimer’s
and Parkinson’s disease: a hypothesis for the role of galanin in
accentuating cholinergic dysfunction in dementia. J Comp Neurol
273(4):543–557.
Chen B, Bromley-Brits K, He G, Cai F, Zhang X, Song W (2010)
Effect of synthetic cannabinoid HU210 on memory deficits and
neuropathology in Alzheimer’s disease mouse model. Curr
Alzheimer Res 7(3):255–261.
Diez M, Koistinaho J, Kahn K, Games D, Hokfelt T (2000)
Neuropeptides in hippocampus and cortex in transgenic mice
overexpressing V717F beta-amyloid precursor protein–initial
observations. Neuroscience 100(2):259–286.
Ehrhart J, Obregon D, Mori T, Hou H, Sun N, Bai Y, Klein T,
Fernandez F, Tan J, Shytle RD (2005) Stimulation of cannabinoid
receptor 2 (CB2) suppresses microglial activation. J
Neuroinflammation 2:29.
Espana J, Gimenez-Llort L, Valero J, Minano A, Rabano A,
Rodriguez-Alvarez J, LaFerla FM, Saura CA (2010)
Intraneuronal beta-amyloid accumulation in the amygdala
enhances fear and anxiety in Alzheimer’s disease transgenic
mice. Biol Psychiatry 67(6):513–521.
Fowler CJ, Rojo ML, Rodriguez-Gaztelumendi A (2010) Modulation of
the endocannabinoid system: neuroprotection or neurotoxicity?
Exp Neurol 224(1):37–47.
Gimenez-Llort L, Blazquez G, Canete T, Johansson B, Oddo S,
Tobena A, LaFerla FM, Fernandez-Teruel A (2007) Modeling
behavioral and neuronal symptoms of Alzheimer’s disease in
mice: a role for intraneuronal amyloid. Neurosci Biobehav Rev 31
(1):125–147.
Girao da Cruz MT1, Jordao J, Dasilva KA, Ayala-Grosso CA,
Ypsilanti A, Weng YQ, LaFerla FM, McLaurin J, Aubert I (2012)
Early increases in soluble amyloid-b levels coincide with
cholinergic degeneration in 3xTg-AD mice. J Alzheimers Dis 32
(2):267–272.
Gonzalez de San Roman E, Manuel I, Giralt MT, Chun J, Estivill-
Torrus G, Rodrıguez de Fonseca F, Santın LJ, Ferrer I,
Rodrıguez-Puertas R (2015) Anatomical location of LPA1
Please cite this article in press as: Manuel I et al. Activity of muscarinic, galanin
triple transgenic mice model of Alzheimer’s disease. Neuroscience (2016), http
activation and LPA phospholipid precursors in rodent and
human brain. J Neurochem 134(3):471–485.
Gonzalez-Maeso J, Torre I, Rodrıguez-Puertas R, Garcıa-Sevilla JA,
Guimon J, Meana JJ (2002) Effects of age, postmortem delay and
storage time on receptor-mediated activation of G-proteins in
human brain. Neuropsychopharmacology 26(4):468–478.
Harrison C, Traynor JR (2003) The [35S]GTPgammaS binding assay:
approaches and applications in pharmacology. Life Sci 74(4).
489-08.
Kawaguchi Y, Kubota Y (1997) GABAergic cell subtypes and their
synaptic connections in rat frontal cortex. Cereb Cortex 7
(6):476–486.
Kinney GA, Emmerson PJ, Miller RJ (1998) Galanin receptor-
mediated inhibition of glutamate release in the arcuate nucleus
of the hypothalamus. J Neurosci 18(10):3489–3500.
Llorente A, Gonzalez de San Roman E, Moreno M, Manuel I, Giralt
MT, Rodrıguez-Puertas R (2014) Activity mediated by neurolipid
(CB1 and LPA1) and neuropeptide (GAL1) receptors in a rat
model with cholinergic basal forebrain lesion. American Society
for Neuroscience Meeting. Washington 2014. USA. 307.25/D71.
Manuel I, Gonzalez de San Roman E, Giralt MT, Ferrer I, Rodrıguez-
Puertas R (2014) Type-1 cannabinoid receptor activity during
Alzheimer’s disease progression. J Alzheimers Dis 42
(3):761–766.
Marcyniuk B, Mann DM, Yates PO (1986) The topography of cell loss
from locus caeruleus in Alzheimer’s disease. J Neurol Sci 76(2–
3):335–345.
Marsicano G, Goodenough S, Monory K, Hermann H, Eder M,
Cannich A, Azad SC, Cascio MG, Ortega-Gutierrez S, Van der
Stelt M, Lopez-Rodrıguez ML, Casanova E, Schutz G,
Zieglgansberger W, Di Marzo V, Behl C, Lutz B (2003) CB1
Cannabinoid receptors and on-demand defense against
excitotoxicity. Science 302:84–88.
Martın-Moreno AM, Brera B, Spuch C, Carro E, Garcıa-Garcıa L,
Delgado M, Pozo MA, Innamorato NG, Charade A, de Ceballos
ML (2012) Prolonged oral cannabinoid administration prevents
neuroinflammation, lowers b-amyloid levels and improves
cognitive performance in Tg APP 2576 mice. J
Neuroinflammation 9:8.
Mastrangelo MA, Bowers WJ (2008) Detailed immunohistochemical
characterization of temporal and spatial progression of
Alzheimer’s disease-related pathologies in male triple-transgenic
mice. BMC Neurosci 2(9):81.
Mazzola C, Micale V, Drago F (2003) Amnesia induced by beta-
amyloid fragments is counteracted by cannabinoid CB1 receptor
blockade. Eur J Pharmacol 477(3):219–225.
Moller C, Sommer W, Thorsell A, Heilig M (1999) Anxiogenic-like
action of galanin after intra-amygdala administration in the rat.
Neuropsychopharmacology 21(4):507–512.
Mufson EJ, Deecher DC, Basile M, Izenwasse S, Mash DC (2000)
Galanin receptor plasticity within the nucleus basalis in early and
late Alzheimer’s disease: an in vitro autoradiographic analysis.
Neuropharmacology 39(8):1404–1412.
Mufson EJ, Counts SE, Perez SE, Binder L (2005) Galanin plasticity
in the cholinergic basal forebrain in Alzheimer’s disease and
transgenic mice. Neuropeptides 39(3):233–237.
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 dysfunction. Neuron 39
(3):409–421.
Palkovits M (2000) Stress-induced expression of co-localized
neuropeptides in hypothalamic and amygdaloid neurons. Eur J
Pharmacol 405(1–3):161–166.
Palmer AM, Francis PT, Benton JS, Sims NR, Mann DM, Neary D,
Snowden JS, Bowen DM (1987) Presynaptic serotonergic
dysfunction in patients with Alzheimer’s disease. J Neurochem
48(1):8–15.
Perez S, Basile M, Mash DC, Mufson EJ (2002) Galanin receptor
over-expression within the amygdala in early Alzheimer’s disease:
and cannabinoid receptors in the prodromal and advanced stages in the
://dx.doi.org/10.1016/j.neuroscience.2016.05.012
670
671
672
673
674
675
676
677
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
695
696
697
698
699
700
701
702
703
704
705
707
708
709
10 I. Manuel et al. / Neuroscience xxx (2016) xxx–xxx
NSC 17098 No. of Pages 10
21 May 2016
an in vitro autoradiographic analysis. J Chem Neuroanat 24
(2):109–116.
Perez SE, Dar S, Ikonomovic MD, DeKosky ST, Mufson EJ (2007)
Cholinergic forebrain degeneration in the APPswe/PS1DeltaE9
transgenic mouse. Neurobiol Dis 28(1):3–15.
Perez SE, He B, Muhammad N, Oh KJ, Fahnestock M, Ikonomovic
MD, Mufson EJ (2011) Cholinotrophic basal forebrain system
alterations in 3xTg-AD transgenic mice. Neurobiol Dis 41
(2):338–352.
Rajarao SJ, Platt B, Sukoff SJ, Lin Q, Bender CN, Nieuwenhuijsen
BW, Ring RH, Schechter LE, Rosenzweig-Lipson S, Beyer CE
(2007) Anxiolytic-like activity of the non-selective galanin receptor
agonist, galnon. Neuropeptides 41(5):307–320.
Rodrıguez-Puertas R, Pascual J, Vilaro T, Pazos A (1997a)
Autoradiographic distribution of M1, M2, M3, and M4 muscarinic
receptor subtypes in Alzheimer’s disease. Synapse 26:341–350.
Rodrıguez-Puertas R, Nilsson S, Pascual J, Pazos A, Hokfelt T
(1997b) 125I-galanin binding sites in Alzheimer’s disease:
706
Please cite this article in press as: Manuel I et al. Activity of muscarinic, galanin
triple transgenic mice model of Alzheimer’s disease. Neuroscience (2016), http
increases in hippocampal subfields and a decrease in the
caudate nucleus. J Neurochem 68(3):1106–1113.
Sperling RA, Karlawish J, Johnson KA (2013) Preclinical Alzheimer’s
disease – the challenges ahead. Nat Rev Neurol 9:54–58.
Steriade M, Descarries L (2006) Cholinergic modulation of cortical
activity. In: Giacobini E, Pepeu G, editors. The brain cholinergic
system in health and disease. Abingdon, UK: Informa Healthcare.
p. 191–207.
Welsh-Bohmer KA (2008) Defining ‘‘prodromal” Alzheimer’s disease,
frontotemporal dementia, and Lewy body dementia: are we there
yet? Neuropsychol Rev 18(1):70–72.
Westlake TM, Howlett AC, Bonner TI, Matsuda LA, Herkenham M
(1994) Cannabinoid receptor binding and messenger RNA
expression in human brain: an in vitro receptor autoradiography
and in situ hybridization histochemistry study of normal aged and
Alzheimer’s brains. Neuroscience 63(3):637–652.
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(4537):1237–1239.
(Accepted 10 May 2016)(Available online xxxx)
and cannabinoid receptors in the prodromal and advanced stages in the
://dx.doi.org/10.1016/j.neuroscience.2016.05.012