Hyposensitivity to the amnesic effects of scopolamine or amyloid beta(25-35) peptide in heterozygous acetylcholinesterase knockout (AChE(+/-)) mice.
ABSTRACT We examined the sensitivity of AChE(+/-) mice to the amnesic effects of scopolamine and amyloid beta peptide. AChE(+/-) and AChE(+/+) littermates, tested at 5-9 weeks of age, failed to show any difference in locomotion, exploration and anxiety in the open-field test, or in-place learning in the water-maze. However, when treated with the muscarinic receptor antagonist scopolamine (0.5, 5mg/kg s.c.) 20 min before each water-maze training session, learning impairments were observed at both doses in AChE(+/+) mice, but only at the highest dose in AChE(+/-) mice. The central injection of Abeta(25-35) peptide (9 nmol) induced learning deficits only in AChE(+/+) but not in AChE(+/-) mice. Therefore, the hyper-activity of cholinergic systems in AChE(+/-) mice did not result in increased memory abilities, but prevented the deleterious effects of muscarinic blockade or amyloid toxicity.
- Internal Medicine News 01/2010; 43(15):22-22.
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ABSTRACT: Decrease in the expression or activity of acetylcholinesterase (AChE) enzymatic activity results in increased cholinergic tonus in the brain and periphery, with concomitant regulations of nicotinic and muscarinic receptors expression. We generated AChE knockout mice and characterized the behavioral phenotype of heterozygous animals, focusing on learning and memory functions. Male and female, AChE+/− and AChE+/+ littermate controls (129sv strain) were tested at 5–9 weeks of age. AChE activity was significantly decreased in the hippocampus and cortex of AChE+/− mice, but butyrylcholinesterase activity was preserved. AChE+/− mice failed to show any difference in terms of locomotion, exploration and anxiety parameters in the open-field test. Animals were then tested for place learning in the water-maze. They were trained using a ‘sustained acquisition’ protocol (3 swim trials per day) or a ‘mild acquisition’ protocol (2 swim trials per day) to locate an invisible platform in fixed position (reference memory procedure). Then, during 3 days, they were trained to locate the platform in a variable position (working memory procedure). Learning profiles and probe test performances were similar for AChE+/− and AChE+/+ mice. Mice were then treated with the muscarinic receptor antagonist scopolamine (0.5, 5 mg/kg) 20 min before each training session. Scopolamine impaired learning at both doses in AChE+/+ mice, but only at the highest dose in AChE+/− mice. Moreover, the intracerebroventricular injection of amyloid-β25–35 peptide, 9 nmol, 7 days before water-maze acquisition, failed to induce learning deficits in AChE+/− mice, but impaired learning in AChE+/+ controls. The peptide failed to be toxic in forebrain structures of AChE+/− mice, since an increase in lipid peroxidation levels was measured in the hippocampus of AChE+/+ but not AChE+/− mice. We conclude that the increase in cholinergic tonus observed in AChE+/− mice did not result in increased memory functions but allowed a significant prevention of the deleterious effects of muscarinic blockade or amyloid toxicity.Behavioural brain research 09/2009; · 3.22 Impact Factor
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ABSTRACT: We have previously identified presenilin-1 (PS1), the active component of the γ-secretase complex, as an interacting protein of the amyloid-associated enzyme acetylcholinesterase (AChE). In this study, we have explored the consequences of AChE-PS1 interactions. Treatment of SH-SY5Y cells with the AChE-inhibitor tacrine decreased PS1 levels, in parallel with increase in the secretion of amyloid precursor protein APPα, whereas the cholinergic agonist carbachol had no effect on PS1. AChE knockdown with siRNA also decreased PS1 levels, while AChE overexpression exerted opposing effect. AChE-deficient also had decreased PS1. Mice administered with tacrine or donepezil displayed lower levels of brain PS1. However, sustained AChE inhibition failed to exert long-term effect on PS1. This limited duration of response may be due to AChE upregulation caused by chronic inhibition. Finally, we exposed SH-SY5Y cells to β-amyloid (Aβ)42 which triggered elevation of both AChE and PS1 levels. The Aβ42-induced PS1 increase was abolished by siRNA AChE pretreatment, suggesting that AChE may participate in the pathological feedback loop between PS1 and Aβ. Our results provide insight into AChE-amyloid interrelationships.Neurobiology of aging 05/2011; 33(3):627.e27-37. · 5.94 Impact Factor
Chemico-Biological Interactions 175 (2008) 131–134
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/chembioint
Hyposensitivity to the amnesic effects of scopolamine or amyloid
?25–35peptide in heterozygous acetylcholinesterase knockout
Julie Espallerguesa,b,c, Laurie Galvana,b,c, Laurence Lepourryc,d,e, B´ eatrice Bonafosc,d,e,
Tangui Mauricea,b,c, Arnaud Chatonnetc,d,e,∗
aINSERM U. 710, 34095 Montpellier, France
bEPHE, 75017 Paris, France
cUniversity of Montpellier II, 34095 Montpellier, France
dINRA UMR 866, 34060 Montpellier, France
eUniversity of Montpellier I, 34967 Montpellier, France
a r t i c l e i n f o
Available online 10 April 2008
Learning and memory
a b s t r a c t
We examined the sensitivity of AChE+/−mice to the amnesic effects of scopolamine and
amyloid ? peptide. AChE+/−and AChE+/+littermates, tested at 5–9 weeks of age, failed to
show any difference in locomotion, exploration and anxiety in the open-field test, or in-
place learning in the water-maze. However, when treated with the muscarinic receptor
antagonist scopolamine (0.5, 5mg/kg s.c.) 20min before each water-maze training session,
learning impairments were observed at both doses in AChE+/+mice, but only at the highest
dose in AChE+/−mice. The central injection of A?25–35peptide (9nmol) induced learning
deficits only in AChE+/+but not in AChE+/−mice. Therefore, the hyper-activity of cholinergic
systems in AChE+/−mice did not result in increased memory abilities, but prevented the
deleterious effects of muscarinic blockade or amyloid toxicity.
© 2008 Elsevier Ireland Ltd. All rights reserved.
Acetylcholinesterase (AChE) is the main catabolic
enzyme of acetylcholine (ACh), responsible for the synap-
tic clearance of the neurotransmitter. Decrease in AChE
expression or activity results in increased cholinergic
tonus in the brain or periphery, with concomittant reg-
ulation of nicotinic and muscarinic receptors expression
. Increasing the cholinergic tonus through inhibition of
AChE activity is the main symptomatic therapy currently
used in Alzheimer’s disease (AD). AD is a progressive neu-
?This work is a project (#03) of the CompAn Behavioral Phenotyping
Facility (Montpellier, France).
∗Corresponding author at: Departement de Physiologie Animale, INRA,
place Viala, 34060 Montpellier cedex 1, France.
E-mail address: firstname.lastname@example.org (A. Chatonnet).
rodegenerative process, due to deposition of senile plaques
containing amyloid proteins and neurofibrillary tangles
formed of hyperphosphorylated Tau protein [2,3]. Amyloid
toxicity severely affects cholinergic pathways, particularly
originating from the magnocellularis basalis nucleus. Sus-
as donepezil, rivastigmine or galantamine, helps to main-
tain the cognitive scores in AD patients . Moreover,
cholinergic receptors may also mediate protection against
amyloid toxicity. Activation of nicotinic ?7receptors, par-
ticularly, has been shown to protect neurons against A?
peptide toxicity, through activation of the PI3kinase/Akt
We used AChE knockout mice [7,8] and characterized
the behavioral phenotype of heterozygous animals, par-
ticularly focusing on memory functions. Male and female,
AChE+/−and AChE+/+littermate controls (129sv strain),
tested at 5–9 weeks of age, failed to show any difference
0009-2797/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.
J. Espallergues et al. / Chemico-Biological Interactions 175 (2008) 131–134
in locomotion, exploration and anxiety, in the open-field
test. Animals were trained to locate an invisible plat-
form in the water-maze, a procedure assessing spatial
reference memory, using either a ‘sustained acquisition’
protocol (3swims/day, 5 days) or a ‘mild acquisition’ proto-
col (2swims/day, 9 days). Learning profiles and probe test
performances were similar in AChE+/−and AChE+/+control
ined whether male AChE+/−mice are sensitive to amnesic
treatments induced by muscarinic receptor blockade or by
central injection of A? peptide. Mice were treated with the
muscarinic receptor antagonist scopolamine (0.5, 5mg/kg
s.c.), 20min before behavioral testing, or received a central
injection of A?25–35peptide (9nmol), 1 week before test-
ing. We observed that the increase in cholinergic tonus did
not result in increased memory abilities in AChE+/−mice,
but provided significant protection against the deleterious
effects of muscarinic blockade or amyloid toxicity.
2. Materials and methods
AChE+/−and wild-type littermates were bred in the lab-
oratory and used at the CompAn behavioral phenotyping
facility (University of Montpellier). Mice were maintained
in a temperature and humidity controlled room, under a
12-h light:12-h dark cycle (lights on at 07:00 a.m.). Animal
procedures were conducted in adherence to the European
Council Directive of 24 Nov. 1986 (86–609).
Scopolamine hydrobromide was from Sigma–Aldrich
(France). The drug was solubilized in physiological saline
and injected subcutaneously (s.c.) in 100?l/20g b.w.,
20min before testing. A?25–35 and scrambled A?25–35
(Sc.A?) peptides were from NeoMPS (France). They were
dissolved in distilled water at 3mg/ml and kept at −20◦C.
peptides were incubated at 37◦C for 4 days and injected
intracerebroventricularly (i.c.v.) in a volume of 3?l per
mouse, as described .
2.3. Water-maze procedures
The water-maze was a circular pool (diameter 150cm,
height 30cm). Water temperature (21±1◦C), light inten-
sity, external cues in the room and water opacity (obtained
out testing. Four departure positions were set at opposite
positions and a transparent Plexiglas platform (diame-
ter 10cm) could be immersed at the centre of each pool
quadrant defined by the departure positions. The quadrant
where the platform was located was termed the training
(T) quadrant and others, opposite (O), adjacent right (AR),
and adjacent left (AL). Swimming was recorded using the
Videotrack®II software (Viewpoint, France), trajectories
being analyzed as latencies and distances.Training con-
sisted of 3 swims per day for 4 or 5 days, with 15min
intertrial time. Start positions were randomly selected.
Mice were allowed a 90s swim to find the platform and left
on it for 20s. Two hours after the last swim, the probe test
was performed. The platform was removed and each ani-
mal was allowed a free 60s swim. The percentage of time
spent in each quadrant was determined. Median latency,
expressed as mean±S.E.M., was calculated for each train-
non-parametric ANOVA, post hoc comparisons being made
using Dunn’s test. Presence in the T quadrant during the
probe test was analyzed vs. chance level (25%) using the
Heterozygous AChE KO mice were first tested for their
sensitivity to the amnesic effect of scopolamine. The mus-
carinic receptor antagonist was injected at 0.5 and 5mg/kg
s.c. Wild-type AChE+/+mice treated with physiological
saline vehicle solution (V) showed a decrease in swimming
latency during acquisition (Fr=4.19, p<0.05; Fig. 1A) with
a significant difference between trial 4 and 1, indicating
that animals correctly learned the platform location in the
maze. Animals treated with either 0.5 or 5mg/kg scopo-
diminution of latency over training trials (Fr=4.15, p>0.05
for Scop (0.5); Fr=2.78, p>0.05 for Scop (5); Fig. 1A). In
scopolamine did not interfere with maze learning, as indi-
cated by similar decreases in acquisition latency over trials
(Fr=11.76, p<0.01 for V; Fr=9.84, p<0.01 for Scop (0.5);
Fig. 1B), with significant differences between trials 3–4 and
1. On the contrary, the scopolamine (5mg/kg) treatment
blocked the decrease in acquisition latencies (Fr=2.12,
p>0.05; Fig. 1B). Interestingly, V-treated AChE+/−mice
showed lower swimming latency than V-treated wild-type
animals, although no significant difference was measured
(p>0.05 for each trial). AChE+/−mice therefore showed
a non-significant tendency to learn faster than wild-type
During the probe test, the scopolamine treatment at
0.5 or 5mg/kg resulted in a decrease of the time spent in
the T quadrant, as compared with V-treated AChE+/+mice
(Fig. 1C). In AChE+/−mice, only the treatment with the
highest dose of scopolamine resulted in a blockade of the
preferential exploration in the T quadrant (Fig. 1C).
amnesic effect of A?25–35peptide. A?25–35or Sc.A? pep-
tide (9nmol) was injected i.c.v. 1 week before the initiation
of place learning in the water-maze. Sc.A?-treated AChE+/+
mice showed a decrease in swimming latency (Fr=16.29,
p<0.01; Fig. 2A) with a significant difference between
trials 4–5 and 1. Animals treated with A?25–35failed to
show a significant decrease in latency over training trials
(Fr= =8.78, p>0.05; Fig. 2A). In AChE+/−mice, both Sc.A?
and A?25–35treated groups showed significant decreases
in acquisition latencies over trials (Fr=10.07, p<0.05 and
Fr=24.63, p<0.0001, respectively; Fig. 2B) with significant
differences between trials 3–5 and 1. During the probe test,
in the T quadrant in AChE+/+mice but not AChE+/−mice
(Fig. 2C), confirming that the latter are insensitive to the
amnesic effect of the amyloid peptide.
J. Espallergues et al. / Chemico-Biological Interactions 175 (2008) 131–134
Fig. 1. AChE+/−mice were less sensitive to the memory impairing effect
of scopolamine in the water-maze test. Acquisition profiles for (A) AChE+/+
and (B) AChE+/−mice and (C) probe test performances. Scopolamine was
administered at 0.5 or 5mg/kg subcutaneously 20min before the first
swim each training day. (A and B) Acquisition profiles, *p<0.05, **p<0.01
vs. trial 1 latency. (C) Probe test, the presence in the T quadrant is shown.
The number of mice per group is shown within the column. *p<0.05,
**p<0.01 vs. chance level (dotted line).
amyloid A?25–35peptide, in the water-maze test. Acquisition profiles for
(A) AChE+/+and (B) AChE+/−mice and (C) probe test performances. Mice
received Sc.A? or A?25–35peptides (9nmol) intracerebroventricularly 7
days before the first training day. (A and B) Acquisition profiles, *p<0.05,
**p<0.01 vs. trial 1. (C) Probe test, the number of mice per group is shown
within the T column. *p<0.05, **p<0.01 vs. chance level (dotted line).
J. Espallergues et al. / Chemico-Biological Interactions 175 (2008) 131–134
We observed that scopolamine was less potent in
inducing learning impairments in AChE+/−mice. Indeed, a
0.5mg/kg dose, effective in wild-type mice, did not pre-
vent maze acquisition in AChE+/−mice, which required a
10× higher dose before showing clear signs of impaired
learning. This effect was observed in male (this data)
as well as female mice (not shown). Therefore, although
the hyper-cholinergy resulting from the inactivation of
AChE did not directly result in learning enhancement, it
appeared sufficient to provoke a shift in the effective dose
of scopolamine. AChE+/−mice appeared less sensitive to
muscarinic receptor blockade. Indeed, Volpicelli-Daley et
al.  reported that M1, M2and M4muscarinic receptors
showed a 50–80% decrease in expression in the hippocam-
pus and cortex of AChE−/−mice, brain regions associated
with memory. In addition, muscarinic receptors showed
decreased presynaptic, cell surface, and dendritic distri-
butions and increased localization to intracellular puncta.
Furthermore, muscarinic agonist-induced activation of
extracellular signal-regulated kinase (ERK), a signaling
pathway associated with synaptic plasticity and amyloido-
genesis, was diminished in the hippocampus and cortex
of AChE−/−mice . Therefore, chronic diminution of ACh
metabolism and resulting hyper-cholinergic tonus results
in adaptations of ACh receptor expression and function and
thus hyposensitivity to muscarinic antagonists.
We also report that central injection of A?25–35pep-
1 week after injection. This observation deserves a more
extensive study, but suggests that adaptations affecting
nicotinic or muscarinic Ach receptors are not sufficient to
conteract the sustained activation of endogenous protec-
tion pathways by ACh. Indeed, protection could be induced
by activation of either ?7nicotinic receptors, through the
phosphatidylinositol 3-kinase (PI3K)-Akt pathway [5,6] or
M1receptors, through the phospholipase C (PLC)/protein
kinase C (PKC) pathway .
In conclusion, AChE+/−mice appear to be a promis-
ing model to analyze the regulations induced by increased
ACh tonus on neurotoxicity and to understand how the
downregulation of nicotinic and muscarinic receptors may
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