Elimination of the Vesicular Acetylcholine Transporter in
the Striatum Reveals Regulation of Behaviour by
Monica S. Guzman1,2., Xavier De Jaeger1,3,4., Sanda Raulic1, Ivana A. Souza1,3,4, Alex X. Li5, Susanne
Schmid3, Ravi S. Menon1,5,6, Raul R. Gainetdinov7,8, Marc G. Caron8, Robert Bartha1,5,6, Vania F.
Prado1,2,3*, Marco A. M. Prado1,2,3*
1Molecular Brain Research Group, Robarts Research Institute, Schulich School of Medicine & Dentistry, University of Western Ontario, London, Ontario, Canada,
2Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, University of Western Ontario, London, Ontario, Canada, 3Department of
Anatomy & Cell Biology, Schulich School of Medicine & Dentistry, University of Western Ontario, London, Ontario, Canada, 4Program in Molecular Pharmacology, Faculty
of Medicine, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil, 5Imaging Research Group, Robarts Research Institute, Schulich School of Medicine &
Dentistry, University of Western Ontario, London, Ontario, Canada, 6Department of Medical Biophysics, Schulich School of Medicine & Dentistry, University of Western
Ontario, London, Ontario, Canada, 7Department of Neuroscience and Brain Technologies, Italian Institute of Technology, Genova, Italy, 8Department of Cell Biology,
Duke University Medical Center, Durham, North Carolina, United States of America
Cholinergic neurons in the striatum are thought to play major regulatory functions in motor behaviour and reward. These
neurons express two vesicular transporters that can load either acetylcholine or glutamate into synaptic vesicles.
Consequently cholinergic neurons can release both neurotransmitters, making it difficult to discern their individual
contributions for the regulation of striatal functions. Here we have dissected the specific roles of acetylcholine release for
striatal-dependent behaviour in mice by selective elimination of the vesicular acetylcholine transporter (VAChT) from striatal
cholinergic neurons. Analysis of several behavioural parameters indicates that elimination of VAChT had only marginal
consequences in striatum-related tasks and did not affect spontaneous locomotion, cocaine-induced hyperactivity, or its
reward properties. However, dopaminergic sensitivity of medium spiny neurons (MSN) and the behavioural outputs in
response to direct dopaminergic agonists were enhanced, likely due to increased expression/function of dopamine
receptors in the striatum. These observations indicate that previous functions attributed to striatal cholinergic neurons in
spontaneous locomotor activity and in the rewarding responses to cocaine are mediated by glutamate and not by
acetylcholine release. Our experiments demonstrate how one population of neurons can use two distinct neurotransmitters
to differentially regulate a given circuitry. The data also raise the possibility of using VAChT as a target to boost
dopaminergic function and decrease high striatal cholinergic activity, common neurochemical alterations in individuals
affected with Parkinson’s disease.
Citation: Guzman MS, De Jaeger X, Raulic S, Souza IA, Li AX, et al. (2011) Elimination of the Vesicular Acetylcholine Transporter in the Striatum Reveals Regulation
of Behaviour by Cholinergic-Glutamatergic Co-Transmission. PLoS Biol 9(11): e1001194. doi:10.1371/journal.pbio.1001194
Academic Editor: Eric Nestler, Mount Sinai School of Medicine, United States of America
Received June 15, 2011; Accepted September 29, 2011; Published November 8, 2011
Copyright: ? 2011 Guzman et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Canadian Institutes of Health Research (CIHR), Canadian Foundation for Innovation (CFI), and the Ontario Research
Fund (ORF). MGC received support from the NIH. XDeJ and IAS received PhD fellowships from CAPES (Brazil). IAS also received support from the Emerging Leaders
of America Program (ELAP) from the Department of Foreign Affairs and International Trade (Canada). The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Abbreviations: BOLD, blood oxygenation level-dependent; ChAT, choline acetyltransferase; CHT1, high-affintiy choline transporter; CPP, conditioned place
preference; phMRI, pharmacological MRI; PPI, pre-pulse inhibition; VAChT, vesicular acetylcholine transporter; VGLUT3, vesicular glutamate transporter 3
* E-mail: firstname.lastname@example.org (MAMP); email@example.com (VFP)
. These authors contributed equally to this work.
The striatum is the major input gateway to the basal ganglia.
Striatal activity plays important roles in controlling motor
functions and goal-directed and reward-related behaviours [1–4].
The striatum is the brain region mostly affected in motor diseases,
such as Parkinson’s disease (PD), Huntington’s disease, and
dystonia . Medium spiny GABAergic neurons (MSN), activated
by corticostriatal glutamatergic inputs, are the major output
neurons for the striatum; these neurons are regulated extensively
by the classical neurotransmitters dopamine and acetylcholine
(ACh) [1,2,4,6]. These two neurotransmitters have reciprocal
relationships, regulating each other’s release at different levels, and
they generally have opposing actions in the direct and indirect
striatal pathways [1,5,7–9]. Regulation of MSNs by dopamine has
received considerable attention, largely due to the well-known
effects of reduced dopamine levels leading to motor symptoms in
PD  and the role of dopamine in the effect of drugs of abuse
In contrast to the widely known effects of dopamine in the
striatum, we know considerably less about how ACh shapes striatal
function. Cholinergic neurons form a small population of aspiny
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and large striatal interneurons that provide the sole source of
cholinergic innervation to MSNs [12,13]. These neurons fire
constantly and therefore ensure relatively high levels of extracel-
lular ACh. To maintain high levels of transmitter release,
cholinergic neurons transport ACh synthesized in the cytoplasm
into synaptic vesicles, a process which requires the activity of the
vesicular acetylcholine transporter (SLC18A3, VAChT [14,15]),
the last cholinergic-specific step for ACh-mediated neurotrans-
mission . A variety of muscarinic receptors , as well as
nicotinic subtypes of receptors [18–21], involved in controlling
striatal function add complexity to unravelling the role of
endogenous ACh in the striatum. To make matters more difficult,
several central cholinergic neurons express both VAChT and
distinct vesicular glutamate transporters (VGLUTs) and thus are
able to store and release both ACh and glutamate . Striatal
cholinergic neurons express VGLUT3 [23–26] and simultaneously
release glutamate and ACh . It is unknown, however, if
cholinergic neurons can use both neurotransmitters to regulate
Elimination of cholinergic neurons in the striatum, using
ablation strategies, indicated that these neurons have a role in
regulating spontaneous and cocaine-induced locomotor activity, as
well as its rewarding properties [28–31]. These neurons have the
capacity to release both ACh and glutamate; therefore, non-
selective manipulations of striatal cholinergic neurons can affect
both VAChT and VGLUT-mediated neurotransmission. Inter-
estingly, mice null for VGLUT3 phenocopy many of the
behavioural alterations found in mice that had their accumbens
cholinergic neurons ablated . However, because VGLUT3-
null mice also presented a 40% decrease on acetylcholine release,
it is difficult to discern the individual effects of these two
neurotransmitters. Therefore, the specific roles of ACh for striatal
function have not yet been addressed.
To investigate the possibility that cholinergic neurons can use
these two distinct neurotransmitters differentially to regulate
striatal circuitry, we generated a novel mouse line in which we
selectively eliminated ACh release by deleting the VAChT gene in
the striatum. Our results reveal specific roles for ACh release in
regulating dopamine receptor-mediated locomotor responses, but
suggest that some of the previous functions attributed to these
neurons are related to their ability to release glutamate.
D2-Cre Mice Express Cre in Striatal Cholinergic Neurons
To address specific roles of ACh release in striatal function we
generated a VAChT floxed mouse line (VAChTflox/flox, ), as
constitutive VAChT knockout mice do not survive birth due to
impaired breathing . The addition of lox P sites did not change
VAChT expression at the mRNA and protein levels when
compared to wild-type control mice. VAChTflox/floxmice had
normal levels of VAChT and other pre-synaptic cholinergic
markers. In addition locomotor activity, grip-strength, and fatigue
were identical in VAChTflox/floxmice and wild-type mice .
In order to selectively eliminate VAChT in the striatum, we
used the D2-Cre bacterial artificial chromosome (BAC) transgenic
mouse line generated by GENSAT , which expresses the
enzyme Cre recombinase under the control of regulatory elements
of the D2 dopamine receptor (D2R). Details related to this mouse
line, including control experiments demonstrating that the
expression of Cre has no effects on the parameters studied here,
are presented in Experimental Procedures and Figure S6. To test
whether Cre was expressed in striatal cholinergic neurons, we
crossed D2-Cre mice to Rosa26 reporter mice (Rosa26-YFP mice),
in which the Rosa26 locus expresses YFP once Cre-mediated
recombination has occurred (Figure 1a). We found that in D2-
Cre;Rosa26-YFP mice almost 100% of striatal cholinergic neurons
identified with an antibody against CHT1 also showed Cre-
recombination (YFP staining 98% co-localization, Table S1). We
did not detect co-localization of YFP in cholinergic neurons in the
penduculopontine nucleus or in motoneurons in the brainstem
(Figure S1 and Table S1). Partial localization of YFP in cholinergic
neurons was detected in the basal forebrain, albeit to a much lower
extent than in the striatum (approx. 50%, Figure 1b and Table
S1). We therefore intercrossed D2-Cre mice to VAChTflox/flox
mice to generate mice with selective elimination of VAChT in the
striatum (VAChTD2-Cre-flox/flox) or control mice (VAChTflox/flox).
Genotyping for these lines is shown in Figure S2. VAChTD2-Cre-flox/flox
mice were born in the expected Mendellian ratio and did not
present overt phenotypes. We found no gross morphological
alterations in the striatum or other brain sections stained with
hematoxylin/eosin in VAChTD2-Cre-flox/floxmice compared to
control mice (unpublished data).
To assess the degree of Cre-mediated recombination we
evaluated the expression of VAChT in the striatum of
VAChTD2-Cre-flox/flox. As expected, based on the observations
with the D2-Cre;Rosa26-YFP mice, both mRNA and protein
levels for VAChT were almost abolished in the striatum of
VAChTD2-Cre-flox/flox(Figure 2a,d,g). In contrast, choline acetyl-
transferase (ChAT) and the high-affinity choline transporter
(CHT1) protein levels were not altered (Figure 2e and f). There
was no difference in VAChT protein expression levels in the
hippocampus of VAChTD2-Cre-flox/floxmice when compared to
controls (Figure 2h and i). Accordingly, release of [3H]-ACh was
abolished in striatal slices from VAChTD2-Cre-flox/flox
depolarized with high KCl, whereas it was identical to controls
in hippocampal slices (Figure 3a and b).
The neurotransmitters dopamine and acetylcholine play
opposite roles in the striatum (a brain region involved in
motor control and reward-related behaviour), and their
balance is thought to be critical for striatal function.
Acetylcholine in the striatum has been linked to a number
of functions, including control of locomotor activity and
response to drugs of abuse. However, striatal cholinergic
interneurons can also release glutamate (in addition to
acetylcholine) and it is presently unclear how these two
neurotransmitters regulate striatal-dependent behaviour.
Previous work has attempted to resolve this issue by
ablating cholinergic neurons in the striatum, but this
causes loss of both cholinergic and glutamatergic neuro-
transmission. In this study, we created a novel genetic
mouse model which allowed us to selectively interfere
with secretion of acetylcholine in the striatum, while
leaving total striatal glutamate release intact. In these
mice, we observed minimally altered behavioural respons-
es to cocaine, suggesting that striatal glutamate, rather
than acetylcholine, is critical for cocaine-induced behav-
ioural manifestations. Furthermore, elimination of striatal
acetylcholine release affects how striatal output neurons
respond to dopamine, by up-regulating dopaminergic
receptors and changing behavioural responses to dopa-
minergic agonists. Our experiments highlight a previously
unappreciated physiological role of cholinergic-glutama-
tergic co-transmission and demonstrate how a population
of neurons can use two distinct neurotransmitters to
differentially regulate behaviour.
Acetylcholine and Striatal-Dependent Behaviours
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Acetylcholine can modulate glutamate release via pre-synaptic
nicotinic receptors in projection glutamatergic nerve-terminals
. In addition, striatal cholinergic neurons can also release
glutamate . Therefore, we examined if there was any effect of
VAChT elimination on glutamate release. Isolated nerve terminals
were obtained from striatal tissue of VAChTD2-Cre-flox/floxand
control mice and glutamate release was stimulated by KCl. We did
not detect changes in glutamate release from isolated nerve
terminals in VAChT-deficient mice compared to controls
(Figure 3c). It should be noted, however, that this method does
not separate terminals containing VGLUT3 from nerve terminals
containing other VGLUTs, and therefore only reflects global
changes in glutamate release. Moreover, VGLUT3 mRNA
expression by qPCR did not differ in VAChTD2-Cre-flox/floxmice
compared to control mice (Figure 3d). These results suggest that
overall glutamate release is not grossly altered in these mice.
VAChTD2-Cre-flox/floxMice Do Not Show Motor Deficits
Because we detected the presence of Cre-mediated recombina-
tion in motoneurons in the spinal cord (Figure S1), which could
affect the behavioural performance in VAChTD2-Cre-flox/floxmice,
we examined the cholinergic system in the spinal cord of
VAChTD2-Cre-flox/floxmice. We did not find alterations in mRNA
levels for VAChT in the spinal cord of VAChTD2-Cre-flox/floxmice
(Figure S3). However, we detected an increase in ChAT mRNA
and protein levels in the spinal cord. Surprisingly, there was also
about a 50% decrease in VAChT protein levels. Previous
experiments showed that up to a 50% decrease in the expression
of VAChT in the spinal cord is well tolerated in mice and does not
alter motor function [35,36]. In agreement with these previous
results, VAChTD2-Cre-flox/floxmice showed no difference in grip-
force strength (Figure S4a, t(47)=1.702, p=0.095) or fatigue
(detected by the Wire-hang task, Figure S4b, Mann-Whitney,
Figure 1. D2-Cre drives the expression of Cre in striatal cholinergic neurons. (a) Expression pattern of Cre detected by staining for YFP in
the brain of D2-Cre;Rosa26-YFP mice. (b) Sections from different regions of the central nervous system were immunostained for CHT1 (Red) and YFP
(Green) in D2-Cre;Rosa26-YFP mice. Arrows show localization of Cre expression (YFP) in cholinergic neurons (CHT1 staining). Arrowheads show
cholinergic neurons that do not express Cre. For additional brain regions, see Figure S1 and Table S1.
Acetylcholine and Striatal-Dependent Behaviours
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Figure 2. Expression of VAChT in the striatum of VAChTD2-Cre-flox/floxmice. (a)VAChT mRNA expression, (b) ChAT mRNA expression, (c) CHT1
mRNA expression, (d) VAChT protein expression, (e) ChaT protein expression, (f) CHT1 protein expression, (g) representative immunoblot of control
and VAChTD2-Cre-flox/floxstriatal tissue, (h) VAChT protein expression in the hippocampus, and (i) representative immunoblot of protein expression in
the hippocampus. ** p,0.01 *** p,0.001. mRNA expression levels were quantified by qPCR using actin to normalize the data, and figures represent
N=5 mice. Protein levels were quantified using synaptophysin as a loading control. N=5 mice. See Figure S2 for VAChT levels in the spinal cord.
Figure 3. Release of acetylcholine and glutamate from VAChTD2-Cre-flox/floxmice. (a) Release of [3H]ACh from striatal slices in response to
depolarization with KCl (33 mM). Basal release was subtracted from stimulated release to obtain only evoked release. *** p,0.001. (b) Release of
[3H]ACh from hippocampal slices performed as in (a). (c) Release of glutamate from striatal isolated nerve terminals and (d) expression of VGLUT 3 in
the striatum. N=5.
Acetylcholine and Striatal-Dependent Behaviours
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T(13)=49, p=0.710). Interestingly, we also found that relative to
controls, VAChTD2-Cre-flox/floxmice showed no deficit in motor
performance or motor learning assessed using the rotarod test
(Figure S4c, Repeated Measures ANOVA reveal no difference
between the two genotypes with respect to time to fall,
F(1,261)=0.0000409, p=0.995; both sets of mice improved their
performance, F(9,261)=41.614, p,0.001; and there was no interac-
tion between genotype and session, F(9,261)=1.333, p=0.220).
These results show that despite a decrease in the levels of VAChT in
the spinal cord there were no detectable changes in motor function.
The rotarod experiments also suggest that VAChTD2-Cre-flox/flox
mice are physically fit and that motor learning does not appear to
depend on striatal cholinergic activity.
VAChTD2-Cre-flox/floxMice Do Not Show Broad Cognitive
Next, as a further control experiment, we determined if the
elimination of ACh release in the striatum could interfere with
cognitive performance that is believed to be generally independent
of striatal function. We used object recognition memory, a task
that is thought to be dependent on the hippocampus [37,38] and
perirhinal cortex , and has been previously shown to be
sensitive to global decreases in VAChT levels [35,36,40]. In this
test VAChTD2-Cre-flox/floxmice performed identically to controls,
suggesting that important cognitive functions are preserved in this
new mouse line (Figure S4d, two-way ANOVA revealed no effect
of genotype, F(1,16)=0.651, p=0.431, a significant effect for object,
F(1,16)=21.559, p,0.001 and no Object 6Genotype interaction,
Previous experiments have shown that the density of cholinergic
neurons in the accumbens, as well as expression of ChAT, is
decreased in the post-mortem brain of schizophrenic individuals
[41,42]. Moreover, partial ablation of cholinergic striatal neurons
caused alterations in sensorimotor gating . Therefore, we used
habituation to acoustic startle and pre-pulse inhibition to assess
sensorimotor gating, but found no effects of elimination of striatal
VAChT on these parameters (Figure S5). These results show that
decreased striatal ACh release does not cause sensorimotor gating
dysfunctions in these animals and likely in schizophrenia as well.
VAChTD2-Cre-flox/floxMice Have Normal Spontaneous
There are controversial views regarding the role of striatal
cholinergic neurons in locomotion. Previous experiments in which
cholinergic neurons in the nucleus accumbens were ablated
indicated that loss of these neurons caused hyperlocomotion and
increased sensitivity to the locomotor effects of cocaine [29–31].
However, more recent experiments using an optogenetics
approach failed to detect an increased locomotor activity in mice
in which striatal cholinergic neurons were acutely silenced . In
agreement with the latter, we found no differences in locomotor
activity when we compared VAChTD2-Cre-flox/floxmice to controls
(Figure 4a). The dynamics of total horizontal activity (Figure 4a
and 4b, t(48)=0.1464; p=0.884) or counts of vertical activity
(unpublished data, t(24)=1.027; p=0.315) were essentially identi-
cal in the two strains. Importantly, in control experiments D2-Cre
mice did not differ in locomotor activity from respective wild type
mice (Figure S6).
It has been shown that VGLUT3-null mice present hyperac-
tivity, which was attributed to decreased ACh release from striatal
cholinergic neurons due to decreased filling of synaptic vesicles
with ACh . Because these experiments with VGLUT3-null
mice were performed in the initial hours of the dark cycle, we
reproduced these conditions with a new cohort of our mice. The
VAChTD2-Cre-flox/floxmice were no more active than their control
counterparts during the first hours of the dark cycle (Figure 4c and
d, repeated measures ANOVA shows no main effect of genotype,
F(1,1593)=0.321, p=0.576, significant effect of time, F(59,1593)=
14.411, p,0.001 and no interaction Genotype 6Time, F(59,1593)
=0.947, p=0.591; total activity was not different, Mann-Whitney,
T(29)=213, p=0.431). Finally, we also tested inter-session
habituation by investigating locomotor activity in 3 consecutive
days in the open-field (Figure 4e). We observed that both
genotypes habituated similarly to the open-field. Repeated
measures ANOVA confirmed that the general activity was the
same for both genotypes (genotype factor, F(1,58)=0.932, p=
0.342). The activity decreased over the day (day factor, F(2,58)=
10.244, p,0.001) and both genotypes habituated to the environ-
ment at comparable rates (interaction between genotype and day,
F(2,58)=1.506, p=0.230). Evidently, deletion of VAChT in the
striatum does not affect general spontaneous activity or compro-
mise the capacity to habituate to a new environment.
Responses of VAChTD2-Cre-flox/floxMice to Cocaine
Previous experiments in mice in which cholinergic interneurons
were ablated suggested that decreased ACh levels increase
sensitivity of mice to the locomotor effects of cocaine [29–31].
However, these experiments did not separate the effects of VAChT
and VGLUT3-mediated transmission. Interestingly, VGLUT3-null
mice are also more sensitive to the locomotor effects of cocaine, a
resultthat wasattributedat leastinpart to a decrease instriatal ACh
activity in VAChT-deficient mice, we investigated the specific
effects of the elimination of VAChT-mediated neurotransmission
on the actions of cocaine. Administration of 5, 20, or 40 mg/kg of
cocaine increased locomotor activity in VAChTflox/floxmice and
VAChTD2-Cre-flox/floxmice (Figure 5c, two-factor ANOVAs show a
significant effect of genotype, F(1,51)=6.531, p=0.014, significant
effect of treatment, F(3,51)=15.611, p,0.001, and no Genotype 6
Treatment interaction, F(3,51)=0.983, p=0.381). There was no
difference between the two genotypes in their ability to increase
activity in response to cocaine-injected i.p. at 5 mg/kg dose
(Figure 5a, 5 mg/kg, repeated measures ANOVAs show no effect
of genotype, F(1,322)=0.201, p=0.661, significant effect of time,
F(23,322)=12.820, p,0.001, and no Time 6 Genotype inter-
action, F(23,322)=1.373, p=0.121). Paradoxically, at 20 mg/kg
VAChTD2-Cre-flox/floxmice showed a smaller effect of cocaine in
locomotor activity than controls (Figure 5b, 20 mg/kg, repeated
measures ANOVA shows significant effect of genotype, F(1,480)=
11.345, p,0.001, significant effect of time, F(23,480)=9.464,
p,0.001, and no Time 6 Genotype interaction, F(23,480)=0.945,
p=0.537). Analysis of total activity counts showed a clear effect of
genotype (Figure 5c, Mann-Whitney, T(23)=166.000, p,0.05). At
40 mg/kg both genotypes showed similar responses (Figure 5c,
t(14)=0.980, p=0.344), suggesting that lack of striatal VAChT
altered the response to 20 mg/kg of cocaine, but overall did not
cause increased sensitivity to locomotor effects of cocaine.
Cocaine increases firing of striatal cholinergic neurons  and
the release of ACh in the striatum [45–47]. Previous experiments
have suggested that striatal cholinergic neurons also play
important roles in the rewarding effects of cocaine. Indeed,
optogenetic silencing of striatal cholinergic neurons seemed to
attenuate the response of cocaine in a conditioned-place
preference (CPP) paradigm. Because these experiments did not
separate the contribution of ACh from that of glutamate and to
determine if there was a causal link between ACh release and
expression of cocaine-induced CPP, we performed CPP experiments
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with VAChTD2-Cre-flox/floxmice. We were unable to obtain reliable
CPPwitheithergenotype at 5 mg/kgofcocaine (unpublished data).
In contrast, at 20 mg/kg we detected robust CPP in both genotypes
(Figure 6a, repeated measures ANOVAs show no effect of geno-
type, F(1,10)=0.443, p=0.521, significant effect of treatment,
F(1,10)=86.033, p,0.001, and no Genotype 6Treatment interac-
tion, F(1,10)=0.0118, p=0.916). In these experiments we used an
extended protocol  with consecutive injections of cocaine in
alternate days. We repeated the short protocol used before in the
optogenetic experiments  with only one injection of cocaine
(20 mg/kg), but we were unable to detect place preference in
control or VAChTD2-Cre-flox/floxmice (unpublished data). In
addition, neither extinction of CPP nor relapse, measured as a
reinstatement of CPP by a priming injection of cocaine after
extinction, were altered in mice without striatal VAChT (Figure 6b,
repeated measures ANOVAs show no effect of genotype,
F(1,7)=0.00057, p=0.982, significant effect of treatment, F(1,7)=
7.457, p=0.029, and no Genotype 6 Treatment interaction,
F(1,7)=9.6761025, p=0.992). Therefore, there was no difference in
CPP response for the two genotypes.
Behavioural sensitization protocols for cocaine likely reflect
altered synaptic plasticity in response to the drug , which
manifests as an increase in the locomotor effects of cocaine. In a
separate group of mice, we measured behavioural sensitization to
10 mg/kg of cocaine (Figure 7) and found that repeated treatment
with this dose of cocaine seems to cause slightly higher locomotor
activity in VAChTD2-Cre-flox/floxmice, but the relative increase in
behavioural sensitization was not different between genotypes
(Figure 7a,b, repeated measures ANOVAs show a significant effect
of genotype, F(1,16)=4.902, p=0.042, significant effect of treat-
ment, F(1,16)=33.855, p,0.001, and no Genotype 6 Treatment
interaction, F(1,16)=0.496, p=0.491). Thus, elimination of striatal
ACh release caused a small change in the dose-response profile of
cocaine-treated mice in intermediate doses: a slight increase in
activity is observed at 10 mg/kg, whereas a decrease in locomotor
response is observed at 20 mg/kg in mutant mice.
Figure 4. Locomotor activity of VAChTD2-Cre-flox/floxmice. (a) Horizontal locomotor activity in an open-field for VAChTD2-Cre-flox/flox(N=24) and
control mice (N=27). (b) Cumulative 2 h locomotion VAChTD2-Cre-flox/flox(N=24) and control mice (N=27) (c) dark cycle activity of VAChTD2-Cre-flox/flox
(N=16) and control mice (N=15). (d) Total locomotion activity during the first initial hours of the dark-cycle. (e) Habituation in the open-field
measured as cumulative 2 h locomotion for VAChTD2-Cre-flox/floxand control mice in 3 consecutive days. ** p,0.01, *** p,0.001 compared to the first
day. VAChTD2-Cre-flox/floxN=10; control mice N=21.
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Figure 5. Cocaine-mediated locomotor activity in VAChTD2-Cre-flox/floxmice. (a) Mice were injected with 5 mg/kg of cocaine after 20 min in
the open-field and horizontal locomotor activity was measured. (b) Locomotor activity before and after injection of 20 mg/kg of cocaine. As with
5 mg/kg the mice were injected with cocaine after 20 min in the open-field. (c) Total locomotion during the 20 min following cocaine injection.
** p,0.01. Injection of saline did not change locomotor activity for either genotype (unpublished data). For 5 mg/kg N=7 for control and 9 for
VAChTD2-Cre-flox/flox. For 20 mg/kg N=10 for control and 15 for VAChTD2-Cre-flox/floxmice. For 40 mg/kg N=8.
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VAChTD2-Cre-flox/floxMice Have Increased Responses to
The balance between acetylcholine-dopamine is important in a
number of conditions, including PD; therefore we further
investigated dopaminergic function in VAChTD2-Cre-flox/floxmice.
For that, we first determined the concentration of dopamine and
metabolites in the striatum of VAChTD2-Cre-flox/floxmice and
compared these to control mice. In general there were no major
changes in dopamine and metabolites in these mutant mice
(Table 1). However, the ratio between dopamine and DOPAC as
well as dopamine and HVA were significantly changed, showing
that dopamine turnover is decreased by 25% (p,0.001), suggesting
potential relatively minor alterations in dopamine dynamics or
To further assess dopaminergic function, we performed qPCR
analysis for D1R and D2R expression in the striatum. We detected
an increase in the expression of D1R and D2R mRNAs in the
striatum of VAChTD2-Cre-flox/floxmice compared to control mice
(Figure 8a and b, D1R, t(12)=2.756, p,0.05, D2R, t(14)=2.300,
p,0.05). In contrast, D2R mRNA expression in the midbrain was
not altered (Figure 8c). G-protein coupled receptors (GPCRs) can
have agonist-independent effects; hence, altered expression of such
receptors could modulate behaviour even in the absence of
neurotransmitter release. We thus also investigated the expression
of cholinergic receptors. Figure 8d–f indicates that expression of
M1 and M2 muscarinc receptors (mAChR1 and mAChR2) was
unchanged, whereas M4 muscarinic receptors (mAChR4) showed
increased expression (t(12)=3.678, p,0.05). Homozygous mice
expressing D2-BAC-GFP construct present some dopaminergic
phenotypes , however control experiments show that the
heterozygous D2-Cre mice used here do not present any of the
phenotypes associated with selective elimination of striatal
VAChT (Figure S6). Because dopamine receptor expression was
normal in D2-Cre mice, we conclude that these molecular
alterations are due to the loss of ACh release.
To confirm the increased alteration of dopamine receptors in
the striatum of VAChTD2-Cre-flox/floxmice we initially performed
Western blots. Unfortunately, we were unable to obtain a reliable
D1 antibody that showed specific detection of D1R (unpublished
data). However, we obtained a D2 antibody that labelled only one
major band with the correct molecular mass (Figure 9a).
Quantification of immunoblots confirmed increased expression of
D2R (Figure 9a,b, t(14)=23.628, p,0.01). In order to provide an
independent measure of D1R activity and test if D1-mediated
responses would be altered in VAChT-eliminated mice, we used
pharmacological magnetic resonance imaging (phMRI) [51,52].
phMRI is a variant of functional magnetic resonance imaging that
indirectly detects neuronal activity using blood oxygenation level-
dependent (BOLD) MRI signal changes  to detect functional
effects of pharmacological agents in intact systems in vivo with
high temporal and spatial resolution. A 9.4T anatomic MRI of the
mouse brain (Figure 9c) was used to outline regions of interest in
the striatum and cortex. The average difference in BOLD effect
between striatum and cortex (Figure 9d) indicates that there is
increased neuronal activation in the striatum in VAChTD2-Cre-flox/flox
mice following injection of SFK 81297 (3 mg/kg, Figure 9d). The
change in the BOLD response after administration of the selective
D1R agonist SKF 81297 relative to baseline (prior to injection) was
then compared between the two genotypes. Saline administration
prior to SKF 81297 did not alter BOLD signal (unpublished data).
In contrast, injectionof SKF 81297 lead to a slow increase in striatal
BOLD response (area under the curve) in VAChTD2-Cre-flox/flox
mice compared to control mice following injection of the D1R
agonist (Figure 9d and e, p,0.01).
To test if the increased expression/sensitivity of D1R and D2R
has direct behavioural consequences, we investigated the effects of
the selective dopaminergic agonists SKF 81297 (D1R agonist) and
quinpirole (D2R agonist) on locomotor activity. VAChTD2-Cre-flox/flox
mice had significantly higher locomotor responses to two doses of
SKF 81297 (Figure 10a and b, two-factor ANOVAs revealed
significant effect of genotype, F(1,66)=11.654, p,0.01, significant
effect of drug concentration, F(3,66)=34.476, p,0.001, and sig-
nificant Drug Concentration 6 Genotype interaction, F(3,66)=
4.277, p,0.01, Tukey post hoc test showed significant differences
with SKF 81297 doses of 3 mg/kg (p,0.01) and 8 mg/kg
(p,0.001)). Moreover, VAChTD2-Cre-flox/floxmice also showed
enhanced inhibition of locomotion in response to low doses of the
D2R-selective agonist quinpirole (Figure 10c and d, ANOVA
showed significant effect of genotype, F(1,111)=12.543, p,0.001,
significant effect of drug, F(4,111)=42.223, p,0.001, but the
interaction was not significant, F(4,111)=2.052, p=0.092). Analysis
of locomotion in response to the individual doses showed a
Figure 6. CPP response of VAChTD2-Cre-flox/floxmice. (a) In the
conditioning phase (days 2–7) mice received alternating injections of
20 mg/kg of cocaine or vehicle and were immediately confined into
one of the two conditioning chambers for 30 min. The CPP response
was measured on day 8, when the animals were allowed to move freely
in the CPP apparatus and the time spent in each compartment was
measured (N=6). (b) Reinstatement to cocaine was tested after
extinction of CPP by pairing of the cocaine paired chamber with saline
injections. Once the extinction was acquired, a prime of 10 mg/kg of
cocaine was injected and the animals re-exposed to the CPP apparatus.
The time spent in each compartment was measured (N=5). * p,0.05,
reinstatement versus extinction. *** p,0.001, cocaine paired versus
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significant difference for 0.005 and for 0.01 mg/kg quinpirole dose
(p,0.01). Taken together, these data reveal important alterations in
the expression and function of striatal dopamine receptors in
Here we present a series of evidence that delineates the role of
released ACh from those of VGLUT3-dependent glutamate
release from striatal cholinergic interneurons. Our data provide
a novel perspective on the function of striatal cholinergic neurons
suggesting the possibility that they can use distinct neurotransmit-
ters to regulate striatal circuitry. We found that elimination of
VAChT in the striatum, without disruption of VGLUT3, did not
cause overt disruptions or alterations in several behavioural tasks
previously thought to be dependent on striatal ACh release, such
as motor learning, sensorimotor gating, and spontaneous locomo-
tor activity. However, we uncovered a novel form of regulation of
MSNs by cholinergic tone, and found that selective silencing of
striatal ACh release results in an increase in the responses to D1R
and D2R agonists. In contrast to the effects of direct dopamine
receptor agonists, we found that overall these mice do not show
increased locomotor response to cocaine. Similarly, sensitization
and rewarding effects of cocaine did not seem to be dependent on
striatal release of ACh. Thus, our results significantly depart from
previous studies in which the specific contributions of striatal ACh
release (mediated by VAChT) were not separated from those of
glutamate release (mediated by VGLUT3). These data suggest
that VGLUT-3 dependent glutamate release may influence
locomotor activity and responses to cocaine considerably more
than VAChT-dependent ACh release. Our data suggest that
targeted approaches aimed at inhibiting VAChT activity in the
striatum may potentially provide a novel strategy to enhance
dopaminergic signalling, without causing other major behavioural
VAChTD2-cre-flox/floxMice Have Normal Motor
Performance, Sensorimotor Gating, and Motor Learning
Our studies in VAChTD2-cre-flox/flox
elimination of ACh release in the striatum does not seem to play
a major role in motor function and motor learning, at least for
acrobatic motor skills in the rotarod test. This observation is also in
agreement with previous experiments in striatal cholinergic
neuron-ablated mice that presented no deficiency in rotarod
performance . However, we cannot completely exclude more
subtle effects of ACh in fine motor tuning and motor tasks. For
example, the chronic nature of elimination of ACh release in our
experiments may lead to adaptations in motor behaviour. Future
experiments using VAChTD2-Cre-flox/floxmice and more sophisti-
cated motor behavioural tests may be necessary to pinpoint
possible roles for striatal ACh in motor learning and performance.
There are multiple lines of evidence that pharmacological
modulation of cholinergic receptors regulates locomotor activity. It
is known that muscarinic antagonists increase locomotor activity
and M1 and M4 muscarinic receptor KO mice are hyperactive
[54–57]. Moreover, we have recently observed that mice with a
significant decrease in VAChT expression in the whole forebrain
show hyperactivity . The present work provides compelling
evidence for more selective roles of the neurotransmitter ACh in
the striatum, indicating that decreased striatal expression of
VAChT does not cause overt motor consequences. These results
mice indicated that
Figure 7. Behavioural sensitization to cocaine. (a) Repeated cocaine injections (10 mg/kg) promoted a progressive increase of locomotor
sensitization (repeated measures ANOVAs show a significant effect of treatment, F(1,16)=33.855, p,0.001). VAChTD2-Cre-flox/floxmice clearly manifested
an enhancement in the locomotor activity in comparison with their control subjects (repeated measures ANOVAs show a significant effect of
genotype, F(1,16)=4.902, * p,0.05). (b) Cumulative 20 min locomotion after cocaine injection (10 mg/kg) of VAChTD2-Cre-flox/floxmice and controls
(*** p,0.001, day 6 versus day 1). Day 0 is the basal activity of the animals (no cocaine was injected).
Table 1. Catecholamine content in the striatum (ng/100 mg of brain tissue).
NE DA DOPACHVA DOPAC/DA HVA/DA(DOPAC+ +HVA)/DA
fx/fx 20.40061.515 358.600639.300 31.48063.523 58.14065.2050.08860.0010.16460.004 0.25060.006
D2-Cre-fx/fx 27.80061.878*365.400627.570 25.16061.78048.54063.2360.06960.002*0.13460.004*0.20060.004*
HPLC analysis of supernatant samples of striatum from VAChTD2-Cre-flox/floxand control mice (n=5). The samples were analyzed for norepinephrine (NE), dopamine (DA),
and its two metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) by NoAb BioDiscoveries. * p,0.05.
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may be of particular importance, since there have been reports
that in Huntington’s disease VAChT levels are decreased in the
striatum . Our data suggest, however, that this alteration is
unlikely to contribute to gross motor symptoms observed in
Huntington’s disease. Cholinergic neurotransmission in brain
regions other than the striatum may still play a role in control of
Previous attempts to assess the function of cholinergic neurons
in the striatum were performed following the ablation of
cholinergic neurons using immunotoxin-mediated cell targeting.
Injection of toxin targeting transgenic cholinergic neuron in the
accumbens led to an 80% decrease in ChAT-positive neurons
. Elimination of cholinergic neurons in the accumbens by this
means inhibited certain forms of reward-related learning;
however, it also induced hyperactivity and increased sensitivity
to the locomotor and the rewarding effects of cocaine, including
increased sensitivity in the CPP test to low doses of cocaine
[28,29,31]. In contrast, recent experiments using an optogenetic
approach to inactivate or activate cholinergic neurons in the
accumbens found no effects of inactivation of these neurons on
locomotor activity, albeit their silencing prevented the response to
cocaine in a CPP test . Thus, elimination of cholinergic
neurons in the accumbens seemed to increase sensitivity to
cocaine-induced CPP , whereas optogenetics silencing of these
neurons blocked cocaine-induced CPP . The reason for the
different outcome in these two experiments is not entirely clear at
the moment, but could be related to the chronic versus acute
nature of the manipulations. Although in our experiments we have
targeted the whole striatum, rather than only the accumbens, we
did not detect major alterations in cocaine-induced CPP,
suggesting that the above effects obtained with neuronal ablation
or by optogenetics manipulation may be linked not to loss of
cholinergic transmission per se but rather to suppression of
glutamate release from cholinergic neurons.
While an optogenetic approach provides a novel paradigm to
acutely activate or inactivate populations of neurons, it is unlikely
Figure 8. Expression of dopamine and acetylcholine muscarinic receptors in VAChTD2-Cre-flox/floxmice. (a) D1R mRNA expression in
striatum, (b) D2R mRNA expression in striatum, (c) D2R mRNA expression in the midbrain, (d) M1 mRNA expression in striatum, (e) M2 mRNA
expression in striatum, and (f) M4 mRNA expression in striatum. * p,0.05 and ** p,0.01.
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that this method can separate VAChT from VGLUT3-dependent
neurotransmission as selectively as that which can be achieved
using VAChTD2-Cre-flox/floxmice. Interestingly, recent data have
shown that cholinergic neurons in the habenula secrete both ACh
and glutamate (mediated by VGLUT1), and release of either of
these neurotransmitters appears to depend on the frequency of
stimulation . Basal forebrain neurons in culture release both
ACh and glutamate . Importantly, recent work shows that
optogenetics stimulation of striatal cholinergic neurons can evoke
synaptic glutamatergic neurotransmission onto MSNs, with
predominant activity over NMDA receptors . The co-release
of glutamate with dopamine has also been described [60,61],
suggesting that interpretation of the roles of dopaminergic neurons
will also need to take into account glutamate co-release. Therefore,
the co-release of glutamate with classical neurotransmitters may be
a more common mechanism than previously appreciated and may
have a broad impact in circuitry control. However, we cannot
discard the possibility that other neuromodulators released from
cholinergic neurons, such as ATP or peptides, could also play a
role as co-transmitters.
The role of VGLUT3 in striatal function is far from being fully
understood . Interestingly, with respect to striatum-related
behaviour, VGLUT3-null mice show hyperactivity and increased
response to the locomotor effects of cocaine . Therefore, mice
lacking VGLUT3 show a phenotype that is remarkably similar to
that of mice in which cholinergic neurons in the accumbens were
targeted by an immunotoxin [29,30]. Experiments in VGLUT3-
null mice suggested that the absence of VGLUT3 causes a
decrease in striatal cholinergic tone. VGLUT3 is used by the
striatal vesicles to facilitate VAChT-mediated ACh storage in
synaptic vesicles [25,62]. However, measurements of ACh release
in VGLUT3-null mice have indicated only a modest reduction, by
30% to 40% , compared to almost 100% inhibition in
VAChTD2-Cre-flox/floxmice. It is unlikely that 40% reduction in
ACh release observed in VGLUT3-null mice can be responsible
for the hyperactive phenotype. Indeed, independent mouse lines
with a 50% decrease in VAChT expression, and concomitant
reduction of ACh release [16,36,63], did not present increased
locomotor activity in the open field [16,35]. We conclude that the
locomotor phenotypes observed previously in striatal cholinergic
neuron-ablated mice [29,31] and in VGLUT3-null mice  are
either a consequence of the disruption of VGLUT3-mediated
neurotransmission or the combination of reducing both glutama-
tergic and cholinergic activity simultaneously from these neurons.
Future experiments using VAChTD2-Cre-flox/floxmice, VGLUT3
floxed mice, and double knockouts will be necessary to provide an
assessment of independent effects of VGLUT3-mediated neuro-
transmission in the striatum.
Although we have focused on striatal-related behaviours, the
extent by which alterations in VAChT expression in other brain
regions in VAChTD2-cre-flox/floxmice may contribute to these
phenotypes should also be taken into account. We did not detect
Figure 9. Increased dopaminereceptorexpressionandactivity in VAChTD2-Cre-flox/floxmice. (a) StriatalD2R proteinlevelsinVAChTD2-Cre-flox/flox
mice and controls (n=8). * p,0.05. (b) D2R representative immunoblot blot. (c) Axial MRI FLASH image (500 mm thick) through the striatum (outlined
region 1) and cerebral cortex (outlined region 2). (d) Average BOLD signal change in the striatum relative to cerebral cortex prior to and following
injection of SFK 81297 at time zero (black arrow). Signal response for VAChTD2-Cre-flox/floxmice (N=5) is shown with black triangles, and response for
control mice (N=4) is shown with open squares. Error bars represent the standard error of the mean. (e) Area under the curve for VAChTD2-Cre-flox/flox
mice (N=5) is shown with black triangles, and response for control mice (N=4) is shown with open squares. The difference between genotypes was
statistically significant (p,0.01).
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Cre-expression in cholinergic neurons in the penduculopontine
area, for example (Figure S2), which harbours groups of
cholinergic neurons that project to the midbrain and thalamus
and could influence striatal function. However, we cannot
completely exclude the possibility that cholinergic neurons in
other brain regions would not be targeted in our mouse line. At the
same time, as the phenotypes described here seem to be mainly
striatal specific and cholinergic interneurons provide the almost
exclusive source of cholinergic tone in the striatum, it is unlikely
that other groups of cholinergic neurons would have contributed
to the observed behaviours.
Elimination of cholinergic neurotransmission in the striatum did
not cause hyperlocomotion, however the responses to direct
activation of dopamine receptors were substantially increased.
Both behavioural and phMRI analysis indicated an increased
response to D1R agonist. Western blot analysis also showed
selective increase of D2R expression in the striatum. Moreover, in
addition to the increased D2R levels in the striatum, which likely
reflect a combination of pre- and post-synaptic receptors, we also
uncovered increased D2-like receptor pre-synaptic activity,
revealed by the increased sensitivity of VAChTD2-Cre-flox/floxmice
to low doses of quinpirole. Certainly, we cannot rule out that
changes at the level of receptors play a more complex role in
regulating locomotor activity in VAChTD2-Cre-flox/flox
Indeed, GPCRs may have agonist-independent activity [64,65].
The locomotor effects of cocaine seem to depend mainly on
inhibition of the dopamine transporter . However, acetylcho-
line can affect release of dopamine via distinct nicotinic receptors
, as well as regulate both dopamine release and activity of
MSNs, via distinct muscarinic receptors [56,57,67]. The fact that
both D1R and D2R had increased expression in the striatum
would suggest that VAChTD2-Cre-flox/floxmice should be more
responsive to dopamine and might present increased spontaneous
locomotor activity or cocaine-induced locomotion or CPP.
However, this was not the case. It is likely that cell-autonomous
compensatory mechanisms related to disrupted cholinergic
function significantly altered striatal circuitry, preventing such a
simple relationship. For example, because M4 muscarinic
receptors seem to specifically regulate D1R-mediated signalling
[56,57,68], it is possible that the increased expression of M4
receptors we detected in the striatum could counterbalance D1R-
mediated responses in vivo, leading to unaltered locomotor
activity. Moreover, because D2-like pre-synaptic receptors may
be more active in VAChTD2-Cre-flox/floxmice, elimination of ACh
release in the striatum may also affect pre-synaptic control of
dopamine release. The slightly decreased turnover of dopamine in
mice without striatal VAChT supports the notion of direct
consequences of reduced cholinergic tone at the level of
VAChTD2-Cre-flox/floxmice indicates that control of locomotor
function and response to cocaine mediated by dopamine might
become more complex in the absence of cholinergic tone. Future
Figure 10. Effect of D1R or D2R agonists on locomotor activity in VAChTD2-Cre-flox/floxmice. (a) Effect of injection of SFK 81297 (3 mg/kg)
20 min after the mice were introduced to the open field, (b) dose-response for SKF 81297, (c) effect of quinpirole (0.01 mg/kg) as in (a), and (d) dose
response for quinpirole. * p,0.05, ** p,0.01, and *** p,0.001. N=10 and 13 for saline, SKF 81297 N=7 and 5 for 0.5 mg/kg, N=9 and 4 for 3 mg/kg,
and N=7 and 9 for 8 mg/kg. For quinpirole N=7 and 13 for 0.005 mg/kg, N=17 and 15 for 0.01 mg/kg, N=11 and 12 for 0.1 mg/kg, and N=11 and
13 for 6 mg/kg for control and VAChTD2-Cre-flox/floxmice, respectively.
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experiments will be needed to evaluate direct consequences of
elimination of either acetylcholine or glutamate neurotransmission
originating from striatal cholinergic neurons on dopamine
The present data provide direct and indirect evidence that
striatal cholinergic neurons can use two different neurotransmit-
ters to regulate striatal function. Hence, re-evaluation of
previously attributed functions of striatal cholinergic tone is
warranted. The data indicate that VGLUT3-mediated glutama-
tergic neurotransmission originating from cholinergic neurons
may have greater influence on striatal function than previously
envisioned. The behavioural consequences of selective elimina-
tion of VAChT, and thus cholinergic transmission, in the
striatum are remarkably minimal, at least for the locomotion
control by the striatal complex. One intriguing phenotype
uncovered in mutant mice is an increase in dopamine receptors’
expression and function without major alterations in cocaine-
induced behaviours. Our experiments provide evidence that
targeting VAChT in the striatum can up-regulate dopamine
receptors and thus could be used in conditions of dopamine
deficiency and abnormally increased cholinergic activity, as found
in individuals with PD.
Materials and Methods
The isolation of a VAChT genomic clone has been described
previously . The genomic clone was used to construct a gene-
targeting vector in which we added LoxP sequences flanking the
VAChT open reading frame and a TK-Neo cassette. Generation
of VAChTflox/floxmice is described elsewhere , and the
construct is shown in Figure S2. Briefly, after removal of the TK-
Neo cassette, one LoxP sequence was present 260 bp upstream
from the VAChT translational initiation codon, and a second
LoxP sequence was located approximately 1.5 kb downstream
from the VAChT stop codon and within the second ChAT intron.
Note that this vector is distinct from that previously used for
generation of VAChT KD mice .
D2-Cre mice (Drd2, Line ER44) were obtained from the
GENSAT project via the mutant mouse regional resource centers.
VAChTD2-Cre-flox/floxmice were generated by crossing VAChTflox/flox
to obtain VAChTD2-Cre-flox/floxmice. Because these mice were
apparently normal and fertile, we bred VAChTD2-Cre-flox/floxmice
and VAChTflox/floxto obtain all the mice used in the present
study. These mice were backcrossed to C57BL/6J mice for five
generations. Unless otherwise stated, all control mice used were
VAChTflox/floxlittermate mice without the Cre transgene.
After the completion of this work we were made aware that the
BAC used to generate D2-Cre mice carried an extra gene, ttc2,
and a recent report suggests that homozygous D2-GFP mice,
generated using the same BAC construct, are hyperactive and
show a number of dopamine-related phenotypes . However, as
these authors point out, their experiments cannot discern if the
phenotypes uncovered are due to the BAC positioning insertion or
to the extra copy of ttc2. We confirmed that the D2-Cre indeed
have increased expression of the TTC2 mRNA (unpublished
data). However, heterozygous D2-Cre mice showed no locomotor
phenotype. Moreover, these mice showed normal levels of
D1R, D2R, and M4-muscarinic receptors (Figure S6). Hence,
neither the phenotypes nor the molecular changes observed in
VAChTD2-Cre-flox/floxmice are due to the BAC transgene.
Rosa26-YFP mice (B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J,
stock number 006148) were obtained from Jackson Laboratories.
Animals were housed in groups of three to four mice per cage
without environment enrichment in a temperature-controlled
room with 12-h light–12-h dark cycles, and food and water were
provided ad libitum. Mouse stocks were SPF, however experi-
mental subjects were kept in a conventional mouse facility.
All studies were conducted in accordance with the NIH and the
Canadian Council of Animal Care (CCAC) guidelines for the care
and use of animals with approved animal protocol from the
Institutional Animal Care and Use Committees at the University
of Western Ontario (protocol number 2008–089). Only male mice
were used for the behavioural studies, and they were at least 12
weeks old. Mice were randomly assigned to distinct experimental
groups. Only mice used for evaluation of spontaneous locomotor
behaviour were used in other tasks.
Immunofluorescence, qPCR, and Western Blot
For the immunofluorescence experiments we followed a
protocol previously described [35,69]. For mRNA analyses tissue
samples were frozen in a mixture of dry ice/ethanol and kept at
280uC until used as described . Immunoblotting was
performed as described elsewhere [36,71,72].
Measurements of [3H]ACh Release from Brain Slices
Slices were obtained from the striatum and hippocampus of
control and test mice, labelled with [3H]methyl-choline, and the
release of labelled ACh was determined essentially as described
 except that 33 mM KCl was used as a depolarizing stimuli.
Preparation of Synaptosomes and Glutamate Release
Striatal synaptosomes were prepared by the method of [74,75]
as previously described . Glutamate release was followed
continuously using a fluorimetric method  exactly as
previously described .
Measurements of Locomotor Activity
All behavioural experiments were performed between 9 a.m.
and 4 p.m. in the light cycle, essentially as previously described
[16,35] except the spontaneous activity of the first hours of the
dark cycle was done from 7 p.m. to 10 p.m.
The dissected brain tissues were homogenized in 0.2 M
perchloric acid with 100 mM EDTA-2Na. Samples were spun in
a microcentrifuge at 12,000 rpm for 15 min at 4uC. Samples of
the supernatant were then analyzed for norepinephrine (NE),
dopamine (DA), and its two metabolites, 3,4-dihydroxyphenyla-
cetic acid (DOPAC) and homovanillic acid (HVA) by NoAb
BioDiscoveries (Mississauga, ON).
The HPLC used was an Eicom EP-700 with electrochemical
detection (Eicom ECD-700). To elute catecholamines from the
reverse phase column (3.06100 mm SC-3ODS column, Eicom), a
mobile phase consisting of 0.1 M citric acetate buffer pH 3.5 with
5 mg/ml EDTA-2Na, 220 mg/L sodium octane sulfonate, and
22% methanol was used.
Acoustic Startle Measurements
These experiments have been described in . Briefly, animals
were acclimatized 3–5 times to the startle boxes (Med Associates).
Habituation of startle was measured using 30 startle pulses (20 ms,
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white noise, 115 dB on a 65 bd white noise background) with an
inter-trial interval of 20 s. Subsequently, prepulse inhibition was
measured by displaying 50 startle stimuli with either no prepulse
(pulse alone), a 75 dB (4 ms white noise) prepulse preceding the
pulse by either 30 ms or 100 ms, or a 85 db prepulse (30 ms or
100 ms interval). Each of the five trial types were displayed 10
times in a pseudorandomized order. PPI is expressed as the
average startle response to the respective prepulse trials in relation
to the pulse alone trials.
Grip-Force and Wire-Hang
A Grip Strength Meter from Columbus Instruments (Columbus,
OH) was used to measure forelimb grip strength essentially as
described . For the wire-hang test each mouse was placed on a
metal wire-grid, which was slowly inverted and suspended 40 cm
above a piece of foam as previously described . The time it
took for each mouse to fall from the cage top was recorded with a
60 s cut-off.
The rotarod task followed a previously described protocol .
The CPP protocol was modified from . Briefly, CPP was
performed in a three chamber apparatus containing two large
compartments with differences in visual and tactile cues, separated
by a neutral area. In day 1 (habituation), mice were placed in the
central compartment and allowed free access to the entire
apparatus for 30 min. The time spent in each compartment was
recorded. On days 2–7 (the conditioning phase), mice received
alternating injections of cocaine or vehicle and were immediately
confined into one of the two large compartments for 30 min. A
combination of unbiased and biased allocation was used. On day 8
(test day) mice were once again allowed free access to all three
compartments for 30 min, and the time spent in each compart-
ment was recorded. For the CPP extinction and reinstatement, the
protocol previously described  was followed.
Behavioural sensitization was performed as described .
Object Recognition Memory
The general procedure was previously described, but for
analysis we used Anymaze .
Pharmacological Magnetic Resonance Imaging (phMRI)
Mice (VAChTD2-cre/flox/flox, N=5; control, N=4) were anes-
thetized with 4% isofluorane and maintained at 1.5% isoflurane
during the MRI scanning. Two intraperitoneal (I.P.) catheters (26
gauge, Abbotcath) were used for injection of saline and SFK. The
catheters were secured in place with subcutaneous sutures.
Catheters were connected to polyethelyne tubing (PE50, VWR,
Canada) and to syringes containing saline and SFK for remote
injection during imaging. Mice were placed in a custom built
frame designed to secure the skull and minimize respiration
induced movement during image acquisition. Mice were imaged
on a 9.4 Tesla small animal MRI scanner (Agilent, Palo Alto, CA)
equipped with a two-channel surface coil (diameter =2 cm). A fast
low angle shot (FLASH) pulse sequence was used to acquire
anatomical images (field of view
=1286128, repetition time =50 ms, echo time =11 ms, flip
angle =11u, and 10 averages). Respiratory gated lower resolution
FLASH images were also acquired for pharmacological imaging
=19.2619.2 mm2, matrix
(field of view =19.2619.2 mm2, data matrix =64664, repetition
time =15 ms, echo time =7 ms, flip angle =11u, and 1 average)
to measure blood oxygen level–dependent (BOLD) signal changes.
Seven contiguous axial slices (500 mm thick) covered the brain.
Each animal received two injections: first, an injection of 0.5 ml
physiological saline (0.9%) administered over a 30 s period
(control), and second, SFK 81297 (3 mg/kg), diluted in 0.5 ml
physiological saline, also administered over a 30 s period (drug).
For the control experiment, images were acquired for 8 min prior
to saline injection and then for 20–50 min following injection. For
the drug experiment, images were acquired for 8 min prior to
drug injection and then for 80–180 min after injection. Through-
out the imaging session, body temperature and respiration rate
was monitored every 10 min using the MR-Compatible, Model
1025 monitoring system (Small Animal Instruments Inc., Stony
Brook, NY). Temperature was maintained at 37.5uC using a warm
air blower, and respiration rate ranged from 45–66 (mean 54
BPM). Following imaging, mice were euthanized by cervical
dislocation while still under isoflurane anaesthesia.
To limit the influence of global motion on the functional result,
the signal intensity difference between striatum and cortex was
used to examine the effect of SFK 81297 on the striatum as a
function of time. A single slice transecting the striatum was chosen
for analysis in each animal (Figure 9c). BOLD signal change was
expressed as the percentage change relative to the average baseline
signal (first 50 images) prior to drug injection.
Data are expressed as mean 6 SEM. Sigmastat 3.1 software was
used for statistical analysis. Comparison between two experimental
groups was done by Student’s t test or Mann-Whitney Rank Sum
Test when the data did not follow a normal distribution. When
several experimental groups were analyzed, we used two-way
analysis of variance (ANOVA). For locomotion experiments we
used ANOVA with repeated measures, and when appropriate, a
Tukey post hoc comparison test was used. For pharmacological
MRI, the area under the curve of the signal time course was
compared between VAChTD2-cre/flox/floxmice and control mice
using a Student’s t test.
cholinergic neurons. (a) Sections from different regions of the
central nervous system were immunostained for CHT1 (Red) and
YFP (Green) in D2-Cre;Rosa26-YFP mice. Arrows show localiza-
tion of Cre expression (YFP) in cholinergic neurons (CHT1
staining). Arrowheads show cholinergic neurons that do not
D2-Cre drives the expression of Cre in striatal
identification by genotyping. (a) Cartoon representing the VAChT
alleles before and after recombination. (b) PCR genotyping for
VAChTflox/floxand VAChTD2-Cre-flox/floxmice. Lanes 1 and 2 WT
PCR product, lane 3 flox/flox PCR product, and lane 4
heterozygous mice PCR product. (c) Lane 1 VAChTflox/flox, lane
2 VAChTD2-Cre-flox/flox, lane 3 VAChTD2-Cre-flox/flox, and lane 4
Representation of VAChTD2-Cre-flox/floxallele and
VAChTD2-Cre-flox/floxmice. (a, b, c) Quantification of mRNA
expression for VAChT, ChAT, and CHT1, respectively, in the
Spinal cord. (d, e, f) Quantification of protein levels in the Spinal
Cholinergic parameters in the spinal cord of
Acetylcholine and Striatal-Dependent Behaviours
PLoS Biology | www.plosbiology.org14 November 2011 | Volume 9 | Issue 11 | e1001194
cord for VAChT, ChAT, and CHT1, respectively. Synaptophysin Download full-text
immunoreactivity was used to correct for protein loading between
experiments. (g) Representative Western blot of VAChT, synapto-
physin, ChAT, CHT1, and actin ** p,0.01 and *** p,0.001.
mice. (a) Grip-force analysis of VAChTflox/floxand VAChTD2-Cre-flox/flox
mice. (b) Time spent hanging upside-down from a grid, to measure
fatigue for VAChTflox/floxmice and VAChTD2-Cre-flox/floxmice
(cut-off time 60 s). (c) Motor learning and acrobatic motor skills of
VAChTD2-Cre-flox/floxmice determined using the rotarod (flox/flox,
N=14; D2-Cre-flox/flox, N=17). (d) Object recognition memory
in VAChTflox/floxand VAChTD2-Cre-flox/floxmice. * p,0.05.
Motor function is not altered in VAChTD2-Cre-flox/flox
mice. (a) Short-term habituation of acoustic startle responses to 30
acoustic startle stimuli of 115 db white noise delivered every 20 s.
(b) Prepulse inhibition of acoustic startle responses with different
prepulse intensities and prepulse-pulse intervals as indicated.
Sensorimotorgatingisnot altered inVAChTD2-Cre-flox/flox
zygous D2-Cre mice. (a) Horizontal activity as described in
Biochemical and behavioural parameters of hetero-
Figure 4. (b) Total ambulance during 2 h. (c) Rearing during 2 h.
(d) D1R mRNA, (e) D2R mRNA, an (f) mAChR-4 mRNA levels.
induced recombination and CHT1.
Percentage of co-localization between cell with Cre-
We thank Jue Fan for animal husbandry and Miranda Bellyou for
assistance with the MRI data collection; Dr. R. Jane Rylett and Dr. Arthur
Brown (University of Western Ontario) for reagents; and Dr. Hymie
Anisman (Carlton University, Ottawa, Canada), Dr. Stephen Ferguson,
and Dr. John F. MacDonald (University of Western Ontario) for comments
on earlier versions of this manuscript.
The author(s) have made the following declarations about their
contributions: Conceived and designed the experiments: RB VFP MAMP
MSG XDeJ SS RRG MGC. Performed the experiments: MSG XDeJ SR
IAS AXL SS. Analyzed the data: MSG XDeJ IAS AXL SS RSM RB VFP
MAMP. Contributed reagents/materials/analysis tools: MGC. Wrote the
paper: VFP MAMP.
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