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Taar1-mediated modulation of presynaptic dopaminergic
neurotransmission: Role of D2 dopamine autoreceptors
D. Leo
a
, L. Mus
a
, S. Espinoza
a
, M.C. Hoener
b
, T.D. Sotnikova
a
, R.R. Gainetdinov
a
,
c
,
d
,
*
a
Department of Neuroscience and Brain Technology, Istituto Italiano di Tecnologia, 16163, Genova, Italy
b
Neuroscience Research, Pharmaceuticals Division, F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland
c
Skolkovo Institute of Science and Technology, Skolkovo, 143025, Moscow Region, Russia
d
Faculty of Biology and Soil Science, St. Petersburg State University, 199034, St. Petersburg, Russia
article info
Article history:
Received 7 January 2014
Received in revised form
12 February 2014
Accepted 12 February 2014
Keywords:
Trace amine-associate receptor 1 (TAAR1)
Dopamine (DA)
Fast scan cyclic voltammetry (FSCV)
Dopamine receptor 2 (D2R)
Schizophrenia
Neuropsychiatric disorders
abstract
Trace Amine-Associated Receptor 1 (TAAR1) is a G protein-coupled receptor (GPCR) expressed in several
mammalian brain areas and activated by “trace amines”(TAs). TAs role is unknown; however, discovery
of their receptors provided an opportunity to investigate their functions. In vivo evidence has indicated
an inhibitory influence of TAAR1 on dopamine (DA) neurotransmission, presumably via modulation of
dopamine transporter (DAT) or interaction with the D2 DA receptor and/or activation of inwardly
rectifying K
þ
channels. To elucidate the mechanisms of TAAR1-dependent modulation, we used TAAR1
knockout mice (TAAR1-KO), a TAAR1 agonist (RO5166017) and a TAAR1 antagonist (EPPTB) in a set of
neurochemical experiments. Analysis of the tissue content of TAAR1-KO revealed increased level of the
DA metabolite homovanillic acid (HVA), and in vivo microdialysis showed increased extracellular DA in
the nucleus accumbens (NAcc) of TAAR1-KO. In fast scan cyclic voltammetry (FSCV) experiments, the
evoked DA release was higher in the TAAR1-KO NAcc. Furthermore, the agonist RO5166017 induced a
decrease in the DA release in wild-type that could be prevented by the application of the TAAR1
antagonist EPPTB. No alterations in DA clearance, which are mediated by the DAT, were observed. To
evaluate the interaction between TAAR1 and D2 autoreceptors, we tested the autoreceptor-mediated
dynamics. Only in wild type mice, the TAAR1 agonist was able to potentiate quinpirole-induced inhib-
itory effect on DA release. Furthermore, the short-term plasticity of DA release following paired pulses
was decreased in TAAR1-KO, indicating less autoinhibition of D2 autoreceptors. These observations
suggest a close interaction between TAAR1 and the D2 autoreceptor regulation.
Ó2014 Elsevier Ltd. All rights reserved.
1. Introduction
Octopamine (OCT), tyramine (TYR) and
b
-phenethylamine (
b
-
PEA), as well as several other non-catechol amines, are metabolites
of aromatic amino acids and are known as trace amines (TAs). TAs
are a family of endogenous compounds with strong structural
similarity to the classical monoamine neurotransmitters and are
present in mammalian tissues at low (nanomolar) concentrations
(Berry, 2004; Grandy, 2007; Lindemann and Hoener, 2005). The
endogenous levels of these compounds are at least two orders of
magnitude below those of classical monoamine neurotransmitters
such as dopamine (DA), noradrenaline (NE) and serotonin (5HT).
TAs are found in many species; in invertebrates, tyramine and
octopamine are well-characterized neurotransmitters that modu-
late movement, feeding, metabolism, muscular tone and other
functions (Axelrod and Saavedra, 1977; Cooper and Venton, 2009).
Trace amines are also produced in bacteria, fungi, and plant cells
and can be found in some food products, most notably in chocolate,
cheese and red wine (Branchek and Blackburn, 2003). Despite be-
ing known for more than a century, the role played by TAs in
mammalian, and particularly human, physiology is still enigmatic.
However, it has been noted that levels of TAs are altered in a variety
of human disorders ranging from schizophrenia, Parkinson’s dis-
ease, attention deficit hyperactivity disorder (ADHD), and
Abbreviations: TAAR1, Trace Amine-Associated Receptor 1; DA, dopamine; DAT,
dopamine transporter; EPPTB, (N-(3-ethoxy-phenyl)-4-pyrrolidin-1-yl-3-
trifluoromethyl-benzamide; HVA, homovanillic acid; FSCV, fast scan cyclic vol-
tammetry; NAcc, nucleus accumbens; DStr, dorsal striatum; D2, dopamine receptor
2.
*Corresponding author. Department of Neuroscience and Brain Technology,
Istituto Italiano di Tecnologia, Genova 16163, Italy. Tel.: þ39 010 71781516.
E-mail addresses: damiana.leo@iit.it (D. Leo), liudmila.mus@iit.it (L. Mus),
stefano.espinoza@iit.it (S. Espinoza), marius.hoener@roche.com (M.C. Hoener),
tatiana.sotnikova@iit.it (T.D. Sotnikova), raul.gainetdinov@iit.it (R.R. Gainetdinov).
Contents lists available at ScienceDirect
Neuropharmacology
journal homepage: www.elsevier.com/locate/neuropharm
http://dx.doi.org/10.1016/j.neuropharm.2014.02.007
0028-3908/Ó2014 Elsevier Ltd. All rights reserved.
Neuropharmacology 81 (2014) 283e291
Tourette’s syndrome to migraines and drug addiction (Boulton,
1980; Sandler et al., 1980). For decades, TAs were considered to
be “false neurotransmitters”that were able to modulate the
signaling of monoamines by displacing them from storage vesicles
and/or by acting on the plasma membrane transporters in an
amphetamine-like manner (Berry, 2004; Parker and Cubeddu,
1988). Interestingly, the rate of synthesis of TAs was found to be
comparable with that of classic monoamines, suggesting that the
low levels of TAs in brain tissue are most likely determined by the
extremely fast rate of metabolism and/or the inability of TAs to be
stored in vesicles as classical neurotransmitters (Grandy, 2007;
Sotnikova et al., 2009).
However, in 2001, a family of novel mammalian G protein-
coupled receptors (GPCRs) were characterized with some mem-
bers of this family showing a high affinity for TAs (Borowsky et al.,
2001). This family of newly discovered receptors was later re-
named the Trace Amine-Associated Receptors (TAARs) family
(Maguire et al., 2009; Lindemann et al., 2005). The TAAR family
includes 6 functional members in humans (TAAR1-9 including 3
members encoded by pseudo-genes) and even more receptors are
found in rodents (Borowsky et al., 2001; Bunzow et al., 2001;
Lindemann and Hoener, 2005). Interestingly, in the TAAR family
only TAAR1 and TAAR4 possess any demonstrable TA responsive-
ness. TA binding to TAAR1 engages G
a
s
-type G proteins that activate
adenylyl cyclases (Berry, 2004). TAAR1 is the best characterized
TAAR member and is found in some areas of the central nervous
system and in certain peripheral tissues (Revel et al., 2013). This
distribution, which includes components of the limbic system, such
as the amygdala, and areas rich in monoaminergic cell bodies, such
as the dorsal raphe nucleus and the ventral tegmental area (VTA)
(Lindemann et al., 2008), makes TAAR1 a promising target for
pharmaceutical treatment of monoamine-related disorders (Revel
et al., 2012a, 2013). Because TAs affect multiple targets including
TAAR1, TAAR4, and DA transporter (DAT), adrenergic and serotonin
receptors, their use in the identification of specific functions of
TAAR1 are limited. Only the generation of TAAR1-deficent and
-overexpressing mice (TAAR1-KO and TAAR1-OE mice) (Lindemann
et al., 2008; Wolinsky et al., 2007; Revel et al., 2012b), the devel-
opment of selective TAAR1 agonists, such as RO5166017 (Revel
et al., 2011), and antagonists, such as ((N-(3-Ethoxy-phenyl)-4-
pyrrolidin-1-yl-3-trifluoromethyl-benzamide EPPTB) (Bradaia
et al., 2009), provided an opportunity to evaluate the specific
roles and mechanisms mediated by TAAR1. TAAR1-KO mice appear
to be similar to control animals at basal state, but they show
enhanced hyperlocomotion and exaggerated striatal release of DA,
NE, and 5-HT when challenged with
D
-amphetamine. Recent
studies on TAAR1-KO mice have demonstrated that TAAR1 is able to
negatively modulate monoaminergic neurotransmission
(Lindemann et al., 2008; Wolinsky et al., 2007). For example, the
genetic ablation of TAAR1 induces an increase in the spontaneous
firing rate of DA neurons (Lindemann et al., 2008), and similar ef-
fects are mediated via application of the selective TAAR1 antagonist
EPPTB in control animals (Bradaia et al., 2009), corroborating the
idea that TAAR1 normally exerts an inhibitory effect on DA neurons.
Although the underlying TAAR1 signaling mechanism remained
unclear, Bradaia et al. clearly showed that TAAR1 activates inwardly
rectifying K
þ
channels (Bradaia et al., 2009). They also demon-
strated that both the acute application of EPPTB and the constitu-
tive genetic lack of TAAR1 increase the potency of DA at D2
receptors in DA neurons (Bradaia et al., 2009). Studies in vitro and
in vivo offered further indication of a physical and functional
interaction between TAAR1 and D2 receptors (Espinoza et al., 2011),
whereas others have suggested that TAAR1 may directly alter DAT
function (Miller et al., 2005). To complement the information about
TAAR1 function, a line of transgenic mice that overexpresses TAAR1
in the brain has been recently generated (Revel et al., 2012b). This
model is hyposensitive to amphetamine and it shows constitutive
hyperactivity of monoaminergic nuclei (Revel et al., 2012b). Overall,
the growing body of evidence suggests a modulatory role of TAAR1
on monoaminergic activity (Reese et al., 2014; Cichero et al., 2013),
particularly on presynaptic function. To determine whether dele-
tion of the Taar1 gene or application of TAAR1 ligands perturbs the
functional presynaptic activity of DA neurons at the level of axon
terminals, we investigated extracellular DA dynamics using fast
scan cyclic voltammetry (FSCV) and in vivo microdialysis tech-
niques in the dorsal striatum (DStr) and nucleus accumbens (NAcc)
of wild type (WT) and TAAR1-KO mice. Furthermore, we applied
FSCV to evaluate the evoked DA release and clearance rates in these
brain regions of WT and TAAR1-KO mice in the presence of the
selective TAAR1 agonist (RO5166017) and a TAAR1 antagonist ((N-
(3-Ethoxy-phenyl)-4-pyrrolidin-1-yl-3-trifluoromethyl-benza-
mide EPPTB).
We found that DA release evoked by a single stimulus was
higher in NAcc, and the basal extracellular level of DA was signifi-
cantly higher in this brain region in TAAR1-KO mice. The TAAR1
agonist RO5166017 decreases DA release in WT mice but not in
TAAR1-KO animals, and application of the TAAR1 antagonist EPPTB
prevented the reduction in the evoked DA release induced by the
TAAR1 agonist in WT animals. We further gained functional evi-
dence suggesting that these presynaptic effects could be mediated
by an interaction between TAAR1 and D2 DA autoreceptors.
2. Materials and methods
2.1. Animals
All experiments were conducted in compliance with the Italian Ministry of
Health (DL 116/92; DL 111/94-B) and European Community (86/609/EEC) directives
regulating animal research. All efforts were made to minimize animal suffering, to
reduce the number of animals used, and to utilize alternatives to in vivo techniques,
if available. Animals were housed under a 12 h light/12 h dark cycle with ad libitum
access to food and water.TAAR1-KO mice of mixed backgrounds (C57BL/6J 129Sv/J
backgrounds) were generated as described previously (Lindemann et al., 2008;
Wolinsky et al., 2007; Espinoza et al., 2011). TAAR1-KO and WT littermates were
obtained from heterozygous matings. Genotyping was performed by PCR. Mice of
both sexes of at least 2.5 months of age were used in FSCV experiments. Three-
month-old males were used for microdialysis studies.
2.2. HPLC measurements of the tissue content of monoamines and their metabolites
Striatal tissue (including both DStr and NAcc) was dissected from WT and
TAAR1-KO mice and homogenized in 40 volumes of 0.1 M HClO
4
. Following
centrifugation and filtration, the samples were analyzed by HPLC as described
below. The protocol for sample preparation for HPLC determination of tissue
monoamines and their metabolites was performed as previously described (Jones
et al., 1998; Gainetdinov et al., 20 03).
2.3. In vivo microdialysis
2.3.1. Surgery
In vivo brain microdialysis was performed in the right dorsal striatum or in the
right NAcc of freely moving mice (Budygin et al., 2004; Jones et al., 1998; Carboni
et al., 2001) using 2 mm (for striatum) or 1 mm (for NAcc) concentric micro-
dialysis probes (membrane length cut off 6000 Da; CMA-11, CMA/Microdialysis,
Solna, Sweden). Stereotaxic coordinates for the position of the probes were chosen
according to the atlas of Franklin and Paxinos (1997) and are relative to the bregma:
AP 0.0; L 2.5; DV 4.4 for the striatum and AP þ1.3; L 0.9; DV 5 for the NAcc. Prior
to fixation in the stereotaxic apparatus, the animals were anesthetized with an
oxygen/isoflurane mixture. The probes were implanted in the brain vertically
through a small drilled aperture in the scull and fixed with dental cement. During
implantation into the brain and for 1 h afterward, the dialysis probes were perfused
with artificial cerebrospinal fluid (aCSF) (NaCl 147 mM, KCl 2.7 mM, CaCl
2
1.2 m M,
MgCl
2
0.85 mM; CMA Microdialysis). 1 h after the operation, the animals were
returned to their home cages.
2.3.2. Sample collecting procedure
Approximately 24 h after surgery, the dialysis probes were connected to a sy-
ringe pump and perfused with the aCSF at 1.0
m
l/min for 60 min (equilibration
period). To reliably determine the basal extracellular DA levels in the DStr vs. the
NAcc of freely moving mice, a quantitative “low perfusion”rate microdialysis
D. Leo et al. / Neuropharmacology 81 (2014) 283e291284
experiment was conducted (Gainetdinov et al., 2003). The perfusate was collected at
a perfusion rate of 0.1
m
l/min every 90 min over a 6 h period into collection tubes
containing 2
m
l of 1 M perchloric acid.
2.3.3. Analytical procedure
Measurements of DA, 5-HT and metabolites in tissue samples and DA in
microdialysis samples were performed by HPLC with electrochemical detection
(ALEXYS LC-EC system, Antec Leyden BV, Netherlands) with a 0.7 mm glass carbon
electrode (Antec; VT-03). The system was equipped with a reverse-phase column
(3
m
m particles, ALB-215 C18, 1 150 mm, Antec) at a flow rate of 200
m
l/min. The
mobile phase contained 50 mM H
3
PO
4
, 50 mM citric acid, 8 mM KCl, 0.1 mM EDTA,
400 mg/l octanesulfonic acid sodium salt and 10% (vol/vol) methanol, pH 3.9. The
sensitivity of this method permitted the detection of w3 fmol DA. Dialyzate samples
(11
m
l) were injected into HPLC without any additional purification.
2.4. Fast scan cyclic voltammetry (FSCV)
Briefly, mice were anesthetized with halothane and decapitated. The brain was
sectioned in cold carboxygenated artificial cerebrospinal fluid (aCSF) (126 mM NaCl,
2.5 mM KCl, 1.2 mM NaH
2
PO
4
, 25 mM NaHCO
3
, 2.4 mM CaCl
2
,11mM
D
-glucose,
1.2 mM MgCl
2
) on a VT1000S vibrating microtome (Leica Microsystems, Nussloch,
Germany) at a thickness of 300
m
m. Coronal slices containing the dorsal striatum and
nucleus accumbens were allowed to recover for at least 1 h at room temperature in
carboxygenated aCSF. For recordings, slices were superfused with 32
C carboxy-
genated aCSF at a flow rate of 1 ml/min. Experimental recordings started 20 min
after transfer to the slice chamber.
Carbon fiber electrodes (7
m
m, Goodfellow, Huntingdon, England) were made as
previously described (Kawagoe et al., 1993; Kuhr and Wightman, 1986). They were
trimmed to obtain a basal current between 140 and 180 nA. The electrodes were
inserted w100
m
m into the dorsal striatal brain slice. The potential of the working
electrode was held at 0.4 V vs. the Ag/AgCl control between scans and was ramped
to þ1.3 V at 300 V/s and back to 0.4 V every 100 ms via an EVA8 amplifier (HEKA
Elektronik, Germany). Axonal DA release in the striatum was evoked using a twisted
bipolar stimulating electrode (Plastics One, Roanoke, VA). Stimulations were deliv-
ered every 2 min by a single electrical pulse (1 ms, monophasic single stimuli). The
stimulus was delivered via a stimulus isolator (AM-system, Carlsborg, WA).
Background-subtracted cyclic voltammograms were obtained by subtracting the
current obtained before the stimulation from all recordings. The peak oxidation
current for DA in each voltammogram was converted into a measure of the DA
concentration by post-calibration of the electrode using 1
m
M DA (SigmaeAldrich, St.
Louis, MO, USA). Data were normalized to the first 5 recordings (10 min) of their
respective control period and graphically plotted against time (means SEM).
2.4.1. FSCV kinetic analysis
TarHeel CV (ESA Biosciences, Inc, Chelmsford, USA) was used for all data analysis.
Computations were based on user defined positions on current traces for baseline
(Pre-Stim cursor), peak (Peak cursor) and return to baseline (Post-Stim cursor) po-
sitions. Tau and half-life values were determined from exponential fit curves based
on Peak cursor and Post-Stim cursor positions using a least squares constrained
exponential fit algorithm (National Instruments, Milan, Italy). Both tau and half-life
are considered to be reliable measures for detecting changes in DA uptake and
accurately represent changes in uptake accordingly with more traditional measures
of the uptake (V
max
and K
m
), as demonstrated by Yorgason et al. (Yorgason et al.,
2011). Particularly, the first-order rate constant (k,or1/
s
) provides a sensitive in-
dex of the efficiency (V
max
/K
m
) of dopamine clearance mediated via the dopamine
transporter at low dopamine concentrations (Chen et al., 2008; Sabeti et al., 2002;
Bass et al., 2010).
2.5. Drugs
RO5166017 and N-(3-Ethoxy-phenyl)-4-pyrrolidin-1-yl-3-trifluoromethyl-ben-
zamide (EPPTB, RO5212773) were generous gifts from F. Hoffmann-La Roche. These
drugs were dissolved in 1% DMSO as described (Revel et al., 2011; Bradaia et al.,
2009). All other drugs were purchased from SigmaeAldrich and were dissolved in
saline immediately before use.
2.6. Statistical analysis
The data are presented as the means SEM. Simple two-group comparisons
were performed using Student’st-test. The minimal significance level for tests was
set at p<0.05. We elected to present normalized current values since multiple
groups were compared with each slice serving as its own precondition control. Thus
maximal DA concentrations were normalized to a respective baseline period rep-
resented the first 10 min of stable recording before drug application.
One-way ANOVA with the pos-hoc Tukey test was used for multiple compari-
sons. All statistical analyses were performed using GraphPad Prism5 software
(GraphPad Software, Inc., La Jolla, CA).
3. Results
3.1. Lack of TAAR1 leads to an increased DA release predominantly
in the nucleus accumbens
Previous studies employing conventional microdialysis have
revealed similar basal levels of dialyzate DA in the NAcc and
striatum (Di Cara et al., 2011) of TAAR1-KO and WT mice, revealing
a difference in monoaminergic transmission only following
amphetamine challenge (Lindemann et al., 2008). To evaluate the
mechanisms of TAAR1-dependent modulation of DA and 5-HT
transmission, we first analyzed the total tissue content of mono-
amines and their metabolites in different brain areas (Fig. 1). HPLC
analysis revealed that monoamines and their metabolite levels in
the prefrontal cortex and hippocampus are generally similar in both
genotypes (Figs. 1B and C, WT ¼10, HET ¼11, KO ¼11; Student’st-
test). However, while DA and its intraneuronal metabolite 3,4-
dihydroxyphenylacetic acid (DOPAC) levels were not altered in
the striatal tissue (including both DStr and NAcc; Fig. 1A) of TAAR1-
KO mice, the levels of predominantly extracellular DA metabolite
homovanillic acid (HVA) were elevated, suggesting an increase in
extracellular DA in mutant mice. Because most of the evidence
indicates a predominant effect of the TAAR1 regulatory effects in
the NAcc in comparison to the DStr (Revel et al., 2012b), we decided
to dissect the contribution of TAAR1 in different striatal subregions,
NAcc and DStr separately, using in vivo microdialysis (Fig. 2). Using
a quantitative low perfusion rate microdialysis approach that, un-
like conventional microdialysis, provides a true measure of extra-
cellular neurotransmitter concentrations (Justice, 1993), we found a
significant increase in extracellular DA in the NAcc of TAAR1-KO
animals (Fig. 2A; WT, n¼5; TAAR1-KO, n¼5; p¼0.0412, Stu-
dent’st-test) while DA levels were not changed in the DStr of
TAAR1-KO mice in comparison to controls (Fig. 2B; WT, n¼9;
TAAR1-KO, n¼7; p¼0.4379, Student’st-test).
Similar results were obtained ex vivo using FSCV performed on
brain slices (Fig. 3). FSCV was used to study the kinetics and the
amount of evoked DA release in the DStr and NAcc brain slices
prepared from TAAR1-KO mice and wild-type littermates. We
recorded DA overflow following single pulses (400
m
A, 1 ms,
monophasic). The oxidation peak occurred at approximately þ0.6 V
and the reduction peak at approximately 0.2 V (Fig. 3A), consis-
tent with the voltammetric characteristics of DA. The maximal
amplitude of DA overflow evoked by single pulses in the DStr was
stable over time and comparable in both the TAAR1-KO and control
mice (Fig. 3E), while in the TAAR1-KO NAcc slices the DA overflow
was significantly increased (Fig. 3B; WT, n¼18 vs. TAAR1-KO,
n¼19; p¼0.0293; Student’st-test). To determine whether the
difference in evoked the DA overflow might be associated with an
unbalanced DA uptake (John and Jones, 2007), we evaluated the
uptake kinetics from an exponential fit curve using a least squares-
constrained exponential fit algorithm (National Instruments in
Demon Voltammetry software, Wake Forest University Health
Sciences, USA) (Yorgason et al., 2011) and quantified the Tau and
half-life parameters for an estimation of DA uptake rates. We
observed that, under basal conditions, the DA uptake as determined
by Tau measurements was similar between the control and TAAR1-
KO animals (see Methods section for details; Fig. 3C and F) Under
basal conditions DA uptake was similar in WT and TAAR-1 KO mice:
NAcc: Tau WT ¼0.6880 0.127 s, N¼18; Ta u
KO ¼0.8927 0.1264 s, N¼19 ; p¼0.2618 Fig. 3C; DStr: Tau WT
0.5087 0.08085 s, N¼12; Tau KO ¼0.6942 0.1490 s, N¼12,
p¼0.2811; Student’st-test. Fig. 3F) suggesting that DAT function is
unaltered in mice lacking TAAR1. Additionally, the DA half life was
similar in both genotypes (see Methods section for details; Fig. 3D
and G) (NAcc: Half life WT ¼0.4751 0.08814 s, N¼18; p¼0.2624;
D. Leo et al. / Neuropharmacology 81 (2014) 283e291 285
Half life KO ¼0.6161 0.08718 s, N¼19; Fig. 3D; DStr: Half life
WT ¼0.3310 0.04690 s, N¼12; Half life KO ¼0.4088 0.06821 s,
N¼24; p¼0.3380; Student t-test. Fig. 3G).
3.2. Selective TAAR1 agonist RO5166017 reduces DA overflow
To further elucidate the role of TAAR1 in DA transmission, we
next evaluated the effect of the selective TAAR1 agonist RO5166017
on electrically evoked DA overflow in the NAcc and DStr in WT and
TAAR1-KO mice (Fig. 4). The effects of two concentrations of TAAR1
agonist (1
m
M and 10
m
M) were tested using single electrical pulses.
While no significant effect was found upon the application of 1
m
M
RO5166017 to brain slices (data not shown), 10
m
M of RO5166017
was able to reduce the evoked DA release selectively in control
animals in both the DStr (Fig. 4A; One-way ANOVA, p<0.0001) and
the NAcc (Fig. 4B; n¼10; One-way ANOVA, p<0.0001) without
significant effects on the Tau and half-life measures of DA uptake
(data not shown). Diminished DA release following RO5166017
seems to be in accordance with a TAAR1 agonist ability to modulate
DA-related functions (Revel et al., 2011).
3.3. TAAR1 antagonist EPPTB increases evoked DA release and
blocks the TAAR1 agonist effect
Previous studies have already demonstrated that treatment
with EPPTB, under current-clamp conditions, significantly
increased the firing frequency of VTA dopaminergic neurons over
the basal level (Bradaia et al., 2009). We tested the effect of EPPTB
on evoked DA release in the NAcc and DStr by applying 10
m
M of the
TAAR1 antagonist to brain slices (Fig. 4C and D). EPPTB failed to
significantly increase DA overflow in the DStr of both control and
TAAR1-KO animals (Fig. 4C; n¼10; One-way ANOVA) but induced
an augmented DA levels in the NAcc of control mice (Fig. 4D, n¼6
One-way ANOVA, p¼0.0210). Thus, the EPPTB effect on evoked DA
release seems to be selective for the NAcc of control animals
(Fig. 4D). Moreover, we evaluated the ability of the TAAR1 antag-
onist to block the effects of the agonist (Fig. 4E). We applied 10
m
M
of EPPTB, and immediately after, 10
m
M of the TAAR1 agonist
(RO5166017) was added. We then measured the amplitude of the
evoked DA release in the WT NAcc. We found that pre-application
of 10
m
M EPPTB is able to block the decrease in the evoked DA
release induced by the TAAR1 agonist (Fig. 4E). None of the drugs
applied changed DA uptake because the Tau and half-life values
were comparable among the naïve and treated brain slices (data
not shown).
3.4. Alterations in the D2 class of DA autoreceptor-mediated
regulation of DA release in TAAR1-KO mice
Because there are several lines of evidence suggesting that
TAAR1 can modulate DA transmission via an interaction with the
DA receptor 2 (D2R) (Espinoza et al., 2011; Ledonne et al., 2010;
Revel et al., 2011), the activity of the D2 class of DA release-
regulating autoreceptors was evaluated in the NAcc of control
and KO animals (Fig. 5). DA autoreceptors of the D2 class regulate
the extracellular levels of DA through a negative feedback mecha-
nism where increasing agonist concentrations result in a reduction
in the firing rate, synthesis and release of DA (Maina and Mathews,
KO
WT
NAcc DStr
0
10
20
30
40
*
DA (nM)
0
10
20
30
40
DA (nM)
AB
Fig. 2. Low perfusion rate (LPR) microdialysis revealed that, while the DA levels are not
altered in the striatum of TAAR1-KO mice (B), the levels of DA in NAcc are elevated in
the mutants (A). *p<0.05, Student’st-test.
A
B
C
0
5
10
15
20
DA DOPAC HVA 5-HIAA 5-HT
*
WT
KO
STRIATUM
ng/mg wet tissue
0.0
0.5
1.0
1.5
NE DA HVA 5-HIAA 5-HT
FRONTAL CORTEX
ng/mg wet tissue
0.0
0.5
1.0
1.5
2.0
NE DA 5-HIAA 5-HT
HIPPOCAMPUS
ng/mg wet tissue
Fig. 1. HPLC analysis of total tissue content in total Str (A), the frontal cortex (B) and
the hippocampus (C). A) In the total striatum, the levels of DA and its intraneuronal
metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) are not altered in TAAR1-KO mice,
but the levels of predominantly extracellular DA metabolite homovanillic acid (HVA)
are elevated in the mutants. (*p<0.05; one-way ANOVA, Tukey’s test). (Band C)
Neither of the analytes were different in the frontal cortex or the hippocampus be-
tween the control and mutant mice.
D. Leo et al. / Neuropharmacology 81 (2014) 283e291286
2010; Gainetdinov et al., 1996). We evaluated the inhibitory effect of
the D2 class receptor agonist quinpirole and the TAAR1 agonist on
DA release in WT (Fig. 5A) and TAAR1-KO (Fig. 5B) mice using FSCV
in NAcc brain slices. In both W T and KO slices treated with 0.1
m
Mof
quinpirole, we observed a well-known decrease in DA release
(Martel et al., 2011; Fawaz et al., 2009; Maina and Mathews, 2010;
Phillips et al., 2003)(Fig. 5A and B). Furthermore, after a washing
period to restore the basal DA levels, we examined the combined
effect of application of the D2 and TAAR1 agonists on DA release. As
shown in Fig. 5, quinpirole and RO5166017 are able to decrease DA
overflow in an additive manner in control animals (WT
quinpirole þRO5166017, vs. WT quinpirole, p¼0.0079, Student’st-
test, Fig. 5A) but not in TAAR1-KO mice (Fig. 5B), suggesting an
altered D2R autoreceptor-mediated regulation of DA release in the
absence of TAAR1.
As an additional approach to confirm the role of DA autor-
eceptors in TAAR1 neuromodulation, a paired-pulse stimulation
test was performed in slices from WT and TAAR1-KO mice (Fig. 5C).
In this voltammetric approach, the autoinhibitory effect of DA
release following the first stimulation is detected by measuring the
magnitude of the decrease in response to the second stimulation
performed 500 ms later. It is believed that this DA release sup-
pression induced by autoinhibition is directly related to D2R class
autoreceptor activation (Phillips et al., 2002). We applied an elec-
trical paired pulse (2 pulses, 400 mA, 500 ms interval), and we
measured the difference between the DA released following the
first stimulus (S1) and the second stimulus (S2) as an indication of
D2R autoreceptor activation (Fig. 5C). We found that the ratio of S2
to S1 is significantly higher in the NAcc slices from TAAR1-KO mice
compared to the control mice, indicating that D2R autoinhibition is
indeed reduced by the lack of TAAR1 (N¼5, p¼0.0029, Student’st-
test). These results further support the role of the D2 class of DA
autoreceptors in TAAR1-mediated modulation of DA release.
4. Discussion
The aim of this study was to understand the mechanism of the
modulatory action of TAAR1 on presynaptic DA transmission. Our
-0.4 V
-0.4 V
1.3 V
V (vs Ag/AgCl)
-0.4 V 1.3 V
Dopamine
15 sec
0.5 μM
A
CB Nucleus Accumbens
Dorsal Striatum
D
FEG
WT KOWT KOWT KO
0.0
0.5
1.0
1.5
DA concentration ( M)
WT KO
WT KO
0.0
0.2
0.4
0.6
0.8
1.0
Tau ( s)
WT KO
0.0
0.2
0.4
0.6
0.8
1.0
Half life (s)
DA concentration (μ
μ
M)
*
0.0
0.5
1.0
1.5
2.0
Tau ( s)
0.0
0.5
1.0
1.5
Half life (s)
0.0
0.2
0.4
0.6
0.8
1.0
μ
Fig. 3. Dopamine (DA) release from mice brain slices. A) The background-subtracted cyclic voltammogram identifies the detected analyte as DA. The color plots represent the
voltammetric currents (encoded in color in the zaxis) plotted against the applied potential (yaxis) and time (xaxis). (A; bottom) Representative traces in the NAcc of control and
TAAR1 mice exemplify the higher peak height in the TAAR1-KO NAcc. B) Quantification of evoked DA release in the NAcc of control and TAAR1-KO mice (*p<0.05, Student’st-test) C
and D) Kinetic analyses of DA reuptake. C) Half-life and Tau measures of DA uptake.Tau and half life were determined from an exponential fit curve using a least squares constrained
exponential fit algorithm (National Instruments in Demon Voltammetry software). The exponential decay constant Tau measures changes in dopamine uptake. TAAR1 mice and
controls have similar uptake kinetics as confirmed by DA half-life measurements (D). E) Quantification of the evoked DA release in the DStr of control and TAAR1-KO mice show
similar DA overflow. FEand GF) Tau (F) and half life (G) of DA uptake do not differ between control and TAAR1-KO mice.
D. Leo et al. / Neuropharmacology 81 (2014) 283e291 287
data support the previous reports showing a close interaction be-
tween TAAR1 and the dopaminergic system (Lindemann et al.,
2008; Revel et al., 2011; Bradaia et al., 2009) and specifically
highlights an interaction between the D2 class of autoreceptors and
TAAR1 receptors. While several recent studies have been per-
formed on understanding the role of TAAR1 in the modulation of
monoaminergic systems in general, we particularly focused on the
presynaptic mechanisms by analyzing the neurochemical effects of
TAAR1 and DA ligands and the consequencesof the lack of TAAR1 in
mice.
First, we found an increased level of the predominantly extra-
cellular DA metabolite HVA in striatal/accumbal tissue that includes
both the DStr and NAcc of TAAR1-KO animals, indirectly indicating
an increase in DA release in this brain area. Furthermore, in
microdialysis studies, we have observed elevated levels of extra-
cellular DA in the NAcc but not in the DStr of mutant mice. In
agreement with these microdialysis studies, voltammetric in-
vestigations of evoked DA release have also demonstrated an
increased DA release selectively in the NAcc of TAAR1-KO mice. To
our knowledge, this is the first report to show a significant increase
in DA release in TAAR1-KO mice. Previous microdialysis studies
have reported markedly enhanced effects of amphetamine and
MDMA on the extracellular levels of monoamines in independently
developed strains of TAAR1-KO mice (Lindemann et al., 2008; Di
Cara et al., 2011; Wolinsky et al., 2007), but not altered basal
levels of DA in the NAcc and DStr. However, all of these measure-
ments were performed by using conventional microdialysis, an
approach known to be limited for assessment of basal absolute
extracellular levels. We therefore employed a low perfusion rate
quantitative microdialysis, providing an opportunity to overcome
the technical limitations of conventional microdialysis in the
assessment of basal neurotransmitter concentrations (Di Chiara
et al., 1996; Chefer et al., 2009; Handbook of Microdialysis, 2007).
Importantly, Lindemann et al. have found an increase in firing rates
Fig. 4. TAAR1-mediated modulation of evoked DA release. Aand B) Effects of
RO5166017 on evoked DA release in the DStr (A) and NAcc (B).10
m
M of TAAR1 agonist
decreases DA overflow in both structures but only in control animals (*p<0.05, One-
way ANOVA, Tukey’s test). Cand D)10
m
MofN-(3-Ethoxy-phenyl)-4-pyrrolidin-1-yl-
3-trifluoromethyl-benzamide (EPPTB) does not influence the evoked DA release in the
DStr (C) but increases the DA overflow in control NAcc slices (D;*p<0.05, One-way
ANOVA, Tukey test). E) Pretreatment with 10
m
M EPPTB blocks the reduction of
evoked DA release mediated by 10
m
M RO5166017.
Fig. 5. 10
m
M of TAAR1 agonist and 0.1
m
M of quinpirole (D2R agonist) have an additive
effect on decreasing DA transmission in control animals (A) but not in TAAR1-KO
animals (B)(*p<0.05, One-way ANOVA with Bonferroni correction) suggesting a
regulatory action of TAAR1 on D2R signaling. C) After using paired stimuli to evoke DA
release in TAAR1-KO and control NAcc slices, TAAR1-KO mice have higher amplitude of
DA release following the second pulse of stimulation (*p<0.05, Student’st-test)
indicating that D2R-mediated autoinhibition is less active in the absence of TAAR1
gene.
D. Leo et al. / Neuropharmacology 81 (2014) 283e291288
in the VTA of TAAR1-KO animals, which is consistent with the
augmented DA release in the NAcc observed in our study. Thus,
TAAR1 seems to be tonically active in physiological conditions,
maintaining a negative control on DA release at least in the NAcc
(Lindemann et al., 2008). The lack of TAAR1 activity then induces a
substantially stronger enhancement in DA overflow in the NAcc. In
agreement, TAAR1 expression was reported in the VTA (Lindemann
et al., 2008) that mainly projects to the NAcc, among other areas.
Importantly, we have documented that neither Tau nor the half-life
of released DA are changed in slices from TAAR1-KO animals,
indicating that TAAR1-KO mice exhibit unaltered DA uptake ability
and thereby normal dopamine transporter (DAT) functionality. It is
believed that Tau and the half-life of released DA are reliable
measures for detecting changes in DA uptake because they are
strongly correlated with changes in the K
m
of DAT mediated DA
uptake (Yorgason et al., 2011). Thus, these neurochemical in vivo
studies, as well as previous demonstrations of the functional ac-
tivity of TAAR1 ligands in mice lacking the DAT (Sotnikova et al.,
2004; Revel et al., 2012a), provide little support for the postu-
lated role of TAAR1 in modulating DAT activity that is based mostly
on in vitro cell culture experiments (Miller et al., 2005; Xie et al.,
2008; Miller, 2011).
We have also found that the TAAR1 agonist is able to reduce
evoked DA release in control animals but that it is ineffective in
TAAR1-KO mice. RO5166017 exhibits a high binding affinity for
mouse TAAR1 and a high potency to stimulate cAMP production,
and it is known to inhibit the VTA firing rate (Revel et al., 2011). In
our ex vivo experiments, 10
m
M RO5166017 exerts an effect on DA
release in both the NAcc and the DStr (Fig. 4), suggesting that while
the influence of TAAR1 over DA transmission is predominant in the
NAcc, it might also occur at the level of the DStr. The concentration
of the TAAR1 agonist seems to be quite high compared to the one
used in in vitro experiments (Revel et al., 2011), but generally,
voltammetric slice studies require application of higher concen-
trations of drugs that differ substantially from those used in the
intact organism or in cultured cells (Borland et al., 2007). As
RO5166017 is able to reduce DA release, TAAR1 antagonist EPPTB
has the opposite effect and not only induces an augmentation of
evoked DA release but also blocks the agonist effect (Bradaia et al.,
2009). Importantly, both the effects of the TAAR1 agonist
(RO5166017) and antagonist (EPPTB) are specific for wild-type
animals and do not affect the kinetics of evoked DA release in
TAAR1-KO mice. Notably, neither of these treatments changed the
kinetics of DA uptake as evidenced by the Tau and DA half-life es-
timations, indicating that DAT-mediated function is not altered by
the action of the drugs on TAAR1. This is in line with the fact that
TAAR1 agonists have a pronounced inhibitory action on DA-
dependent hyperactivity in mice lacking the DAT (Revel et al.,
2011), ruling out the contribution of DAT at least these functional
effects of selective TAAR1 ligands.
Because we have not found evidence of the postulated
involvement of DAT in the effects of TAAR1 in our in vivo studies, we
explored an alternative potential mechanism of action by which
TAAR1 can influence DA release. Thus, we focused our attention on
the status of the DA D2 class receptor (D2R) autoreceptors, per-
forming FSCV in the presence of the D2R agonist quinpirole.
Importantly, while D2 DA autoreceptors have a predominant role in
presynaptic autoinhibition of firing rates, synthesis and release
(Anzalone et al., 2012), an important contribution of the DA D3
autoreceptors on DA release regulation has also been demonstrated
(Accili et al., 1996; Joseph et al., 2002; Gainetdinov et al., 1996).
While we are not aware of any studies focused on the interaction
between TAAR1 and the DA D3 receptor, there are several lines of
evidence indicating a role of D2 receptors in functions mediated by
TAAR1. Our group has previously demonstrated that TAAR1 is able
to physically and functionally interact with the D2 receptors,
potentially via heterodimerization both in vitro and in vivo
(Espinoza et al., 2011). Moreover, it has been reported that TAAR1-
KO mice have a larger proportion of striatal D2 receptors that are in
a high-affinity state (Wolinsky et al., 2007). Because FSCV provides
an opportunity to follow real-time inhibition of DA release by
autoreceptors (Palij et al., 1990; Kennedy et al., 1992), we used an
application of 0.1
m
M quinpirole to NAcc slices, which has a well-
known effect of decreasing the evoked DA release (Yorgason
et al., 2011). A significant quinpirole-induced reduction in DA
release was observed in both TAAR1-KO and WT mice, indicating a
preserved D2R autoreceptor-mediation in the absence of TAAR1.
However, the D2R agonist showed an additive effect when com-
bined with the TAAR1 agonist RO5166017 in WT mice, but not in
TAAR1-deficient animals. Presumably, there is an increase in the
D2R-mediated autoinhibition of DA neurons under tonic activation
of TAAR1 that supports the role of TAAR1 as a homeostatic regu-
latory mechanism preventing the excess activity of DA neurons. DA
autoreceptors are known to exert their effects by down-regulating
adenylate cyclase via a G protein-coupled mechanism (Missale
et al., 1998), and it can decrease tyrosine hydroxylase activity by
phosphorylation. Importantly, it has been reported that TAAR1-KO
mice have an increased basal phosphorylation state of tyrosine
hydroxylase at Ser19, Ser31, and Ser40 (Di Cara et al., 2011).
Moreover, it has been proposed that the D2R autoreceptor-
mediated modulation of secretion might occur via modulation of
potassium and possibly calcium channels in the adenylate cyclase-
independent pathway (Beaulieu and Gainetdinov, 2011). TAAR1
induces a G protein-dependent inwardly rectifying K
þ
current that
is inhibited by Ba
2þ
and tertiapin, suggesting that TAAR1 reduces
the firing rate of DA neurons by activating Kir3 channels (Bradaia
et al., 2009). G protein-mediated activation of K
þ
channels gener-
ally hyperpolarizes the membrane, thus decreasing the probability
of release.
Finally, to further investigate the relationship between the level
of autoreceptor stimulation and the resulting inhibition of DA
release, we used paired stimuli to evoke DA release in TAAR1-KO
and control NAcc slices. In this paradigm, DA released by the first
pulse activates D2R autoreceptors and thus results in less amount
of DA release evoked by the following pulse. In this test, TAAR1-KO
animals have a higher amplitude of DA release following the second
pulse of stimulation, thus directly indicating that D2R-mediated
autoinhibition is less active in the absence of TAAR1 (Phillips
et al., 2002). Importantly, while both presynaptic and post-
synaptic D2 receptors are present in the recording site and there is
growing evidence that both these populations could be regulated
by TAAR1 (Borowsky et al., 2001; Espinoza et al., 2011) only the
former are involved in DA autoinhibition. Indeed, DA autoinhibition
is still observed in the striatum of mice lacking only postsynaptic
D2 receptors (Benoit-Marand et al., 2001; Usiello et al., 2000).
These observations, demonstrating ability of TAAR1 to modulate
presynaptic DA autoreceptor function suggest intriguing new pos-
sibility how TAs may influence monoaminergic transmission in
general. Thus, not only direct activation of D2R autoreceptors by
released DA but also action of endogenous TAAR1 ligands such as
TAs and the extracellular DA metabolite 3-methoxytyramine on
TAAR1 (Bunzow et al., 2001; Sotnikova et al., 2010) may exert fine-
tuning regulatory action on presynaptic dopaminergic trans-
mission. It would be interesting to explore if this presynaptic
autoreceptor-based mechanism of action could be extended to
other TAs and monoaminergic systems.
In conclusion, we report that TAAR1 is able to regulate DA
release predominantly in the NAcc by exerting a negative modu-
lation of DA tone. Thus, TAAR1-KO mice have an increased evoked
DA release compared to control mice. The uptake rates are similar
D. Leo et al. / Neuropharmacology 81 (2014) 283e291 289
between the two groups of mice, excluding an involvement of DAT
in the modulatory action of TAAR1. We also observed a functional
link between TAAR1 and D2R autoreceptors localized on DA ter-
minals, further indicating that TAAR1 and D2R can modulate each
other’s activity. These observations uncover a mechanism of close
interaction between TAAR1 and the DA system at the level of the
presynaptic neurons and autoreceptor regulation that further
promotes the general strategy of targeting TAAR1 to modulate the
dopaminergic system in a range of neuropsychiatric disorders.
Acknowledgments
This work was supported in part by research awards to RRG from
F. Hoffmann-La Roche Ltd. (Basel, Switzerland) and Fondazione
Compagnia di San Paolo (Torino, Italy). We are grateful to Lundbeck
A/G and Lundbeck USA for generously providing the TAAR1
knockout mice. We thank Dr. M. Morini, D. Cantatore and F. Piccardi
for their excellent technical assistance.
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