International Journal of Neuropsychopharmacology (2012), 15, 1457–1471. f CINP 2011
doi:10.1017/S1461145711001568
ARTICLE
Glutamate input to noradrenergic neurons
plays an essential role in the development of
morphine dependence and psychomotor
sensitization
Jan Rodriguez Parkitna1, Wojciech Solecki1, Krystyna Gołembiowska2,
Krzysztof Tokarski3, Jakub Kubik1, Sławomir Gołda1, Martin Novak4, Rosanna Parlato4,
Grzegorz Hess3, Rolf Sprengel5 and Ryszard Przewłocki1
1
Department of Molecular Neuropharmacology, Institute of Pharmacology of the Polish Academy of Sciences, Cracow, Poland
Department of Pharmacology, Institute of Pharmacology of the Polish Academy of Sciences, Cracow, Poland
3
Department of Physiology, Institute of Pharmacology of the Polish Academy of Sciences, Cracow, Poland
4
Department of Molecular Biology of the Cell I, German Cancer Research Center, Heidelberg, Germany
5
Department of Molecular Neurobiology, Max Planck Institute for Medical Research, Heidelberg, Germany
2
Abstract
The brain’s noradrenergic system is involved in the development of behaviours induced by drugs of
abuse, e.g. dependence and withdrawal, and also reward or psychomotor effects. To investigate how
noradrenergic system activity is controlled in the context associated with drug-induced behaviours, we
generated a Cre/loxP mouse model in which the essential glutamate NMDA receptor subunit NR1 is
ablated in cells expressing dopamine b-hydroxylase (Dbh). As a result, the noradrenergic cells in NR1DbhCre
mice lack the NMDA receptor-dependent component of excitatory post-synaptic currents. The mutant
mice displayed no obvious behavioural alterations, had unchanged noradrenaline content and mild increase in dopamine levels in the nucleus accumbens. Interestingly, NR1DbhCre animals did not develop
morphine-induced psychomotor sensitization. However, when the morphine injections were preceded by
treatment with RX821002, an antagonist of a2-adrenergic receptors, the development of sensitization was
restored. Conversely, pretreatment with clonidine, an agonist of a2-adrenergic receptors, blocked development of sensitization in wild-type mice. We also found that while the development of tolerance to
morphine was normal in mutant mice, withdrawal symptoms were attenuated. These data reveal that
NMDA receptors on noradrenergic neurons regulate development of opiate dependence and psychomotor sensitization, by controlling drug-induced noradrenaline signalling.
Received 11 April 2011 ; Reviewed 9 June 2011 ; Revised 19 September 2011 ; Accepted 22 September 2011 ;
First published online 1 November 2011
Key words : Morphine, NMDA receptor, noradrenaline, reinforcement, sensitization, withdrawal.
Introduction
The noradrenergic system of the brain controls
arousal, selective attention, response to stress, and
plays an important role in learning and memory
(Aston-Jones & Cohen, 2005 ; Sara, 2009). It also plays a
central role in drug dependence, becoming strongly
activated during opiate withdrawal (Aghajanian,
1978 ; Gold et al. 1978 ; Rasmussen et al. 1990).
Address for correspondence : Professor R. Przewłocki, Institute
of Pharmacology of the Polish Academy of Sciences, Sme˛tna 12,
31-343 Cracow, Poland.
Tel. : +4812 6623 218 Fax : +4812 6374 500
Email : [email protected]
However, beyond physical dependence, the role of the
noradrenaline (NA) system in the development of behaviours associated with drugs of abuse has been a
matter of controversy (Sofuoglu & Sewell, 2009 ;
Tassin, 2008 ; Weinshenker & Schroeder, 2007 ; Wise,
1978). Although drugs of abuse interfere with both NA
and dopamine (DA) signalling, the accumulated evidence points to the latter as the main substrate for the
actions of drugs of abuse (Hyman et al. 2006 ; Kauer &
Malenka, 2007 ; Tzschentke, 1998).
Studies with genetically modified mice have reopened the discussion on the respective roles of NA
and DA in drug-conditioned behaviours. It was found
that the ablation of the DA transporter (Sora et al. 1998,
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J. R. Parkitna et al.
but see Chen et al. 2006), D1 (Karasinska et al. 2005) or
D2L dopamine receptors (Smith et al. 2002) did not
abolish cocaine-conditioned place preference (CPP),
suggesting that DA-dependent signalling is not the
only system involved. In contrast, the ablation of
adrenergic a1b receptors (Adra1b) completely prevented the development of drug-induced psychomotor sensitization or CPP (Drouin et al. 2002).
Furthermore, in mice lacking dopamine b-hydroxylase
(Dbh), an enzyme necessary for NA synthesis, morphine CPP and psychomotor activation were abolished (Olson et al. 2006). When mutant mice were
subjected to injections of virus vector expressing Dbh
into the solitary tract nucleus (NTS), morphineinduced behaviours were rescued. In addition, morphine-induced CPP was still possible in DA-deficient
mice (Hnasko et al. 2005). Thus the NA system, acting
independently of DA, could support development of
opiate-induced behaviours.
While drug-induced long-term adaptations in DA
pathways were found to be associated with the plasticity of glutamatergic transmission in the ventral
tegmental area (VTA) (Chen et al. 2010 ; Ungless et al.
2001), the mechanisms controlling NA pathwayrelated behaviours are poorly understood. The cells
involved receive glutamatergic afferents originating
mainly from the nucleus paragigantocellularis of the
medulla as well as descending projections from the
forebrain including the paraventricular nucleus of
the hypothalamus and the insular and limbic cortices
(Aston-Jones et al. 1986 ; Baude et al. 2009 ; Reyes et al.
2005). A large proportion of these glutamatergic
fibres in the locus coeruleus (area A6, LC) were found
to contain opioid peptides and/or corticotrophinreleasing factor, potentially allowing interaction between the stress axis and the endogenous enkephalin
system (Van Bockstaele et al. 2010).
In our study, we sought to define the role of the
brain’s NA system in relation to the development of
opiate-induced behaviours. We reasoned that in the
absence of functional NMDA receptors, the NA neurons would display disrupted long-term excitatory
modulation, thereby altering development of druginduced behaviours dependent on changes in NA
signalling. Therefore, we generated a transgenic
mouse, NR1DbhCre, through the ablation of the essential
NMDA receptor subunit NR1 (Grin1) in NA cells.
Methods
Animals
The DbhCre and NR1 flox strains have been
described previously (Niewoehner et al. 2007 ; Parlato
et al. 2007). Transgenic animals from both parental
strains were crossed into the C57BL/6N background
for at least six generations. Animals aged 8–20 wk
at the beginning of experimental procedures were
housed in Plexiglas home cages (30r40r20 cm)
(n=2–6 animals per cage) on a 12-h light/dark
cycle (lights on 08 : 00 hours) in a temperaturecontrolled room (24¡1 xC) at 55–65 % humidity.
Standard laboratory chow (Labofeed H, WPiK,
Poland) and water were available ad libitum. Behavioural tests started 2 wk after the arrival of the
animals at the laboratory and were conducted during
the light phase (08 : 00–20 : 00 hours) by experimenters
who were blinded to the genotype and drug treatment. NR1loxP/loxP or NR1loxP/wild-type animals were
used as controls. Behavioural phenotyping was performed on four cohorts of male mice, totalling
93 animals and including 47 NR1DbhCre mice. Three
additional separate groups of mice were used in
electrophysiology, microdialysis and tissue neurotransmitter measurements. The size of each experimental group is given in the figure legends.
Experiments were conducted in accordance with European Union guidelines for the care and use of laboratory animals (ECC Directive of 24 November 1986)
and were approved by the II Local Bioethics Committee (Poland).
Immunostaining
Two control and two NR1DbhCre mice were deeply anaesthetized and transcardially perfused with saline
followed by 4 % paraformaldehyde in phosphatebuffered saline (PBS). Dissected brains were fixed for
an additional 12 h in 4 % paraformaldehyde and then
cut with a vibratome (Leica, Germany) at 40 or 50 mm.
For immunohistochemistry, coronal sections were
incubated with antibodies against tyrosine hydroxylase (TH ; Millipore, USA, MAB318, 1 : 2000) or
NR1 (Sigma, USA, G8913, 1 : 100) and stained with
diaminobenzidine (DAB) using the ABC kit (Vector
Laboratories, USA) according to the manufacturer’s
instructions. For each animal 4–6 sections were
stained at the levels of the aqueduct, LC and NTS.
Images of stained sections were acquired using a
Leica DMNB microscope equipped with a Basler
‘ Scout ’ digital camera and Visiopharm software
(Denmark).
Whole-cell recording
Recordings were performed as previously described
(Tokarski et al. 2003). Briefly, five control and five
NR1DbhCre mice aged 8–12 wk were decapitated, the
Glutamate inputs to NA cells regulate opiate effects
brains were quickly removed and immersed in icecold artificial cerebrospinal fluid (aCSF) containing
(mM) 130 NaCl, 5 KCl, 2.5 CaCl2, 1.3 MgSO 4, 1.25
KH2PO4, 26 NaHCO3, 10 D-glucose and bubbled with a
mixture of 95 % O2/5 % CO2. Horizontal slices (350 mm
thick) were cut on a vibrating microtome (Leica).
Slices were placed in the recording chamber
mounted on the Zeiss Axioskop microscope (Zeiss,
Germany) and superfused (3 ml/min) with modified,
Mg-free aCSF (32¡0.5 xC). Neurons were visualized
using Nomarski optics and a 40r lens. NA cells of the
LC were identified by their position in relation to the
IV ventricle, the shape of the soma and the response to
a depolarizing current pulse (Andrade & Aghajanian,
1984). Only cells with a resting membrane potential of
at least x50 mV and overshooting action potentials
were accepted for analysis. Series resistance did not
change appreciably during the experiments, indicating stable recording conditions. Stimulus–response
characteristics of recorded neurons were evaluated
using rectangular current pulses (500 ms) of increasing intensity in 20 pA steps (60–300 pA). Then, cells
were depolarized with current injection to x50 mV
and spontaneous spiking activity was recorded for
4 min in the current-clamp mode. Next, cells were
voltage-clamped at x76 mV, and spontaneous excitatory post-synaptic currents (sEPSCs) were recorded
for 12 min (3r4 min). To block NMDA receptormediated currents, a specific antagonist, CGP37849
(5 mM), was added to the aCSF. After 20 min perfusion,
sEPSCs were again recorded for 12 min. Spontaneous
EPSCs were detected offline and analysed using Mini
Analysis software (Synaptosoft, USA). The amplitude
and area thresholds for the detection of an event were
set to 7 pA and 25 fC, respectively. Recorded traces
were visually verified following automated analysis.
Tissue levels of neurotransmitters
After each experiment was completed, mice were decapitated, brains were dissected and brain regions
were separated on ice. Tissue samples were weighed
and homogenized in ice-cold 0.1 M perchloric acid.
Then, homogenates were centrifuged at 10 000 g.
Supernatants were filtered through membrane filters
(0.1 mm pore size) and injected into HPLC for the determination of tissue levels of NA, DA, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid
(HVA).
Microdialysis in freely moving animals
Microdialysis probes were constructed by inserting
two fused silica tubes (30 and 35 mm long, 150 mm
1459
outside diameter (o.d.) ; Polymicro Technologies Inc.,
USA) into a microdialysis fibre (220 mm o.d. ; AN69,
Hospal, Italy). The tube assembly was placed in a
Peek cannula (0.3 mm o.d., 6 mm long) to form the
shaft of the probe. Portions of the inlet and outlet
tubes were individually placed inside polyethylene
PE-10 tubing and glued. The free end of the dialysis
fiber was sealed, and 2 mm of the exposed length was
used for dialysis. The molecular cut-off value of the
membrane was 6000 Da. Recovery rate was determined by an in-vitro experiment ; the probe was
placed in a solution containing 50 pg/ml DA and the
DA concentration was measured in dialysate fractions. The recovery efficiency was between 10 % and
15 %.
Mice were anaesthetized with ketamine (7.5 mg/kg
i.p.) and xylazine (1 mg/kg i.p.) and placed into a
stereotaxic apparatus (David Kopf Instruments, USA).
The skulls were exposed, and small holes were drilled
for the insertion of the microdialysis probes using the
following coordinates : 1.5 mm anterior to bregma,
1.0 mm lateral to the sagittal suture, x4.0 mm ventral
to the dural surface [nucleus accumbens (NAc)].
Animals were allowed to recuperate until the next day
(24 h). Then probes were connected to a syringe pump
(BAS, USA), which delivered aCSF composed of (mM)
145 NaCl, 2.7 KCl, 1.0 MgCl2, and 1.2 CaCl2 (pH 7.4) at
a flow rate of 1.5 ml/min. After a 2-h washout period,
baseline samples were collected every 30 min over 2 h.
Next, mice were injected subcutaneously with morphine (10 mg/kg), and dialysate fractions were collected every 30 min for 4 h. Elution times were not
corrected for the volume of the tubing and swivel. At
the end of the experiment, the mice were sacrificed,
and their brains examined histologically to validate
probe placement.
Neurotransmitter analysis
NA, DA and metabolites (DOPAC and HVA) were
analysed by HPLC with coulochemical detection.
Chromatography was performed using an Ultimate
3000 System (Dionex, USA), coulochemical detector
Coulochem III (model 5300, ESA, USA) with 5020
guard cell, 5014B microdialysis cell and Hypersil
Gold-C18 analytical columns (3r100 mm). The mobile
phase was composed of 0.05 M potassium phosphate
buffer adjusted to pH 3.9, 0.5 mM EDTA, 13 mg/l
1-octanesulfonic acid sodium salt, 3.1 % methanol and
0.93 % acetonitrile. The flow rate during analysis
was 0.7 ml/min. The applied potential of a guard cell
was +600 mV, whereas that of the microdialysis cell
was E1=x50 mV, E2=+300 mV. Sensitivity was set
1460
J. R. Parkitna et al.
at 50 nA/V. The chromatographic data were processed by Chromeleon v. 6.80 software (Dionex). The
values were not corrected for in-vitro probe recovery,
which was approximately 15 %.
CPP/conditioned place aversion (CPA) and
locomotor activity measurements
The procedure for studying changes in locomotor
activity and CPP/CPA was adapted from (Itzhak &
Martin, 2002). Doses of morphine hydrochloride (10 or
7.5 mg/kg i.p., Polfa, Poland ; dissolved at 0.75 or
1 mg/ml in saline) and naloxone (1.5 or 2 mg/kg i.p.,
Sigma Aldrich, USA, dissolved at 0.15 or 0.2 mg/ml in
saline) were selected based on their ability to reveal
genotype-dependent differences in behaviour as observed in a previous study from our laboratory
(Solecki et al. 2009). Clonidine hydrochloride (a2adrenergic receptor agonist ; Tocris, UK) and
RX821002 hydrochloride (a2-adrenergic receptor antagonist ; Tocris) were dissolved in sterile water at
0.003 and 0.05 mg/ml, and administered i.p. at 0.03 or
0.5 mg/kg, respectively, 30 min prior morphine or
vehicle. Final injected volume was always 0.1 ml per
10 g of animal weight.
CPP/CPA and locomotor activity measurements
were conducted in eight automatic boxes (Med
Associates, USA) consisting of two conditioning
chambers separated by guillotine doors and a central
platform. The design of the experiment is shown
in Fig. 5 a. During the pre-conditioning and postconditioning tests, mice were placed individually on
the central platform of the apparatus and had free
access to both arms for 20 min. The time spent in
each arm and movements were recorded with the
use of an infrared beam system. After the preconditioning phase, one arm was paired with drug
administration, and the other with saline. The assignment of treatments to the arms was counterbalanced. During conditioning, the mice were treated
with vehicle, morphine, cocaine or naloxone (i.p.)
immediately before being placed in the appropriate
arm for 40 min. Locomotor activity measured on
conditioning sessions was used to assess the development of psychomotor sensitization. The extinction
of CPP/CPA consisted of 10 conditioning sessions
during which mice were treated with saline (i.p.) in
both previously drug- and vehicle-paired arms. To
induce reinstatement of CPP/CPA mice were treated
with 75 % of the drug dose used for conditioning,
either i.p. morphine (7.5 mg/kg) or naloxone
(1.5 mg/kg) and immediately placed in the apparatus for 20 min.
Physical dependence after chronic morphine
treatment
Physical dependence was evaluated by measuring
behavioural manifestations of naloxone-precipitated
withdrawal in mice treated with chronic morphine
(Fig. 7 a). Three hours after the last morphine treatment,
each mouse was injected s.c. with naloxone (4 mg/kg
dissolved at 0.4 mg/ml in saline) and then placed in
a Plexiglas tube. The number of jumps (defined as
no contact with the surface for all four paws), paw
tremors, teeth chattering and defecations were scored
by observers blind to the genotype and treatment.
Data analysis
CPP score was defined as the difference in time spent
in the drug- and vehicle-paired arms during the test
day. In general data were analysed by factorial analysis of variance (ANOVA) followed by a post-hoc test or
a t test. Results from microdialysis, electrophysiology
and conditioned reinforcement were analysed by twoway ANOVA with repeated measures with variables
(between-subjects : genotype ; within-subjects : time,
treatment or CPP stage). Locomotor activity data was
analysed in two separate tests. Effects of saline treatment and first drug treatment on locomotor activity
were analysed by two-way ANOVA with repeatedmeasures between-subjects genotype or treatment (i.e.
clonidine or RX821002) and within-subjects treatment
(i.e. saline or morphine) with activity counted in 5-min
bins over a 30-min session. Effects of intermittent
drug treatment on locomotor activity were analysed
by two-way ANOVA with repeated-measures between-subjects genotype or treatment (i.e. clonidine or
RX821002) and within-subjects day, with activity
counted as mean values from 30-min test sessions on
drug-injection days. Frequencies of morphine withdrawal symptoms were analysed using two-way
ANOVA (genotype, treatment).
Additional description of behavioural phenotyping
methods is included in the Supplementary material
(available online).
Results
Generation of NR1DbhCre mice
The ablation of the NR1 (Grin1) gene was restricted to
cells expressing DA b-hydroxylase (Dbh) using the
Cre/loxP system. Transgenic mice harbouring the Cre
recombinase under the control of the PAC-derived
Dbh gene promoter were crossed with NR1 flox mice,
in which loxP sequences flank exons 11–18 of the gene
Glutamate inputs to NA cells regulate opiate effects
(a)
DbhCre
NR1 (Grin1)
10
exons 11–18
19
iCre
Exon 1
loxP
loxP
recombination
10
(b)
Control
(c)
19
NR1DbhCre
1461
(Fig. 1a). Resulting NR1 flox/flox ; DbhCre Tg/0
(NR1DbhCre) animals were born at the expected ratio
and did not differ from their NR1loxP/loxP littermates or
wild-type C57BL6/N mice of similar age. The loss of
NR1 protein was restricted to NA cells, as observed
in the LC by immunostaining with NR1-specific antibodies (Fig. 1b–e). The mutation did not cause
any gross morphological changes in the LC or NTS
(Fig. 1 f–i). These results are in agreement with the
previously reported specificity and efficiency of Cre/
loxP-driven deletions using the DbhCre line (Parlato
et al. 2007, 2010).
Phenotypic characterization of NR1DbhCre mice
NR1
(d)
NR1
Control
NR1
(f )
(e)
NR1DbhCre
NR1
Control
(g)
NR1DbhCre
The NR1DbhCre animals displayed no impairments or
major phenotypic alterations. They had normal muscular strength and coordination as assessed by the
wire-hang test (Fig. 2 a, b). The activity pattern of
NR1DbhCre animals in the open field was similar to that
of control littermates ; i.e. animals habituated to the
environment at comparable rates and avoided the
illuminated centre of the field (Fig. 2 c, d). Accordingly,
exploration of the light-dark box apparatus by
NR1DbhCre mice did not differ from that of control
animals, indicating typical anxiety-like behaviours
(Fig. 2 e–g). Finally, the NR1DbhCre animals demonstrated the same pattern of Y-maze exploration as
controls, alternating the explored arms of the apparatus with normal frequency and thus indicating
normal spatial memory performance (Fig. 2 h–j).
In conclusion, the loss of NMDA receptors on NA
neurons did not cause observable impairments,
abnormal anxiety levels or learning deficits.
Loss of the NR1 subunit in noradrenergic cells
blocks the function of NMDA receptor channels
TH
(h)
TH
Control
TH
(i)
NR1DbhCre
TH
Fig. 1. Generation of mice with NR1 gene ablation restricted
to noradrenergic cells. (a) Schematic representation of the
bacterial artificial chromosome-derived transgene (DbhCre)
expressing the Cre under the control of the Dbh gene
promoter used for the recombination of a ‘ floxed’ variant of
the NR1 gene. (b–e) Micrographs show immunohistological
staining of the NR1 protein (brown) on representative coronal
sections from control (b, d) and NR1DbhCre mice (c, e).
Panels (d, e) are higher magnifications of the corresponding
areas from (b, c) marked by dashed line rectangles. ( f–i) The
In order to characterize the functional consequences
of NR1 ablation at the cellular level, we performed
whole-cell patch-clamp recordings from LC neurons
under Mg2+-free conditions, which promote NMDA
receptor-dependent glutamatergic transmission. The
stimulus–response characteristic of NA neurons in
NR1DbhCre mice was normal and similar to that of
controls (Fig. 3 a). Noradrenergic cells from mutant
mice exhibited a non-significant trend towards higher
spontaneous activity when depolarized to x50 mV
loss of NR1 was not associated with any apparent anatomical
alterations in the locus coeruleus ( f, g) or solitary tract
nucleus (h, i) as shown on representative tyrosine
hydroxylase immunostainings (brown). Scale bars :
(b, c) 25 mm ; (d, e) 8.3 mm ; ( f, g) 200 mm ; (h, i) 400 mm.
J. R. Parkitna et al.
(a)
(b)
300
Walking time (s)
Time to fall (s)
300
200
100
200
Control
100
0
NR1
(c)
(d)
100
Time in the inside zone (s)
Distance (m)
60
40
20
0
Day
(e)
1
2
3
4
80
60
40
20
0
Day
(f)
40
20
Transitions
60
% time in the
light box
DbhCre
0
0
1
2
3
4
(g)
20
20
15
15
Rearings
1462
10
10
5
5
0
0
Light
(i)
20
10
Alternater
arm returns
Arm entries
30
(j)
100
100
80
80
Spontaneous
alteration
(h)
60
40
20
0
Dark
0
60
40
20
0
DbhCre
Fig. 2. Basic behavioural phenotyping of NR1
mice. (a, b) The motor performance of the NR1DbhCre animals (n=14) and
controls (n=11) was similar in the wire hang test, including the ability of mice to hang on to the wire (a, |t23|<1, n.s.) and time
spent taking steps (b, t23=1.4, n.s.). (c, d) Locomotor activity of NR1DbhCre animals (n=8) and controls (n=8) in the open field.
There was no genotype effect on locomotor activity ; all mice showed decreased activity in subsequent sessions over 4 d
(c, genotype : F1,42=1.9, n.s. ; time : F3,42=68.7, p<0.001 ; genotypertime : F3,42<1, n.s.). The loss of NR1 had no effect on the
exploration of the illuminated centre of the field (d, genotype : F1,42<1, n.s. ; time : F3,42=5.6, p<0.01 ; genotypertime : F3,42<1,
n.s.). (e–g) NR1DbhCre mice (n=10) and littermate controls (n=8) did not differ with regard to anxiety-related behaviours in the
light-dark box test. Irrespective of genotype, mice spent the same amount of time in the light side of the box (e, |t16|<1, n.s.), with
a similar number of transitions between compartments ( f, t16=1.05, n.s.) and rearings (g, compartment : F1,30=10.36, p<0.01 ;
genotype : F1,30<1, n.s. ; genotypercompartment : F1,30<1, n.s.). (h–j) Spatial memory-dependent performance in the Y-maze was
normal in NR1DbhCre mice (h, |t14|<1, n.s.). Both groups (n=8 mice per group) explored the maze and tended not to re-enter
previously visited arms (i, j, t14=1.7 and |t14|<1 respectively, both n.s.).
Glutamate inputs to NA cells regulate opiate effects
(Fig. 3 b ; genotype : F1,13=2.43, n.s.). Similar to
controls, neurons lacking NR1 showed a reduction in
spiking frequency after administration of CGP37849,
a NMDA receptor antagonist (Fig. 3c ; treatment :
F1,13=13.60, p<0.01). When the neurons were voltageclamped at x76 mV, we observed increased frequency of sEPSCs in NR1DbhCre animals compared to
controls (Fig. 3 d, e ; genotype : F1,16=4.52, p<0.05),
without a significant difference in the mean sEPSC
amplitude (Fig. 3 f ).
The administration of CGP37849, an antagonist of
NMDA receptors, had no effect on the sEPSC decay
time constant in mutant NA neurons (Fig. 3 g–i),
whereas in controls it resulted in a faster decay of the
synaptic current (genotypertreatment : F1,16=28.81,
p<0.001). All noradrenergic cells recorded from
NR1DbhCre mice were insensitive to CGP37849 treatment and exhibited sEPSC decay times in the same
range as those of LC neurons in wild-type mice after
administration of the NMDA antagonist (Fig. 3 i). At
the same time, the rise time and amplitude were
not affected, indicating that AMPA/kainate receptordependent components of sEPSCs were not altered by
the mutation.
Notably, the mean frequency of sEPSCs recorded
from NR1DbhCre cells was higher than that observed in
controls. Because the frequency of sEPSCs is determined mainly by presynaptic factors, this observation
suggests a strengthening of the glutamatergic input,
which might partly compensate for the reduced reactivity of the post-synaptic side due to NMDA receptor
ablation. This result could also explain the trend towards higher spontaneous activity observed in LC
cells from NR1DbhCre mice when held at x50 mV, and
resembles the up-scaling of activity that occurred after
the ablation of NR1 in DA cells (Engblom et al. 2008 ;
Zweifel et al. 2008). The observed NMDA receptor
antagonist-induced decrease in the spontaneous activity of LC neurons in NR1DbhCre mice can be explained by the fact that the preparations used for the
recording contained functioning excitatory neurons
(or their parts) that project to the LC. It is conceivable
that CGP37849 administration led to reduced activity
of those cells, which in turn resulted in a decreased
excitatory input to LC neurons, and consequently a
reduction in their activity. Finally, the presence of
presynaptic NMDA receptors on afferents to the LC
was reported (Van Bockstaele et al. 2000), and their
inhibition could also contribute to the genotypeindependent decrease in activity observed after
administration of the antagonist. Taken together, the
data confirm loss of functional NMDA receptors in
NA neurons upon NR1 deletion.
1463
Catecholamine release in the NAc
We analysed the content and extracellular levels of
catecholamines in the medial NAc including parts of
both the core and shell subdivisions (Fig. 4 a). There
was a trend towards higher DA content (t10=2.21,
p=0.051), without differences in levels of DA metabolites (Fig. 4 b). Then, we assessed extracellular levels
of catecholamines after a single s.c. injection of 10 mg/
kg morphine. Neurotransmitter levels were monitored
using custom-made probes with 2-mm-long microdialysis windows and tips extending into the medial
NAc (Fig. 4 a ; Supplementary Fig. S1). We found a robust morphine-induced increase of NA in the medial
NAc/striatum of both control and NR1DbhCre mice
(Fig. 4 c ; time : F8,80=3.3, p<0.01 ; genotype : F1,10=3.4,
p=0.096). The injection of morphine caused a slight
increase of DA (n.s., Fig. 4 d), accompanied by a robust
y1.5-fold increase in the extracellular concentrations
of DA metabolites (Fig. 4e, f ; time : F8,80=4.2 and 5.8,
both p<0.001) without differences between control
and mutant mice. There were no significant differences between NR1DbhCre mice and littermate controls
in terms of basal extracellular concentrations of catecholamines and related metabolites (Supplementary
Table S1). In conclusion, neurotransmitter measurements demonstrated that morphine treatment robustly
increases extracellular NA levels in the medial NAc/
striatum.
Psychomotor and reinforcing effects of morphine
and naloxone
In order to test the role of NMDA receptor inputs
in development of drug-conditioned behaviours we
tested the development of psychomotor sensitization
and CPP in NR1DbhCre mice and control animals. A
schematic diagram of the procedure is shown in Fig. 5 a.
We found that acute drug treatments caused an expected increase in activity in both mutant and control
mice (Fig. 5, Supplementary Table S2). However, locomotor activity of NR1DbhCre mice was not further increased upon intermittent treatment with morphine
(10 mg/kg i.p.), while controls developed normal psychomotor sensitization (Fig. 5 b ; genotype : F1,48=15.1,
p<0.01 ; day : F4,48=2.4, p=0.065 ; genotyperday :
F4,48=4.6, p<0.01 ; for complete statistics see Table S2).
This observation was validated in an independent cohort of animals (Supplementary Table S2), confirming
that the phenotype was stable in different generations
of mutant mice. We reasoned that if NMDA receptordependent signalling was necessary for morphineinduced NA release, then pharmacological blockade of
NA release should replicate the mutant phenotype.
J. R. Parkitna et al.
1464
(b)
10
NR1 DbhCre
Control
8
Spikes
(c)
Spontaneous activity (Hz)
(a)
6
Control
4
2
0
100
200
NR1DbhCre
300
(d)
Control
(e)
0.2
0
NR1DbhCre
8
4
2s
–16
–20
–24
–28
Mg free +CGP
Mg free +CGP
Mg free +CGP
Control
NR1DbhCre
Control
NR1DbhCre
(h)
(i)
8
4
**
6
Tau (ms)
Rise (ms)
–12
Mg free +CGP
5
10 ms
NR1DbhCre
–32
0
5 pA
Mg free +CGP
Control
–8
12
20 pA
NR1DbhCre
Mg free +CGP
(f)
Amplitude (pA)
Frequency (Hz)
NR1DbhCre
Mg2+ free
+5 µM CGP37849
0.4
10 mV
16
Control
0.6
10 s
Current step (pA)
(g)
0.8
3
2
4
2
1
0
0
Mg free +CGP
Mg free +CGP
Mg free +CGP
Mg free +CGP
Control
NR1DbhCre
Control
NR1DbhCre
Fig. 3. Whole-cell patch-clamp recordings of neurons in the locus coeruleus (LC). (a) Stimulus–response curves of LC putative
NA neurons in slices obtained from NR1DbhCre mice ($) and control animals (#). Data points represent the mean number of
spikes evoked by 500 ms depolarizing current pulses¡S.E.M. Analysis was performed on six NA neurons from five control mice
and 10 NA neurons from five NR1DbhCre animals. (b, c) The spontaneous activity of LC neurons maintained at x50 mV in the
absence of Mg2+ ions, before and after administration of the NMDA receptor antagonist CGP37849. Examples shown in (b) were
recorded before CGP37849 administration. Several recorded neurons displayed no spontaneous activity, and overlap at 0
frequency (c). Data were collected from eight NA neurons from five control mice and 13 neurons from five NR1DbhCre animals.
(d–f ) Spontaneous excitatory post-synaptic current (sEPSC) recordings in LC neurons under Mg2+-free conditions indicate
higher frequencies in cells from NR1DbhCre mice compared to controls (d, e) without a significant difference in amplitude ( f ).
CGP37849 treatment had no effects on either frequency or amplitude. The examples shown in (d) were recorded before
CGP37849 administration. Recordings were performed on eight NA neurons from five control mice and 13 NA neurons from
five NR1DbhCre animals. (g–i) Spontaneous EPSCs from the LC of control (left) and NR1DbhCre (right) mice are differentially
affected by administration of the NMDA receptor antagonist CGP37849 (g). While the ‘rise ’ intervals (time elapsed from 10 % to
90 % of amplitude) are similar (h), the ‘tau’ (time elapsed from 100 % to 37 % of amplitude) is reduced by administration of
CGP37849 in control mice (i) but not NR1DbhCre animals. Analyses were performed on eight NA neurons derived from five
control mice and 13 NA neurons from five NR1DbhCre animals. Significant difference (paired t test with Bonferroni’s correction
p<0.01) in mean tau time in control neurons before and after CGP37849 administration is indicated by ‘**’.
Glutamate inputs to NA cells regulate opiate effects
(a)
1465
(b)
M2
M1
9000
Cg1
DbhCre
NR1
S1J
Cg2
S1ULp
Control
7000
IG
GI
DP
DI
LSD
CPu
AID
LSV
CI
AIV
SHi
LST
pg/mg
IG
1000
M
DEn
500
Pir
LAcbSh
5000
1500
AcbC
VI
LSS
0
VP
(c)
(d)
DbhCre
NRI
*
#
400
DA
NA
Control
#
#
#
200
% basal value
600
% basal value
HV
A
ICj
DA
DO
PA
C
+1.18
NA
VP
200
100
100
–60 0
60 120 180 240
–60 0
Drug administration (min)
Drug administration (min)
(e)
60 120 180 240
(f)
HVA
*
200
% basal value
% basal value
DOPAC
100
–60 0
60 120 180 240
Drug administration (min)
#
200
100
–60 0
60 120 180 240
Drug administration (min)
Fig. 4. Content and extracellular levels of catecholamines in the nucleus accumbens. (a) Diagram of a coronal brain section shows
NAc area used for catecholamine analysis. Grey circle indicates the piece punched out for tissue content measurements. The
thick black line indicates approximate microdialysis probe placement, the length of the line corresponds to the y2-mm-long
dialysis window extending from the tip. The number below the diagram (+1.18) is the distance from bregma (Paxinos &
Franklin, 2001). (b) Catecholamine content (pg/mg of tissue). Values are mean¡S.E.M. (n=6 mice per group). (c–f ) Relative levels
of extracellular catecholamines and their metabolites measured after s.c. injection of 10 mg/kg morphine (at minute 0). The lines
on the graphs connect points representing mean values normalized to the median of the four measurements performed prior to
morphine injection. Dotted line and open symbols (#) correspond to controls, solid line and solid symbols ($) represent
NR1DbhCre animals. (c) Measured relative changes in extracellular NA levels, (d) corresponds to DA, (e) DOPAC and ( f ) HVA.
Values are mean¡S.E.M. (n=6 animals per group). Significant differences (Dunnett’s post-hoc test p<0.05) vs. basal levels
(minute 0) are indicated by ‘*’ for control and ‘#’ for NR1DbhCre mice.
Indeed, when 30 min before morphine injections wildtype C57BL/6 mice were pretreated with clonidine
(0.03 mg/kg i.p.), an a2-adrenergic receptor agonist,
psychomotor sensitization did not develop (Fig. 5c ;
treatment : F1,72=12.6, p<0.01 ; day : F4,72=4.3, p<0.01 ;
dayrtreatment : F4,72=2.1, p=0.08). Clonidine treatment at this dose significantly decreased basal locomotor activity (treatment : F1,56=71.32, p<0.001).
Conversely, a drug that activates NA release independently of NMDA receptor signalling rescued
J. R. Parkitna et al.
1466
Drug
Sal
(a)
Day
1
Test
2
3
Prime
4–7
8
(b)
13
Test
6
Control
4
**
2
*
***
5
7
***
Beam crossings x103
NR1DbhCre
29
Test
WT + clonidine 0.03 mg/kg
WT + saline
4
*
* ***
2
0
0
Sal
3
10
12
Sal
3
Morphine
10 mg/kg
(d)
5 7 10 12
Morphine
10 mg/kg
(e)
6
6
NR1DbhCre + RX 0.5 mg/kg
Beam crossings x103
Control + saline
Beam crossings x103
28
Test
(c)
6
Beam crossings x103
9–12
10 x
14–23
4
2
WT + RX 0.5 mg/kg
WT + saline
4
2
0
0
Sal
3
5
7
10
12
Sal
3
5
7
10 12
Morphine
10 mg/kg
Fig. 5. Drug-induced psychomotor sensitization. (a) Experimental design. The boxes represent sessions in the conditioned place
preference (CPP) apparatus. Drugs or saline were administered on days marked by syringe symbols. On ‘test’ days the animals
were allowed to explore the entire CPP cage. (b–e) Locomotor activity after saline injection (day 2), first drug injection (day 3),
and after 5 d repeated treatment (day 12), (b) with 10 mg/kg morphine (n=7 mice per group), (c) 0.03 mg/kg clonidine 30 min
prior to 10 mg/kg morphine (n=10 mice per group), (d) 0.5 mg/kg RX 821002 30 min prior to 10 mg/kg morphine (n=8
mutants, 11 controls) and (e) 0.5 mg/kg RX 821002 alone (n=6 mice per group). Data shown are mean¡S.E.M., significant
differences (Bonferroni’s post-test) between mutant and control mice are indicated by : * p<0.05, ** p<0.01 and *** p<0.001.
WT, Wild-type.
the sensitization in NR1DbhCre mice. Psychomotor sensitization was rescued when, 30 min before morphine
injections, NR1DbhCre animals were pre-treated with
RX821002 (i.p. 0.5 mg/kg), an a2-adrenergic receptor
antagonist (Fig. 5d ; genotype : F1,68<1, n.s. ; treatment :
F4,68=9.2, p<0.001 ; genotypertreatment : F4,68<1,
n.s.). At the same time, pre-treatment with RX821002
had no further effect on ambulation in control mice,
and injections of RX821002 (1 mg/kg) alone had no
effect on activity of wild-type C57BL6 mice (Fig. 5 e).
Thus, morphine-induced psychomotor sensitization
did not develop in NR1DbhCre animals, but the
phenotype could be rescued by facilitating NA release
at the synapse.
To assess the consequences of the mutation with
regard to behavioural reinforcement we measured
morphine-induced CPP and naloxone-induced CPA.
Both NR1DbhCre and control mice acquired similar
morphine CPP, extinction and reinstatement (Fig. 6 a ;
genotype : F1,12=1.7, n.s. ; CPP stage : F2,24=16.05,
p<0.001). The trend towards stronger preference
for the CS+ compartment observed in NR1DbhCre mice
after CPP and reinstatement was not significant.
Naloxone-induced CPA was normal in NR1DbhCre mice
Glutamate inputs to NA cells regulate opiate effects
(a)
NR1DbhCre
Control
Morphine
400
#
CPP score
##
200
*
0
–100
13
28
29
Day
Naloxone
(b)
100
CPA score
0
1467
on pain sensitivity, opioid induced-analgesia and
withdrawal. Sensitivity to pain, as measured by tailflick and hot-plate tests, was similar in control and
NR1DbhCre mice (Supplementary Fig. S2). The analgesic
effects of morphine [analgesic dose (AD50) with 95 %
confidence intervals] and the development of tolerance to morphine analgesia after repeated treatment
with increasing doses of morphine did not differ between genotypes (Supplementary Fig. S2). After treatment with morphine and injection of the opioid
antagonist naloxone (4 mg/kg i.p.) to induce precipitated withdrawal (Fig. 7, Supplementary Table S3),
mutant mice displayed less jumping (genotype : F1,19=
12.04, p<0.01 ; treatment : F1,19=193.54, p<0.001 ; genotypertreatment : F1,19=12.04, p<0.01), paw tremor
(genotype : F1,19=3.5, p=0.07 ; treatment : F1,19=31.42,
p<0.001 ; genotypertreatment : F1,19=3.59, p=0.07)
and teeth chattering (genotype : F1,19=3.8, p=0.06 ;
treatment : F1,19=21.96, p<0.001 ; genotypertreatment : F1,19=3.81, p=0.06). Thus, NMDA receptors on
NA neurons act as an important modulator of behavioural symptoms of morphine withdrawal.
–200
Discussion
13
–400
28
29
Day
Fig. 6. Drug-induced conditioned place preference (CPP) or
conditioned place aversion (CPA). (a) Injections of 10 mg/kg
morphine induced CPP, while (b) treatment with 2 mg/kg
naloxone induced CPA. The values (‘scores ’) represent the
difference in time spent in the drug-paired compartment vs.
the saline-paired compartment, after conditioning (day 13),
after 10 sessions of extinction (day 28) and after reinstatement
of preference by drug injection (day 29). Data shown are
mean¡S.E.M. ; n=7 mice per group in the CPP experiment
and n=6 mice per group in the CPA experiment. Significant
differences (Bonferroni’s post-test) in time spent in the
drug-paired compartment after conditioning compared to
extinction is indicated by * p<0.05 and between extinction
and reinstatement by # p<0.05 or ## p<0.01.
(Fig. 6b ; genotype : F1, 10<1, n.s. ; CPP stage : F2,20=
5.24, p<0.05) Therefore, we conclude that the loss of
functional NMDA receptors in NA neurons does not
impair positive or negative reinforcement.
Morphine tolerance, physical dependence and
withdrawal in NR1DbhCre mice
The activity of NA neurons regulates endogenous analgesic systems and is strongly affected during opiate
physical dependence (Rasmussen et al. 1990). Therefore, we examined the consequences of the mutation
We show that NMDA-dependent input to NA cells
controls their involvement in the development of specific morphine-induced behaviours. NR1DbhCre mice do
not develop psychomotor sensitization after intermittent treatment, and show attenuated withdrawal
symptoms. However, the loss of functional NMDA
receptors in noradrenergic cells had no apparent effects on the anxiety levels, learning ability or sensitivity to reinforcement in mutant mice.
The lack of morphine-induced psychomotor sensitization in NR1DbhCre mice indicates a role for the noradrenergic system in incentive sensitization (Berridge
& Robinson, 1998). This phenotype was stable in two
independently bred cohorts of mutant mice, which
excludes generation-dependent effects. Moreover,
this phenotype could be reproduced using a pharmacological approach. When wild-type mice were preinjected with clonidine, an a2-adrenoceptor antagonist, which suppresses the brain’s NA system,
psychomotor sensitization to morphine did not develop. It should be noted that clonidine at the dose
of 0.03 mg/kg may be causing sedation, and we
observed a decrease in basal locomotor activity
in clonidine-treated mice. Moreover, although a2adrenoceptor ligands act mainly at presynaptic sites,
involvement of a post-synaptic NA mechanism or interaction with other neurotransmitter systems may not
be completely excluded. Conversely, NR1DbhCre mice
J. R. Parkitna et al.
1468
(a)
Nlx or sal
Morphine
3h
Day 1
Day 2
Day 3
Day 4
Day 5
10 mg/kg
s.c.
20 mg/kg
s.c.
40 mg/kg
s.c.
40 mg/kg
s.c.
40 mg/kg
s.c.
NR1DbhCre
Control
(b)
***
25
0
Nlx
Sal
Teeth chatering
Paw tremor
Jumps
50
18
15
75
10
*
5
0
12
*
6
0
Nlx
Sal
Nlx
Sal
Fig. 7. Morphine withdrawal. (a) Morphine was administered s.c. at increasing doses for 5 d, and then withdrawal was
precipitated by injection of 4 mg/kg naloxone (Nlx). Control groups of animals were challenged with saline. (b) Bar graphs
summarize behavioural symptoms of morphine withdrawal : numbers of jumps, teeth chattering and paw tremors. Data are
shown as mean¡S.E.M., control (n=7) and mutant (n=6) mice were treated with naloxone, and five per genotype with saline.
Significant differences (Bonferroni’s post-hoc test) between naloxone-treated mutant and control mice are indicated by * p<0.05
and *** p<0.001.
had normal activity in the open-field apparatus, and
furthermore, when they were treated with RX821002, a
selective a2-adrenoceptor antagonist which augments
NA release, morphine psychomotor sensitization was
rescued. Together, this strongly indicates that NMDA
receptors are necessary for morphine-induced NA release, which in turn is essential for the development of
psychomotor sensitization.
Here we report for the first time that the administration of morphine produced a robust, y3-fold increase of extracellular NA concentration in the medial
NAc/striatum. Based on previously published data,
this implies activation of NA inputs from the NTS and
area A1, sole sources of NA in the NAc/striatum,
chiefly projecting to the medial part of the NAc shell
(Delfs et al., 1998, 2000 ; Olson et al. 2006). It is not clear
what the mechanism responsible for the NA increase
could be, especially since direct administration of
opioids inhibits activity of neurons (e.g. Williams et al.
1982). However, previous microdialysis studies found
an increase in extracellular NA in the prefrontal cortex
after morphine injection (Ventura et al. 2005). Thus,
although direct action of morphine on NA cells is inhibitory, the net effect is an increase in their activity. In
regard to our study, there is sparse data on origins of
excitatory inputs on NA cells in the NTS or A1 with
ascending projections to the striatum/NAc. Likely
candidates are glutamatergic neurons from the brainstem and also excitatory afferents originating from the
limbic brain areas or the hypothalamus (Baude et al.
2009).
We found a mild increase in DA tissue content in
the NAc of NR1DbhCre mice, which was not associated
with changes in its metabolite levels. Conversely, we
observed that both in control and mutant mice morphine treatment caused a significant increase in extracellular levels of DA metabolites in the NAc (DOPAC
and HVA), but levels of DA itself were only slightly
increased (<30 % compared to basal level). The
change in extracellular DA levels is lower than expected based on previous reports (Chefer et al. 2003 ;
Murphy et al. 2001). It should be noted, however, that
the construction of the probe, its placement and the
protocol used here were not identical to previous reports. Since a clear morphine-induced increase in
extracellular levels of DA metabolites was observed,
which implies a rise in extracellular DA levels, we
believe that our data is in agreement with previous
reports.
Interestingly, our data shows that the NMDA receptor-dependent inputs to NA neurons are either not
involved or contribute weakly to the development of
Glutamate inputs to NA cells regulate opiate effects
CPP or CPA. This is surprising because psychostimulant- or morphine-induced CPP was altered or abolished in Dbh and Adra1b knockout mice (Drouin et al.
2002 ; Olson et al. 2006). Moreover, there is ample evidence from pharmacological studies of NA system
involvement in morphine-induced reinforcement
(Hand et al. 1989 ; Mantsch et al. 2010 ; Tzschentke,
1998 ; Zarrindast et al. 2002). In addition, morphine
and other drugs induced NA release to the medial
prefrontal cortex, and this increase was correlated to
an increase in extracellular DA levels in the NAc as
well as the development of CPP (Ventura et al. 2005).
Therefore, although NA signalling is involved in
drug-conditioned responses, the NMDA receptordependent inputs are not essential in these mechanisms. Furthermore, based on previous reports and
data reported here, we hypothesize that DA pathways
have a dominant role in positive reinforcement,
whereas NA signalling controls psychomotor sensitization (Airio & Ahtee, 1997 ; Tassin, 2008). Indeed,
loss of functional NMDA receptors on DA neurons in
NR1DATCre mutants interferes with CPP, but has no
effect on psychomotor sensitization (Engblom et al.
2008). The complementarities between phenotypes
observed previously in NR1DATCre mice and reported
here for NR1DbhCre animals strongly indicate distinct
behavioural roles of NMDA receptor-dependent plasticity in DA vs. NA neurons.
It should also be noted that the NA neurons play
a central role in drug physical dependence, becoming strongly activated during opiate withdrawal
(Aghajanian, 1978 ; Gold et al. 1978 ; Rasmussen et al.
1990). At the cellular level, the development of physical dependence was found to involve adaptive
changes in intracellular cAMP-dependent signalling
in LC NA neurons (Duman et al. 1988). However, the
exact role of this adaptation remains unclear, as it was
reported that lesions of the LC do not attenuate
symptoms of opiate withdrawal (Christie et al. 1997).
Furthermore, a specific deletion of cAMP-response element binding protein (CREB) in NA cells had minor
effects on withdrawal symptoms (Parlato et al. 2010). It
was even suggested that the anatomical loci of withdrawal are non-NA neurons, and other brain areas,
particularly the GABAergic neurons of the periaqueductal grey matter, are the actual substrates (Christie
et al. 1997). Our data indicate that the NMDA receptormediated signalling in NA neurons modulates opiate
dependence, as the withdrawal symptoms were alleviated in NR1DbhCre mice. This result is in agreement
with the proposed role of the NA system and the glutamatergic afferents in mediating opioid withdrawal
(Akaoka & Aston-Jones, 1991 ; Nestler et al. 1999). We
1469
suggest a more significant role of NMDA receptors
than previously attributed (Rasmussen, 1995), perhaps
partially accounting for the observed alleviation of
withdrawal symptoms following treatment with
NMDA antagonists such as MK-801 (Trujillo & Akil,
1991).
Our results indicate that the importance of NA signalling in development of drug-conditioned behaviours has been underappreciated. Further efforts
will focus on the significance of NMDA receptordependent plasticity of NA neurons in control of
motivated behaviours.
Note
Supplementary material accompanies this paper on
the Journal’s website (http ://journals.cambridge.org/
pnp).
Acknowledgements
We thank Professor Günther Schütz for his support.
J.R.P., W.S., J.K., S.G. and R.Prz. were supported
by grants from the EU LSHM-CT-2004-005166 GENADDICT and LSHM-CT-2007-037669 PHECOMP, as
well as the Polish Ministry of Science and Higher
Education (MSHE) subsidiary grants 26/E-40/6.PR
UE/DIE 305/2005-2008, 936/6.PR UE/2009/7 and
478/6.PR UE/2007/7. J.R.P. and R.Prz. were also
supported by grant N405 143238 from the MSHE.
W.S. received support from the Foundation for Polish
Science individual grant ‘START ’. K.G., K.T. and G.H.
were supported by statutory funds from the MSHE
awarded to the Institute of Pharmacology. R.S. was
supported by the ‘Deutsche Forschungsgemeinschaft ’
through the Collaborative Research Center SFB636/
A4. M.N. and R.Pa. were supported by the Helmholtz
Gemeinschaft Deutscher Forschungszentren through
the Helmholtz Alliance for Mental Health in an Ageing Society (HelmA, HA-215). Language revision of
one of the versions of the manuscript was performed
by an external editor at ‘American Journal Experts ’.
Statement of Interest
None.
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