International Journal of Neuropsychopharmacology (2010), 13, 891–903.

Copyright f CINP 2009 ARTICLE

doi:10.1017/S1461145709990794

Adjunctive a2-adrenoceptor blockade enhances

the antipsychotic-like effect of risperidone
and facilitates cortical dopaminergic and
glutamatergic, NMDA receptor-mediated
transmission

Monica M. Marcus1, Charlotte Wiker1, Olivia Frånberg1, Åsa Konradsson-Geuken1,

Xavier Langlois2, Kent Jardemark1 and Torgny H. Svensson1

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1
Department of Physiology and Pharmacology, Section of Neuropsychopharmacology, Karolinska Institutet, Stockholm, Sweden
2
Neuroscience, RED Europe, Johnson & Johnson Pharmaceutical Research and Development, Turnhouseweg, Beerse, Belgium

Abstract
Compared to both first- and second-generation antipsychotic drugs (APDs), clozapine shows superior
efficacy in treatment-resistant schizophrenia. In contrast to most APDs clozapine possesses high affinity
for a2-adrenoceptors, and clinical and preclinical studies provide evidence that the a2-adrenoceptor an-
tagonist idazoxan enhances the antipsychotic efficacy of typical D2 receptor antagonists as well as olan-
zapine. Risperidone has lower affinity for a2-adrenoceptors than clozapine but higher than most other
APDs. Here we examined, in rats, the effects of adding idazoxan to risperidone on antipsychotic effect
using the conditioned avoidance response (CAR) test, extrapyramidal side-effect (EPS) liability using the
catalepsy test, brain dopamine efflux using in-vivo microdialysis in freely moving animals, cortical N-
methyl-D-aspartate (NMDA) receptor-mediated transmission using intracellular electrophysiological re-
cording in vitro, and ex-vivo autoradiography to assess the in-vivo a2A- and a2C-adrenoceptor occupancies
by risperidone. The dose of risperidone needed for antipsychotic effect in the CAR test was y0.4 mg/kg,
which produced 11 % and 17 % in-vivo receptor occupancy at a2A- and a2C-adrenoceptors, respectively.
Addition of idazoxan (1.5 mg/kg) to a low dose of risperidone (0.25 mg/kg) enhanced the suppression of
CAR, but did not enhance catalepsy. Both cortical dopamine release and NMDA receptor-mediated re-
sponses were enhanced. These data propose that the therapeutic effect of risperidone in schizophrenia can
be enhanced and its EPS liability reduced by adjunctive treatment with an a2-adrenoceptor antagonist,
and generally support the notion that the potent a2-adrenoceptor antagonistic action of clozapine may be
highly important for its unique efficacy in schizophrenia.
Received 31 March 2009 ; Reviewed 18 May 2009 ; Revised 10 September 2009 ; Accepted 15 September 2009 ;
First published online 19 October 2009

Key words : a2-adrenoceptor, dopamine, glutamate, receptor occupancy, schizophrenia.

Introduction 1997), and also, in comparison with second-generation

Clinical studies have shown that clozapine exerts
superior clinical efficacy compared to first-generation
antipsychotic drugs (FGAs) in treatment-resistant
schizophrenia, including positive, negative and cog-
nitive symptoms (Kane et al. 1988 ; Rosenheck et al.
Address for correspondence : Professor T. H. Svensson, Department
of Physiology and Pharmacology, Section of Neuro-
psychopharmacology, Karolinska Institutet, S-171 77 Stockholm,
Sweden.
Tel. : +46 8 524 8 7921 Fax : +46 8 30 84 24
Email : Torgny.Svensson@ki.se
antipsychotic drugs (SGAs) clozapine is the most
effective drug for individuals with a poor symptom
response to previous antipsychotic drug (APD) treat-
ment (Davies et al. 2008 ; Lewis et al. 2006 ; McEvoy et al.
2006 ; Swartz et al. 2008). In addition, clozapine has
shown superiority compared to FGAs and the SGA
olanzapine in reducing suicidality in schizoaffective
disorders and schizophrenia (Meltzer et al. 2003).
Indeed, the efficacy of clozapine seems to be superior
to both FGAs and most, if not all SGAs (Davis et al.
2003 ; Leucht, 2009). Besides its modest affinity for the
D2 receptor, clozapine possesses high affinity for a
892 M. M. Marcus et al.
number of other receptors, including a potent a2-
adrenoceptor antagonistic action (Ashby & Wang,
1996 ; Shahid et al. 2009). Interestingly, adjunctive
treatment with idazoxan, a selective a2-adrenoceptor
antagonist, has been found to enhance the effect of
FGAs in treatment-resistant schizophrenia (Litman
et al. 1993, 1996).
Both negative symptoms and cognitive dysfunction
in schizophrenia are thought to be related to a hypo-
dopaminerigic state in the prefrontal cortex (PFC ;
e.g. Stone et al. 2007). Prefrontal dopamine, specifically
via dopamine D1 receptors, has been shown to be
importantly involved with normal cognitive function
(Goldman-Rakic et al. 2000). In schizophrenia, both
clinical and preclinical results indicate an impairment
of prefrontal dopamine function (see Tan et al.
2007 ; Weinberger et al. 2001), and D1 receptor dys-
regulation is suggested to be critically involved in the
cognitive impairments associated with this disease
(Abi-Dargham & Moore, 2003 ; Abi-Dargham et al.
2002). Experimentally, SGAs, e.g. clozapine, increase
dopamine output preferentially in the PFC, whereas
FGAs, e.g. haloperidol, induce higher dopamine
output in subcortical areas such as the nucleus ac-
cumbens (NAc) and the striatum (Kuroki et al. 1999 ;
Moghaddam & Bunney, 1990 ; Nomikos et al. 1994 ;
Svensson, 2003). In accordance with these experimen-
tal observations some clinical studies suggest that
SGAs, but not FGAs, may improve various aspects of
cognitive dysfunction in schizophrenia (Davis et al.
2003 ; Meltzer & McGurk, 1999).
Glutamate, the main transmitter in cortical pyrami-
dal cells, is involved in higher mental functions, such
as cognition, memory and learning. Thus, antagonists
at the glutamatergic N-methyl-D-aspartate (NMDA)
receptors impair learning and memory, while NMDA
receptor agonists and facilitators may improve
memory (Francis, 2003 ; Francis et al. 1993). In addition,
NMDA receptor antagonists have, in healthy volun-
teers, been shown to be capable of inducing both
positive and negative symptoms as well as the formal
thought disorder that is a distinct feature of schizo-
phrenia (Javitt & Zukin, 1991), and to generate cog-
nitive impairment, e.g. verbal working memory
(Honey et al. 2003). Therefore, a glutamatergic synaptic
hypofunction in schizophrenia has been proposed
(see Coyle et al. 2003), a notion recently supported by
a reduced prefrontal expression of the NMDA recep-
tor subunits NR1, NR2A and NR2C in schizophrenia
(Beneyto & Meador-Woodruff, 2008). Significantly,
SGAs, but not FGAs have been found to facilitate
cortical NMDA receptor-mediated transmission
(Jardemark et al. 2002 ; Ninan et al. 2003).
Although the 5-HT2A/D2 ratio has been proposed as
a critical determinant of atypicality among the SGAs
(Meltzer, 1989 ; Meltzer et al. 1989), which may have
a bearing on their extrapyramidal side-effect (EPS)
liability, little attention has so far been focused on the
a2/D2 ratio of various APDs (but see Nutt, 1994).
Previous work from our group has shown that the
a2-adrenoceptor antagonist idazoxan enhances the
effect of a low dose of raclopride, a selective D2/3 re-
ceptor antagonist, on conditioned avoidance response
(CAR), a preclinical test with high predictive validity
for clinical antipsychotic effect (Wadenberg & Hicks,
1999), and similarly with SGAs, but not FGAs, pro-
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duces an increased dopamine efflux in the medial PFC
(mPFC ; Hertel et al. 1999a). Both clozapine and the
combination of idazoxan+raclopride have also been
shown to enhance cortical NMDA receptor-mediated
transmission, effects mediated by dopamine acting at
D1 receptors, as well as to reverse cognitive impair-
ment in rats induced by NMDA receptor blockade
(Marcus et al. 2005). Addition of idazoxan to low
doses of the FGA haloperidol and the SGA olanzapine,
both possessing low affinity for the a2-adrenoceptor
(Schotte et al. 1996 ; Shahid et al. 2009), also enhances
the effect of these APDs in the CAR test as well as on
cortical dopamine release (Wadenberg et al. 2007).
Risperidone possesses lower affinity for the
a2-adrenoceptor than clozapine but higher than most
other SGAs (Schotte et al. 1996 ; Shahid et al. 2009).
Therefore, in the present study, we investigated the
significance of additional a2-adrenoceptor blockade
by adding idazoxan to a low dose of risperidone and
examined the effect on antipsychotic efficacy using the
CAR test, EPS liability using a catalepsy test, dopa-
mine efflux in the mPFC and NAc using the micro-
dialysis technique in freely moving rats, and cortical
NMDA receptor-mediated transmission, using intra-
cellular electrophysiological recording in pyramidal
neurons in vitro. In addition, we assessed the in-vivo
a2A- and a2C-adrenoceptor occupancies of risperidone,
using ex-vivo autoradiography.
Methods
Animals
Adult male Wistar rats (200–270 g upon arrival) were
used for behavioural and microdialysis experiments,
whereas male Sprague–Dawley rats (y70 g upon
arrival ; B&K Universal, Sweden) were used for
in-vitro electrophysiological experiments. The animals
were housed under standard laboratory conditions
with food and water available ad libitum. For the

behavioural tests the animals were kept on a reversed
12 h light/dark cycle (lights off 06:00 hours), whereas
for the other experiments, animals were maintained
on a 12 h light/dark cycle (lights on 06:00 hours).
Experiments were approved by, and conducted in ac-
cordance with, the local Animal Ethics Committee,
Stockholm North and the Karolinska Institutet,
Sweden.
For the ex-vivo radioligand binding experiments
male Wistar rats (180–220 g) were housed in a rat
raising facility with food and water available ad libi-
tum. All procedures were conducted in strict accord-
ance with the European Communities Council
Directive of 24 November 1986 (86/609/EEC) and
were approved by the animal care and use committee
of Johnson and Johnson Pharmaceutical Research and
Development.
CAR behaviour
Rats were trained and tested in conventional shuttle
boxes (530r250r225 mm), divided into two com-
partments of equal size by a partition with an opening
(Salmi et al. 1994). Upon presentation of the 80-dB
white noise (Lafayette Instruments, USA) conditioned
stimulus (CS), the rats had 10 s to move from one
compartment of the shuttle box into the other. If the rat
remained in the same compartment for more than 10 s,
an intermittent electric shock (intershock interval 2.5 s,
shock duration 0.5 s) of y0.4 mA, i.e. the uncondi-
tioned stimulus (UCS), was presented to the grid floor
until an escape was performed. If the rat did not re-
spond within 50 s, the trial was terminated, i.e. escape
failure. Avoidance (response to CS within 10 s), escape
(response to CS+UCS) and escape failure (failure to
respond within 50 s) were recorded. The animals were
trained for 5 d, each session consisted of y20 trials
randomly distributed over 15 min. Only animals re-
liably performing >85 % avoidance were included in
the study. Experiments were preceded by a pre-test
and experiment sessions, lasting 10 min, were con-
ducted at 20, 90 and 240 min. Experimental days were
separated by at least two non-experimental days. The
animals were tested in a counterbalanced change-over
design serving as their own controls (Li, 1964).
Catalepsy measurements
Catalepsy was observed in a dimly lit room by placing
the animals on an inclined grid (60x) for a maximum of
2.5 min. The animals were allowed 30 s of adaptation
on the grid before observation started. The different
treatments were unknown to the observer. Catalepsy
was scored from 0 to 5, according to the time
a2 blockade enhances effects of risperidone 893
(square-root transformation) the rat remained immo-
bile (min) : 0=0.00–0.08 ; 1=0.09–0.35 ; 2=0.36–0.80 ;
3=0.81–1.42 ; 4=1.43–2.24 ; 5o2.25 (Ahlenius &
Hillegaart, 1986).
In-vivo microdialysis
Procedures for microdialysis were as previously de-
scribed (Schilström et al. 1998). Rats were anaes-
thetized with a cocktail of Hypnorm1 (0.315 mg/ml
fentanyl citrate and 10 mg/ml fluanisone ; Janssen-
Cilag Ltd, UK) and Dormicum1 (5 mg/ml mid-
azolam ; Roche AB, Sweden) diluted in distilled water
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[1 : 1 : 2 ; 5 ml/kg, intraperitoneal (i.p.)] mounted in a
stereotaxic frame, and implanted with dialysis probes
in the mPFC or NAc [AP (mm) : +2.6, +1.4 ; ML :
x0.6, x1.4 ; DV : x5.2, x8.2], respectively, relative to
bregma and dural surface (Paxinos & Watson, 1998).
Dialysis occurred through a semi-permeable mem-
brane (Filtral AN69, Hospal Industrie, France) with
an active surface length of 4 mm (mPFC) or 2.25 mm
(NAc). Dialysis experiments were conducted y48 h
after surgery in awake, freely moving rats. The dialy-
sis probe was perfused with a physiological perfusion
solution [in mM : 147 NaCl, 3.0 KCl, 1.3 CaCl2,
1.0 MgCl2, 1.0 NaHPO4 (pH 7.4)] at a rate of 2.5 ml/
min, set by a microinfusion pump (Harvard Ap-
paratus, USA). Dialysate samples were collected over
30 min (mPFC) or 15 min (NAc). Online quantification
of dopamine was accomplished by high performance
liquid chromatography (HPLC) coupled to electro-
chemical detection (ESA Bioscience, USA), with a de-
tection limit of y0.2 fmol/min. The injector (Valco
Instruments, USA) was directed by a computerized
system, Totalcrom WS version 6.3 (PerkinElmer,
USA). Separation of dopamine and metabolites was
achieved by reversed-phase liquid chromatography.
The mobile phase consisted of 55 mM sodium acetate
buffer (pH 4.0), 12 % methanol and 0.55 mM octane-
sulfonic acid delivered by an HPLC pump (model
2150, Pharmacia LKB, Sweden) on a C-18 column
(Nucleocil 150/75r4.6 mm, 5 mm), flow rate 0.8 ml/
min. After separation, the analyte was passed through
a guard cell with an applied oxidizing potential of
50 mV to reduce baseline. Samples were quantified by
sequential oxidation and reduction in a high sensitive
analytical cell (model 5011 ; ESA Bioscience) that was
controlled by a potentiostat (Coulochem II model
5200 ; ESA Bioscience) with applied potentials of
400 mV and x200 mV for detection of metabolites and
dopamine. Injection of drug was performed after a
stable outflow (<10 % variation) of dopamine and
metabolites. Baseline was calculated as the average of
894 M. M. Marcus et al.

the last two (mPFC) or four (NAc) pre-injection values.
The placement of the probe was later verified in slices
stained with Neutral Red.
Electrophysiological experiments
Procedures for electrophysiological experiments were
as previously described (Konradsson et al. 2006).
Briefly, the rats were decapitated while under
halothane anaesthesia (AstraZeneca AB, Sweden). The
brains were quickly removed and cooled in ice-cold
Ringer’s solution [in mM : 126 NaCl, 2.5 KCl, 2.4 CaCl2,
1.3 MgCl2, 1.2 NaH2PO4, 10 D-glucose, 18 NaHCO3
the Ringer’s solution. All drugs were diluted in
Ringer’s solution and administered via bath perfusion.
The effects of the drug/drug combination on the
NMDA-induced current were calculated as percent of
NMDA-induced current, before and after bath appli-
cation of the drug/drug combination.
Ex-vivo receptor binding
Rats were treated by vehicle or risperidone [0.16–
10 mg/kg subcutaneously (s.c.)]. Six animals per dose
were used. The animals were decapitated 1 h after
drug administration. Brains were immediately re-
moved from the skull and rapidly frozen in dry-ice

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(pH 7.4)] aerated by 95 % O2 : 5 % CO2. The brains were
cooled 2-methylbutane (x40 xC). Sections (20-mm
cut coronally into 450-mm slices, using a Vibroslice
thick) were cut using a Leica CM 3050 cryostat-
(Campden model MA 752, World Precision
microtome (van Hopplynus, Belgium), and thaw-
Instruments, USA), and kept submerged in aerated
mounted on microscope slides (SuperFrost Plus,
Ringer’s solution at room temperature for >1 h to al-
LaboNord, France). The sections were kept at x20 xC
low for recovery. A slice containing mPFC (approxi-
until required.
mately AP +3.2 mm from bregma) was transferred to
The occupancy of a2A- and a2C-adrenoceptors by
a recording chamber (32 xC), and held submerged be-
risperidone was measured by ex-vivo autoradiog-
tween two nylon nets. The chamber was continuously
raphy using the radioligands [3H]RS79948-197 and
perfused with aerated Ringer’s solution, flow rate
[3H]rauwolscine as previously described (Marcus et al.
1.5–2.5 ml/min. Electrodes were pulled from boro-
2005). Quantitative autoradiography analysis was
silicate glass capillaries (i.d. 0.58 mm ; Clark
performed with the b-imager (Biospace, Paris) accord-
Electromedical Instruments, UK) by using a horizontal
ing to our standard protocol (Langlois et al. 2001). a2A-
electrode puller (model P-87, Sutter Instruments,
and a2C-adrenoceptor occupancy were measured by
USA). Recording electrodes were filled with 2 M KAc,
quantifying the specific binding of [3H]RS79948-197 in
tip resistance 55–120 MV, and used for recording with
the septum and the specific binding of [3H]rauwolscine
an Axoclamp 2A amplifier (Molecular Devices, USA).
in the striatum, respectively. The percentage of recep-
Penetration of cells by a sharp electrode was per-
tor occupancy was plotted against dosage and the sig-
formed blindly. The electrophysiological criteria for
moidal log dose–effect curve of best fit was calculated
distinguishing presumed pyramidal from non-pyr-
by nonlinear regression analysis, using the GraphPad
amidal neurons have been described previously
Prism program (USA). From these dose–response
(Arvanov et al. 1997). It is very rare to impale fast-
curves, the ED50 (the drug dose producing 50 % recep-
spiking interneurons with a relatively low resistance
tor occupancy) were calculated, with their 95 % confi-
microelectrode (McCormick et al. 1985), which might
dence limits.
account for not recording fast-spiking non-pyramidal
cells. The presumed pyramidal cells of the mPFC
Statistics
have relatively long spike duration (1–3 ms at half-
maximum spike amplitude) and show a pronounced Data from the behavioural experiments are not nor-
spike-frequency adaptation in response to constant- mally distributed and accordingly non-parametric
current depolarization pulses. Single electrode tests are used. Thus, for the CAR data we used the
voltage-clamp (holding potential x60 mV) was per- Friedman two-way analysis of variance (ANOVA)
formed in the discontinuous mode with a sampling followed by Wilcoxon matched-pairs signed-ranks
rate of 5–6.2 kHz. The voltage-clamp recordings were test, and for the catalepsy data the Kruskal–Wallis
acquired using digital/analogue sampling and acqui- one-way ANOVA followed by Mann–Whitney U test.
sition software (Clampex version 9.2, Molecular Statistical evaluation of microdialysis data over time
Devices, USA). During the voltage-clamp recordings was performed by two-way ANOVA for repeated
of NMDA (10–15 mM)-evoked currents, tetrodotoxin measures followed by planned comparison test. To
(0.5 mM, to block action potentials), glycine (1 mM, to analyse differences between different treatments, we
enhance NMDA-induced responses), and bicuculline also measured the overall effects (AUC=area under
(5 mM, to block GABAA responses) were included in curve), i.e. interval 60–180 min for mPFC and

45–180 min for NAc. The overall effects were statisti-
cally evaluated by one-way ANOVA, followed by
planned comparison test. One-way ANOVA was
used to detect differences between baseline values.
Statistical evaluation of the electrophysiological ex-
periments was performed by one-way ANOVA fol-
lowed by post-hoc least significant difference (LSD)
test. In all statistical measures p<0.05 was considered
significant. The statistical evaluations were performed
by using Statistica version 8.0 (StatSoft Inc., USA).
Drugs
Risperidone was provided by Johnson & Johnson
Pharmaceutical Research & Development, division of
Janssen Pharmaceutica NV, Belgium. Idazoxan HCl,
bicuculline methiodide, glycine and NMDA were
purchased from Sigma-Aldrich, USA. Tetrodotoxin
was obtained from Tocris, UK. For in-vivo experiments
risperidone was dissolved in a minimal amount of
acetic acid with a 5.5 % glucose solution added to
volume and idazoxan was dissolved in saline (0.9 %
NaCl). For electrophysiological experiments, stock
solutions of risperidone (dissolved in dimethyl sulf-
oxide) and idazoxan (dissolved in purified water)
were prepared.
Results
Effects of risperidone alone and in combination
with idazoxan on CAR behaviour
The dose–response relationship for the antipsychotic-
like effect of risperidone was established. Risperidone
(0.25, 0.3, 0.4, 0.5 mg/kg, n=11) produced a sup-
pression of CAR [20 min : x2(4)=32.84, p<0.001,
Fig. 1 a ; 90 min : x2(4)=16.07, p<0.01, Fig. 1 b] in a
dose-dependent manner. All doses of risperidone
produced statistically significant suppression of CAR
at 20 min (p<0.01–0.001) and 0.4 and 0.5 mg/kg at
90 min (p<0.05). At 240 min all animals were back to
baseline performance. The results show that the dose
needed for antipsychotic effect, i.e. y80 % suppression
of CAR (Wadenberg et al. 2001) is between 0.4 and
0.5 mg/kg. However, at 20 min both 0.4 and 0.5 mg/
kg risperidone induced some escape failures, 4 and 5
of 11 rats, respectively, indicating that these doses also
produce non-specific behavioural effects. ED50 was
0.28 mg/kg.
In another set of animals (n=12), pre-treatment
with 1.5 mg/kg idazoxan was added to a dose of ris-
peridone, that by itself did not produce a suppression
of CAR sufficient for antipsychotic effect, i.e. 0.25 mg/
kg. A statistically significant suppression of CAR was
a2 blockade enhances effects of risperidone 895
(a)
100
**
% Avoidance
***
50 ***
***
0
Vehicle 0.25 0.3 0.4 0.5
Risperidone (mg/kg i.p.), 20 min
(b)
100 *
*
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% Avoidance
50
0
Vehicle 0.25 0.3 0.4 0.5
Risperidone (mg/kg i.p.), 90 min
Fig. 1. Effects of risperidone (0.25, 0.3, 0.4, and 0.5 mg/kg i.p.)
on conditioned avoidance response (CAR) behaviour in rats
at (a) 20 and (b) 90 min after administration of drug. The
results are presented as median (avoidance %)¡semi-
interquartile range. Animals (n=11) are serving as their
own control in a change-over design (Li, 1964). * p<0.05,
** p<0.01, *** p<0.001 for vehicle vs. risperidone-treated
animals.
found at 20 min [x2(3)=22.88, p<0.001, Fig. 2 a], but
not at 90 min (Fig. 2 b). No escape failures were in-
duced. Compared to vehicle, 0.25 mg/kg risperidone
enhanced suppression of CAR (p<0.01). Compared
to risperidone alone, the combination of risperidone+
idazoxan also significantly enhanced the suppression
of CAR (p<0.01).
The relatively transient effect of risperidone in the
CAR test, i.e. that it declined after 90 min and returned
to baseline at 240 min, is in all probability related to
the concomitantly declining degree of D2 occupancy,
since this has been shown to peak at 30 min and begin
to decline after 1 h (Olsen et al. 2008).
Effects of risperidone alone and in combination
with idazoxan in the catalepsy test
Risperidone (0.3, 0.5 and 1.0 mg/kg) was tested in the
catalepsy test [30 min : x2(3)=9.50, p<0.05 ; 60 min :
x2(3)=11.61, p<0.01 ; 120 min : x2(3)=2.58, p=0.46 ;
Table 1a]. Compared to vehicle, risperidone (0.3 and
0.5 mg/kg) reached statistically significant levels at
30 min (p<0.05–0.01), and 1.0 mg/kg risperidone
at both 30 and 90 min (p<0.05–0.01). However, the
896 M. M. Marcus et al.
(a) **
100
##
++
% Avoidance
50
0 there were a statistically significant difference between
Vehicle Risperidone
0.25 mg/kg i.p.
+ saline
+ idazoxan 1.5 mg/kg s.c.
(b)
100
% Avoidance
50
0 vehicle, all treatments were significantly higher (p<
Vehicle Risperidone
0.25 mg/kg i.p.
Fig. 2. Effects of risperidone (0.25 mg/kg i.p.) or idazoxan
(1.5 mg/kg s.c.), alone and in combination with risperidone,
on conditioned avoidance response (CAR) behaviour in rats
at (a) 20 and (b) 90 min after administration of vehicle or
risperidone (saline/idazoxan was administered 10 min
before vehicle/risperidone). The results are presented as
median (avoidance %)¡semi-interquartile range. Animals
(n=12) are serving as their own control in a change-over
design (Li, 1964). ## p<0.01 risperidone vs. vehicle ;
++
p<0.01 idazoxan vs. the combination of idazoxan and
risperidone ; ** p<0.01 risperidone vs. the combination of
idazoxan+risperidone.
median catalepsy scores were all <2, the level at
which catalepsy is considered to begin (Wadenberg
et al. 2001). In addition, neither 1.5 mg/kg idazoxan,
0.25 mg/kg risperidone nor the combination of these
drugs showed any catalepsy (Table 1 b).
Effects of risperidone alone and in combination
with idazoxan on dopamine output in the mPFC
and the NAc
The mean¡S.E.M. basal dopamine level in the mPFC
and NAc were 0.34¡0.03 fmol/min (n=24) and
3.18¡0.43 fmol/min (n=25), respectively. No statisti-
cally significant differences between mean baseline
concentrations of dopamine output were found be-
tween different treatment groups within the same
brain region studied. Vehicle injections had no stat-
istically significant effect on dopamine output.
Statistical evaluation for dopamine output in the
mPFC (Fig. 3 a) revealed a significant treatment
(F3,20=8.07, p<0.001), time (F8,160=22.00, p<0.001)
as well as interaction effect (F24,160=3.02,
p<0.001). Compared to baseline, 0.25 mg/kg risper-
idone increased dopamine efflux in the mPFC at 60–90
and 150 min (p<0.05–0.01), 1.5 mg/kg idazoxan at
60–150 min (p<0.05) and the combination at
30–210 min (p<0.05–0.01). For the interaction effect,
vehicle and risperidone at 90 and 150 min (p<0.05), ve-
hicle and idazoxan at 90 and 150 min (p<0.05), vehicle
and the combination at 60–180 min (p<0.01–0.001).
There was also a statistically significant difference
between risperidone and the combination at 60 and
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120 min (p<0.05). In order to assess the effects of the
two drugs in combination, we analysed the overall
effect following injection of the second drug, AUC
(60–180 min). The overall effect was statistically sig-
nificant (F3,20=9.06, p<0.001). Compared to saline/
0.01–0.001) and the combination of risperidone+
idazoxan was statistically higher than both risper-
idone (p<0.05) and idazoxan (p<0.05), when given
alone.
Statistical evaluation for dopamine output in the
NAc (Fig. 3 b) revealed a significant treatment (F3,21=
4.73, p<0.01), time (F16,336=14.77, p<0.001) as well as
interaction effect (F48,336=4.73, p<0.001). Compared to
baseline, 1.5 mg/kg idazoxan had no significant effect
on dopamine efflux in the NAc, whereas 0.25 mg/kg
risperidone increased dopamine efflux at 30–90 min
(p<0.05–0.01) and the combination at 15–180 min
(p<0.05–0.01). For the interaction effect, there was a
statistically significant difference between vehicle and
risperidone at 45–75 min (p<0.05–0.001), vehicle and
idazoxan at 15 min (p<0.05), vehicle and the combi-
nation at 30–120 and 150–180 min (p<0.05–0.001).
There was also a statistically significant difference
between risperidone and the combination at 60 min
(p<0.05) and between idazoxan and the combination
at 45–120 and 165 min (p<0.05–0.001). The overall
effect, AUC (45–180 min), was statistically significant
(F3,21=6.43, p<0.01). Compared to saline/vehicle,
only the combination of idazoxan+risperidone was
significantly higher (p<0.001), and the combination
was statistically higher than idazoxan (p<0.01), but
not compared to risperidone.
Effects of risperidone alone and in combination
with idazoxan on NMDA-induced currents in
pyramidal cells
Intracellular voltage-clamp recordings were per-
formed in pyramidal cells in layers V and VI in the
 a2 blockade enhances effects of risperidone 897

Table 1. Effects of (a) risperidone and (b) idazoxan+risperidone on catalepsy in rats

Score at 30 min Score at 60 min Score at 120 min

(a) Effects of risperidone (0.3, 0.5. 1.0 mg/kg i.p.)

Vehicle 0¡0 0¡0 0¡0.5
Risperidone
0.3 mg/kg 0.5¡0.5* 0¡0.5 0¡0.25
0.5 mg/kg 1.5¡1.5** 0¡0.25 0.5¡0.5
1.0 mg/kg 0.5¡1.5* 1.5¡1.0** 1.0¡1.25
(b) Effects of idazoxan (1.5 mg/kg s.c.)+risperidone (0.25 mg/kg i.p.)
Saline+vehicle 0¡0.25 1¡0.5 1¡0.5
Saline+risperidone (0.25 mg/kg) 0¡0.5 0¡0.5 0¡0.25
Idazoxan (1.5 mg/kg)+vehicle 0.5¡0.5 0¡0.5 0¡0.5

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Idazoxan (1.5 mg/kg)+risperidone (0.25 mg/kg) 0¡0 0¡0.5 0¡0

The results are shown as medians¡semi-interquartile range based on observations of eight animals per treatment group.
* p<0.5, ** p<0.01 vehicle vs. risperidone-treated animals.

prelimbic region of the rat mPFC. The characterization Discussion

of pyramidal cells was performed according to criteria
The present study shows that adjunctive treatment
previously described (see above).
with the selective a2-adrenoceptor antagonist idazox-
Concentration–response curves for both clozapine
an when added to a low dose of risperidone signifi-
and risperidone on NMDA-induced currents have
cantly enhances the risperidone-induced suppression
previously been established in our laboratory
of CAR in rats, indicating an enhanced antipsychotic
(Konradsson et al. 2006), showing biphasic response
effect. In addition, both increased cortical dopamine
curves with a maximal effect at 100 nM for
release and enhanced NMDA receptor-mediated re-
clozapine [214¡16 % (mean¡S.E.M.)] and 20 nM for
sponses in cortical pyramidal cells were obtained.
risperidone (147¡26 %), i.e. a concentration that
Previous studies suggest that such effects may be as-
has been found to generate functionally effective a2-
sociated with an improvement of negative symptoms
adrenoceptor blockade in rat cortical slices (Leysen et al.
and cognitive impairment in schizophrenia. Our data
1988). For comparative reasons these data are included
generally propose that the a2/D2 ratio for risperidone
in Fig. 4. To investigate the effect of adjunctive a2-
is not necessarily optimal and that its therapeutic ef-
blockade, a submaximal concentration of risperidone
fect in schizophrenia can be enhanced by adjunctive
was chosen, i.e. 10 nM. Here we show (Fig. 4) that
treatment with an a2-adrenoceptor antagonist such as
whereas neither 1 mM idazoxan (96¡8 %, n=4) nor
idazoxan in spite of a dose reduction of risperidone.
10 nM risperidone (101¡7 %, n=6) had any effect on
The CAR test provides a highly predictive and
their own, the combination of 1 mM idazoxan+10 nM
reliable animal assay of antipsychotic potential
risperidone enhanced NMDA-induced currents
(Wadenberg & Hicks, 1999). Thus, within a certain
(177¡26 %, n=5). Group comparison (F2,12=7.55,
dose range, all APDs so far tested have been found to
p<0.01) revealed a statistically significant difference
effectively suppress CAR without affecting escape
between risperidone and the combination of risper-
capability, whereas compounds with a general seda-
idone+idazoxan (p<0.01) and between idazoxan and
tive but not antipsychotic action do not show this se-
the combination of risperidone+idazoxan (p<0.01).
lectively at any dose. Previous data also show that
the CAR test does not reflect amnesic effects, since
a2A- and a2C-adrenoceptor occupancies by risperidone
anticholinergic drugs such as scopolamine, which
The dose-dependent occupancy of a2A- and a2C- induces amnesia in animals and causes memory dis-
adrenoceptors by risperidone in the rat brain is illu- turbances in man, do not suppress CAR (Shannon et al.
strated in Fig. 5. The ED50 value (95 % confidence limit) 1999).
generated from the curves is 3.5 mg/kg (2.9–4.3) Dopamine D2 receptor blockade is a common
for a2A-adrenoceptors and 1.6 mg/kg (1.4–1.9) for denominator of all clinically available APDs. How-
a2C-adrenoceptors 1 h after s.c. administration. ever, the degree of receptor blockade differs between
898 M. M. Marcus et al.

(a) (a)
300 300

NMDA induced current

*
* **

(% of baseline)
Dopamine (% of baseline)
250 **
* 200
*
# *
*
200 *
* *
* *
*
* 100
150 # *
* *
*
100
0
risperidone 10 nM clozapine 100 nM
50
idazoxan 1 µM risperidone 20 nM
risperidone 10 nM
0 + idazoxan 1 µM
–30 0 30 60 90 120 150 180 210 240

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Time (min) (b)
Control risperidone 10 nM + idazoxan 1 µM
saline/vehicle
saline/risperidone 0.25 mg/kg i.p. NMDA NMDA
idazoxan 1.5 mg/kg s.c./vehicle
idazoxan 1.5 mg/kg s.c./risperidone 0.25 mg/kg i.p.

(b) 10 pA
300 60 s
* *
* *
Dopamine (% of baseline)

* * Fig. 4. (a) The effects of 10 nM risperidone, 1 mM idazoxan and

250 *
*
* * the combination of 10 nM risperidone+1 mM idazoxan on
*
200 # * NMDA-induced currents in pyramidal cells of the mPFC. The
* *
* * * * results are presented as mean¡S.E.M. ** p<0.01 between
* * * * *
150 * * different treatments. For comparison, the maximal effects of
clozapine (100 nM) and risperidone (20 nM ; Konradsson et al.
100
2006) are included in the figure. (b) Representative traces
50 illustrating NMDA-induced inward currents in a pyramidal
cell of the mPFC, before (left) and after (right) administration
0 of the combination of 10 nM risperidone+1 mM idazoxan.
–30 0 30 60 90 120 150 180 210 240
Time (min)

Fig. 3. The effects of risperidone (0.25 mg/kg i.p.)

administration on dopamine output in (a) the mPFC and 100

(b) the NAc, respectively, in rats pretreated with saline or

Receptor occupancy

idazoxan (1.5 mg/kg s.c.). Arrows indicate saline/idazoxan 75

(% of control)

and vehicle/risperidone injections. The results are

presented as mean¡S.E.M. * p<0.05, ** p<0.01, *** p<0.001 50
compared to vehicle, # p<0.05 risperidone vs.
idazoxan+risperidone (n=5–7). 25

0
different APDs. Whereas FGAs, such as haloperidol,
0.01 0.04 0.16 0.63 2.5 10 40
typically produce a D2 receptor occupancy of y70 %,
clozapine generates only y45 % at clinically effective Risperidone (mg/kg)
dosage (Farde & Nordström, 1992 ; Farde et al. 1989). Fig. 5. In-vivo a2A-adrenoceptor (%) and a2C-adrenoceptor
The D2 receptor occupancy generated by risperidone ($) occupancy measured by ex-vivo autoradiography 1 h
is approximately in the same range as that produced after subcutaneous administration of risperidone.
by FGAs (Kapur et al. 1995, 1999 ; Nyberg et al. 1999).
In rodents, a dose of clozapine that shows a robust
antipsychotic effect in the CAR test, i.e. 5 mg/kg (Ol- achieve a robust antipsychotic-like effect was between
sen et al. 2006), produces y45 % D2-, 65 % a2A- and 0.4 and 0.5 mg/kg, corresponding to y55 % D2 re-
95 % a2C-receptor occupancy (Marcus et al. 2005). In ceptor occupancy (Wadenberg et al. 2001) and to
the present study, the dose of risperidone required to y12 % a2A- and y19 % a2C-adrenoceptor occupancy.

However, already at this dose level some escape fail-
ures were registered, indicating a contribution of
unspecific effects of the drug. Significantly, the com-
bination of a low dose of risperidone (0.25 mg/kg),
which produces y50 % D2 receptor occupancy
(Wadenberg et al. 2001), and idazoxan (1.5 mg/kg),
which generates 75 % a2A- and 90 % a2C-adrenoceptor
occupancy (Marcus et al. 2005), thus resulted in re-
ceptor occupancies that are similar to those of cloza-
pine and also produced a robust antipsychotic effect in
the CAR test. Given the superior efficacy of clozapine
in schizophrenia, it is therefore of considerable interest
that we here, by adding idazoxan, obtained a robust
antipsychotic activity by a low dose of risperidone,
which by itself was not sufficient to generate such an
effect. Yet, no catalepsy was observed with this drug
combination, thus suggesting a very low EPS liability.
Besides being an a2-adrenoceptor antagonist, idazox-
an also has affinity for 5-HT1A and imidazoline re-
ceptors. However, its affinity for a2-adrenoceptors is
much higher than for both 5-HT1A and imidazoline
receptors (Ernsberger et al. 1990 ; Kleven et al. 2005),
indicating that the effects seen with idaxozan are, in
all probability, largely due to a2-adrenoceptor block-
ade.
Addition of idazoxan to a low dose of risperidone
significantly enhanced dopamine output in the mPFC,
and also in the NAc. Addition of idazoxan to a selec-
tive D2 antagonist and also to haloperidol has pre-
viously been shown to increase dopamine output in
the mPFC but not in NAc (Hertel et al. 1999a ;
Wadenberg et al. 2007). However, the effect in the NAc
by the combination of idazoxan+risperidone is com-
parable to the effects produced by adding idazoxan to
olanzapine, which also produced a marked increase in
prefrontal dopamine output as well as an enhanced
antipsychotic-like effect (Wadenberg et al. 2007). Both
olanzapine and risperidone possess higher affinity for
serotonergic receptors, e.g. 5-HT2A and 5-HT2C, than
for D2 receptors (Shahid et al. 2009). Clinically, rit-
anserin, a 5-HT2A/2C receptor antagonist has been
found to enhance the effect of FGAs in schizophrenia
(Gelders et al. 1986 ; Reyntjens et al. 1986). However,
when added to the selective D2 receptor antagonist
raclopride, ritanserin not only enhanced the anti-
psychotic-like effect (Wadenberg et al. 1996) but also
increased the raclopride-induced dopamine outflow
in both the mPFC and NAc (Andersson et al. 1995).
Moreover, asenapine, a SGA with higher affinity for a
range of serotonergic receptors and a2-adrenoceptors
than for D2 receptors (Shahid et al. 2009) increases do-
pamine output in mPFC as well as in both NAc and
striatum (Frånberg et al. 2008). Thus, both preclinical
a2 blockade enhances effects of risperidone 899
and clinical data seem to indicate that the increased
accumbal dopamine output induced by adding a
5-HT2A/C receptor antagonist or idazoxan to various
APDs does not prevent a concomitantly enhanced
antipsychotic effect of the drug combinations. Indeed,
previous data propose that a marked enhancement of
dopamine outflow in the mPFC per se is sufficient to
produce an enhanced antipsychotic effect of APDs
(Eltayb et al. 2005 ; Hertel et al. 1999a), a notion that is
indirectly supported by several studies showing that
cortical dopamine may regulate and functionally in-
hibit subcortical dopamine transmission (Murase et al.
1993 ; Saunders et al. 1998). An enhanced dopamine
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output in the NAc might seem counteractive to the
antipsychotic-like effect of APDs, which has been
shown to be mediated in the ventral striatum
(Wadenberg et al. 1990). The increased dopamine out-
put in the NAc does not seem to have any functional
consequences, at least it does not prevent the anti-
psychotic-like effect seen in this model. The increased
dopamine outflow in striatal areas might still contrib-
ute to reduce catalepsy. The combination of idazox-
an+risperidone, in concentrations that had no effect
by themselves, facilitated NMDA receptor-induced
currents in the mPFC. Thus, both enhanced cortical
dopaminergic and glutamatergic NMDA receptor-
mediated transmission were observed following com-
bined treatment with risperidone+idazoxan. In our
previous study (Marcus et al. 2005), the facilitation of
cortical NMDA receptor-mediated neurotransmission
by the combination of D2 receptor and a2-adrenoceptor
blockade was found to be mediated by dopamine and
executed via the D1 receptor. Moreover, evidence for
dopaminergic D1 dysregulation as well as glutama-
tergic NMDA receptor hypofunction in schizophrenia
has been reported (see Introduction) and cognitive
processes per se seem to be dependent on normally
functioning D1 receptors as well as NMDA receptors
(Aura & Riekkinen, 1999 ; Goldman-Rakic et al. 2000).
Consequently, the facilitation of cortical NMDA re-
ceptor-mediated transmission generated by a combi-
nation of idazoxan and a low dose of risperidone
might contribute to improve cognitive deficits, e.g.
impaired working memory, in schizophrenia, an effect
that may contribute to improve treatment outcome
(Green, 1996).
Clozapine has been shown to reduce suicidality
in schizophrenia and schizoaffective disorder (see
Introduction). The a2-adrenoceptor blocking effect
of clozapine may well contribute to its suicide-
preventing effect, since a2-adrenoceptor blockade rep-
resents one of the mechanisms of action of clinically
effective antidepressant drugs such as mianserin and
900 M. M. Marcus et al.
mirtazapine, and blockade of a2-auto- and hetero-
receptors on nerve terminals results in an increased
release of both noradrenaline and serotonin in the
brain (de Boer, 1995 ; Haddjeri et al. 1995 ; Hertel et al.
1997, 1999b). Indeed, the antidepressant drug mian-
serin has been found to enhance the effect of FGAs on
positive and negative symptoms as well as cognitive
dysfunction in schizophrenia (cf. above), thereby
generating a clozapine-like effect. Therefore, adjunct
treatment with an a2-adrenoceptor antagonist may
improve the efficacy of risperidone on depressive
symptoms in schizophrenia and potentially even re-
duce suicidality.
Consequently, the enhanced prefrontal dopaminer-
gic as well as cortical NMDA receptor-mediated
transmission induced by adjunct idazoxan when ad-
ded to a low dose of risperidone may improve its effect
on negative symptoms and cognitive deficits in
schizophrenia. In addition, the lower D2 receptor
occupancy, which is achieved by a reduced dose of
risperidone, might in itself contribute to such im-
provement, since a high degree D2 blockade per se
has been shown to generate detrimental effects on
both cognition and mood in healthy volunteers (Saeedi
et al. 2006). In fact, a high degree of D2 blockade may
in itself serve to exacerbate cognitive as well as nega-
tive symptoms in schizophrenia (Carpenter, 1996).
Finally, a reduced EPS liability would, in principle,
also be expected by the combination of idazoxan
and a low dose of risperidone. The present results
provide further evidence for the notion that the
potent a2-adrenoceptor antagonistic action of cloza-
pine may significantly contribute to its unique efficacy
in schizophrenia, as originally proposed by Nutt
(1994).
Acknowledgements
This work was supported by the Swedish Research
Council (grant no. 4747), Torsten och Ragnar
Söderbergs Stiftelser and The Karolinska Institutet,
Stockholm, Sweden. The expert technical assistance
of Mrs Anna Malmerfelt and Mrs Ann-Chatrine
Samuelsson is gratefully acknowledged. We also
thank Johnson & Johnson Pharmaceutical Research &
Development, division of Janssen Pharmaceutica NV,
Beerse, Belgium for generously providing us with
risperidone.
Statement of Interest
Dr X. Langlois is an employee of Johnson & Johnson
Pharmaceutical Research and Development, Beerse,
Belgium.
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