The difference in effect of mGlu2/3 and mGlu5 receptor agonists on cognitive impairment induced by MK-801.

Abstract

The manipulation of glutamate neurotransmission could represent a potential strategy for the pharmacotherapy of schizophrenic symptoms. Preclinical studies suggest that two subtypes of metabotropic glutamate (mGlu) receptors such as mGlu2/3 and mGlu5 receptors have the potential to ameliorate deficits in schizophrenia. In our study we evaluated the role of a non-specific mGlu receptor agonist ((1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid; 1S,3R-ACPD), mGlu5 receptor agonist or positive modulators ((RS)-2-Chloro-5-hydroxyphenylglycine;CHPG; [(3-Fluoro-phenyl)methylene]hydrazone-3-fluorobenzaldehyde; DFB; 3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide; CDPPB) and a mGlu2/3 receptor agonist (2,2,2-Trifluoro-N-[4-(2-methoxyphenoxy)phenyl]-N-(3-pyrdinylmethyl)ethanesulfonamide hydrochloride; LY-487379) on performance in a cognitive task (Active Allothetic Place Avoidance) after sub-chronic administration of 5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)-cyclo-hepten-5,10-imine; MK-801 . The Active Allothetic Place Avoidance task is suitable for assessing the executive function and attention of animals and was previously validated for testing the effect of anti-psychotics. Application of the mGlu2/3 receptor agonist had no effect on cognitive impairment induced by MK-801. However, the mGlu5 receptor agonists ameliorated cognitive impairment induced by MK-801 without affecting locomotion. In conclusion, the mGlu5 receptor agonists could be effective in the treatment of cognitive deficits in patients with schizophrenia. However, the pro-cognitive effect of the agonist of mGlu2/3 receptors was not demonstrated in the present study.

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Behavioural Pharmacology
The difference in effect of mGlu2/3 and mGlu5 receptor agonists on cognitive
impairment induced by MK-801
Karel Valesa,⁎, Jan Svobodaa, Kristina Benkovicovaa, Vera Bubenikova-Valesovab, Ales Stuchlika
aInstitute of Physiology, Academy of Sciences of the Czech Republic, Videnska 1083, Prague 4, Czech Republic
bPrague Psychiatric Center, Ustavni 91, Prague 8, Czech Republic
a b s t r a c t a r t i c l e i n f o
Article history:
Received 22 June 2009
Received in revised form 12 November 2009
Accepted 23 November 2009
Available online 2 April 2010
Keywords:
MK-801
Animal model of schizophrenia
mGlu receptor
Cognition
Behavior
AAPA (Active Allothetic Place Avoidance)
The manipulation of glutamate neurotransmission could represent a potential strategy for the pharmacother-
apy of schizophrenic symptoms. Preclinical studies suggest that two subtypes of metabotropic glutamate
(mGlu) receptors such as mGlu2/3 and mGlu5 receptors have the potential to ameliorate deficits in
schizophrenia. In our study we evaluated the role of a non-specific mGlu receptor agonist ((1S,3R)-1-
aminocyclopentane-1,3-dicarboxylicacid;1S,3R-ACPD),mGlu5receptoragonistorpositivemodulators((RS)-
2-Chloro-5-hydroxyphenylglycine;CHPG; [(3-Fluoro-phenyl)methylene]hydrazone-3-fluorobenzaldehyde;
DFB; 3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide; CDPPB) and a mGlu2/3 receptor agonist (2,2,2-
Trifluoro-N-[4-(2-methoxyphenoxy)phenyl]-N-(3-pyrdinylmethyl)ethanesulfonamide hydrochloride; LY-
487379) on performance in a cognitive task (Active Allothetic Place Avoidance) after sub-chronic
administration of 5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)-cyclo-hepten-5,10-imine; MK-
801 . The Active Allothetic Place Avoidance task is suitable for assessing the executive function and attention
of animals and was previously validated for testing the effect of anti-psychotics. Application of the mGlu2/3
receptor agonist had no effect on cognitive impairment induced by MK-801. However, the mGlu5 receptor
agonists ameliorated cognitive impairment induced by MK-801 without affecting locomotion. In conclusion,
the mGlu5 receptor agonists could be effective in the treatment of cognitive deficits in patients with
schizophrenia.However, the pro-cognitive effect ofthe agonist ofmGlu2/3 receptors was not demonstrated in
the present study.
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
Schizophrenia is a chronic neuropsychiatric disorder prevailing in
approximately 1% of the human population. Its symptoms can be
described as positive (e.g. hallucinations and delusions), negative (e.g.
emotional blunting and anhedonia), and cognitively deficient. It is
believed that cognitive symptoms of schizophrenia are primary, as they
are relatively stable from the onset of thediseasedespite thefluctuation
of other symptoms (Moghaddam, 2004; Nilsson et al., 2001).
Glutamate is the main excitatory neurotransmitter in the brain of
vertebrates. Glutamatergic neurotransmission is executed via two
families of receptors: ionotropic and metabotropic receptors. Eight
subtypes of mGlu receptors are known. These are classified into three
groups: group I (mGlu1, 5 receptors), coupled with inositol
phosphate, group II (mGlu2,3 receptors) and III (mGlu4,6,7,8
receptors), coupled with adenylyl cyclase (Schoepp et al., 1999).
A number of studies point to the disruption of ionotropic NMDA
receptors as the primary phenomenon in the pathophysiology of
schizophrenia. Firstly, non-competitive antagonists of NMDA recep-
tors impair cognitive function in healthy subjects. These drugs also
exacerbate psychotic symptoms when given to schizophrenic patients
(Krystal et al., 1994). Also, alterations in the expression of NMDA
receptors have been found in the cortex, hippocampus and thalamus
of schizophrenic patients (Meador-Woodruff and Healy, 2000). In
addition, the application of the high-affinity non-competitive NMDA
-receptor antagonist (5R,10S)-(+)5-methyl-10,11-dihydro-5H-
dibenzo(a,d)-cyclo-hepten-5,10-imine; MK-801) has been proposed
in an animalmodelof schizophrenia (Andine etal., 1999;Bubenikova-
Valesova et al., 2008a; Maj et al., 1991). Animals treated with MK-801
exhibit typical changes in behavior including hyperactivity (Maj et al.,
1991), stereotypic behaviors, cognitive deficits and other alterations
(Nilsson et al., 2001).
In light of this, the manipulation of glutamate neurotransmission
could represent a potential strategy for the pharmacotherapy of the
symptoms of schizophrenia. Since ionotropic glutamate receptors are
present in nearly all subtypes of neurons, direct pharmacological
manipulation of these receptors may produce a global disruption in
brain function and profound side effects. Indirect modulation of
European Journal of Pharmacology 639 (2010) 91–98
⁎ Corresponding author. Institute of Physiology of the Czech Academy of Sciences,
v.v.i., Videnska 1083, Prague 4, Czech Republic. Tel.: +420 24106 2713; fax: +420
24106 2488.
E-mail address: vales@biomed.cas.cz (K. Vales).
0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejphar.2009.11.067
Contents lists available at ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar

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glutamatergic neurotransmission receptors may also be achieved by
targeting mGlu receptors. Preclinical studies suggest that two
subtypes of mGlu receptors have the potential for ameliorating
deficits in schizophrenia. These include mGlu5 receptors (group I),
which can directly modulate the function of NMDA receptors, and the
mGlu2/3 receptors (group II), which presynaptically modulate the
release of glutamate (Moghaddam, 2004). The mGlu5 receptors can
directly modulate the function of the NMDA channel through
interaction with anchoring and Homer proteins (Tu et al., 1999).
The mGlu2/3 receptors regulate glutamate release in the pre-frontal
cortex.
In our study we evaluated the role of mGlu5 and mGlu2/3
receptors on performance in a cognitive task (Active Allothetic Place
Avoidance task) after administration of MK-801. The Active Allothetic
Place Avoidance task is a spatial memory test that can determine the
ability of animals to organize their behavior efficiently in both normal
and pathological conditions. Successful performance in the task
requires the rat to solve a conflict between two discordant subsets
of spatial stimuli (extramaze and intramaze cues). We believe that
successful performance in the Active Allothetic Place Avoidance task,
where the rat has to differentiate between relevant and irrelevant
stimuli, depends on the mode of information processing disturbed in
schizophrenic patients (Bubenikova-Valesova et al., 2008b).
Since the pharmacological activation of mGlu receptors may be a
potential strategy for the alleviation of cognitive impairments in
animal models, the present study examined the interaction between
MK-801 and mGlu2/3 receptor agonist, mGlu5 receptor agonist,
mGlu5 receptor allosteric positive modulators and non-specific mGluI
and II receptor agonist.
2. Methods
2.1. Subjects
One hundred fifty-four 3–4 month-old male rats of the Long
Evans strain were obtained from the accredited breeding colony of
the Institute of Physiology, Academy of Science, Prague. Rats were
housed in pairs in transparent plastic cages (30×30×40 cm) in an
air-conditioned animal room with a constant temperature of 21 °C
and with 12/12 light/dark cycle (lights on at 7:00). Food and water
were freely available. Experiments were pursued during daylight
hours. All animals were implanted with a low-impedance connector
made from a hypodermic needle piercing the rat's skin between the
shoulders. The sharp end of the needle was cut off and bent with
tweezers to form a small loop, which prevented the connector
from slipping out and provided a link for an alligator clip connected
to a wire delivering electric shocks through to the grounded
floor. All procedures were in accordance with Czech and Europe-
an (86/609/EEC) legislation regarding treatment of laboratory
animals.
2.2. Drugs
MK-801 ((5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo(a,
d)-cyclo-hepten-5,10-imine) was obtained from SigmaAldrich,
Czech Republic. ACPD ((1S,3R)-1-aminocyclopentane-1,3-dicarbox-
ylic acid), CHPG ((RS)-2-Chloro-5-hydroxyphenylglycine) and DFB
([(3-Fluoro-phenyl)methylene]hydrazone-3-fluorobenzaldehyde)
were purchased from Tocris Bioscience, United Kingdom, CDPPB (3-
Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide, LY-487379
(2,2,2-Trifluoro-N-[4-(2-methoxyphenoxy)phenyl]-N-(3-pyrdinyl-
methyl)ethanesulfonamide hydrochloride) were acquired as a gen-
erous gift from Abbott, Germany. MK-801 was dissolved in saline in
concentrations of 0.1 mg/ml and injected intraperitoneally (i.p.)
40 min prior to behavioral testing at doses 0.1 mg/kg of body weight
(b.w.). As a control for MK-801, saline was injected at 1 ml/kg b.w.
ACPD was dissolved in saline at concentrations of 0.01 mg/ml and
0.1 mg/ml and injected i.p. 40 min prior to testing at doses 0.01 mg/kg
and 0.1 mg/kg at a volume of 1 ml/kg. As a control for ACPD injection,
saline was injected 40 min prior to training at a volume of 1 ml/kg.
The doses were selected on the basis of previous studies. CHPG was
dissolved in saline at concentrations of 0.01, 0.1 and 1 mg/ml and
injected i.p. 40 min prior to testing at doses 0.01, 0.1 and 1 mg/kg at a
volume of 1 ml/kg. As a control for CHPG injection, saline was injected
40 min prior to training at a volume of 1 ml/kg. DFB was dissolved in
saline at concentrations of 0.01, 0.1 and 1 mg/ml and injected i.p.
40 min prior to testing at doses 0.01, 0.1 and 1 mg/kg at a volume of
1 ml/kg. As a control for DFB injection, saline was injected 40 min
prior to training at a volume of 1 ml/kg. CDPPB was dissolved in saline
at concentrations of 0.5 mg/ml and 5 mg/ml and injected i.p. 40 min
prior to testing at doses 0.5 and 5 mg/kg at a volume of 1 ml/kg. As a
control for CDPPB injection, saline was injected 40 min prior to
training at a volume of 1 ml/kg. Therefore, all animals received the
same volume of liquid per 1 kg of body weight. The doses of mGlu5
receptor agonists and allosteric positive modulators were selected on
the base of previous studies (Kinney et al., 2005; Lecourtier et al, 2007,
Uslaner et al., 2009). LY-487379 was dissolved in saline at concentra-
tions of 0.1 mg/ml and 10 mg/ml and injected i.p. 40 min prior to
testing at doses 0.1 and 10 mg/kg at a volume of 1 ml/kg. As a control
for LY-487379 injection, saline was injected 40 min prior to training at
a volume of 1 ml/kg. The doses were selected on the base of previous
studies (Harich et al., 2007).
2.3. Apparatus and behavioral procedures
The Active Allothetic Place Avoidance arena was described in
detail in previous papers (Cimadevilla et al., 2001). Briefly, it
consisted of a smooth metallic circular arena (82 cm in diameter),
enclosed by a 30 cm high transparent Plexiglas wall. The arena was
elevated 1 m above the floor of 4×5 m experimental room containing
an abundance of extramaze landmarks. The arena together with the
wall was rotated (1 rpm) by an electric motor placed underneath the
arena disc. At the beginning of each training session, a rat was placed
in the arena at a location opposite an imperceptible 60° to-be-
avoided sector (shock sector), which was defined by the computer-
based tracking system (iTrack, Biosignal Group, USA), located in an
adjacent room. The arena rotation started immediately after
placement of the rat. The location of the shock sector could be
determined solely by its spatial relation to the distal orienting cues
located in the room. Rats were wearing a light latex harness, to which
an infrared light-emitting diode (LED) was attached between the rat's
shoulders. Its position was tracked every 40 ms and recorded into a
computer track file, allowing subsequent reconstruction of the track
by an off-line analysis program (TrackAnalysis, Biosignal Group, USA).
Whenever the rat entered the to-be-avoided sector for more than
500 ms, the tracking system delivered a mild, constant-current shock
(50 Hz, 0.5 s, 0.4―0.7 mA) and counted an entrance. If the rat did not
leave the sector, additional shocks were given every 1200 ms, but no
more entrances were counted until the rat left the sector for more
than 300 ms. The shocks were delivered through the subcutaneous
low-impedance connector on the rat standing on the grounded floor.
We used a compact floor instead of grid, in order to allow an
accumulation of intramaze scent cues, which enhanced the conflict
between arena and room frames. This procedure using shocks has
previously been shown to be effective and safe for animals, leading to
rapid avoidance behavior (Cimadevilla et al., 2001). The appropriate
shock current (ranging between 0.4 and 0.7 mA) was carefully
adjusted for each rat to evoke a rapid escape reaction but prevent
freezing. Most animals responded appropriately to a shock intensity
of 0.5 mA. The arena surface was carefully cleaned by a detergent
solution between rats.
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2.4. Design of experiments
Each mGlu receptor agonist was assessed in a separate experi-
ment. Therefore rats were split up into 5 batches (ACPD, LY-487379,
CHPG, DFB, and CPPB). Within each batch, two doses of mGlu receptor
agonist were used (ACPD 0.01 and 0.1 mg/kg, LY-354740. 0.1 and
10 mg/kg, CHPG 0.01 and 1 mg/kg, DFB 0.01 and 1 mg/kg, CDPPB 0.5
and 5 mg/kg). For each dose, rats were assigned to a group either with
the mGlu receptor agonist only or with a combination of mGlu
receptor+MK-801 0.1 mg/kg. For each of the groups, the same
control and MK-801 treated rats were used as a reference.
Animals were trained in four consecutive daily sessions in the
Active Allothetic Place Avoidance task, with the shock sector
stretching from the center of the arena to its north circumference.
Experimental sessions in Active Allothetic Place Avoidance task lasted
20 min, and each rat had one session every day, carried out during
daylight hours.
2.5. Data analysis and statistics
For analysis, the data were collected from the last day (day 4) of
Active Allothetic Place Avoidance task training, when they reached
their asymptotic value. Three variables were assessed: Total path,
measured as a session sum of linear distances between successive
animal locations sampled at 1 Hz. This variable reflects locomotor
activity and represents a non-cognitive measure. Number of
Entrances into the punishment region during a session reflects
efficiency of spatial avoidance behavior in Active Allothetic Place
Avoidance task. Maximum time (Maximum Time Avoided) between
twoconsecutiveentries intothepunishment regionreflectsthe ability
to remember the shock region and avoid it.
Each mGlu receptor agonist was assessed in a separate experi-
ment. Therefore rats were split up into 5 batches (ACPD, LY-487379,
CHPG, DFB, and CPPB). Within each batch, two doses of the mGlu
receptor agonists were used (ACPD 0.01 and 0.1 mg/kg, LY-487379.
0.1 and 10 mg/kg, CHPG 0.01 and 1 mg/kg, DFB 0.01 and 1 mg/kg,
CDPPB 0.5 and 5 mg/kg). For each dose, rats were assigned to a group
either with the mGlu receptor agonist only or with a combination of
mGlu receptor+MK-801 0.1 mg/kg. In addition the mGlu receptor
agonist only groups (“saline” groups), a group treated with saline only
served as a reference (control group). In addition to the combined
mGlu receptor agonist and MK-801 0.1 mg/kg groups (“MK-801”
groups), a group treated with MK-801 0.1 mg/kg served as a
reference. The same two reference groups were used throughout
the study.
3. Results
3.1. Non-specific mGlu receptor agonist: ACPD
3.1.1. Locomotor activity
ACPD treatment did not affect locomotion. Although the effect of
MK-801 just reached the level of significance F(1,42)=4.14, P=0.05,
the interaction remained insignificant and subsequent Newman–
Keuls post-hoc test failed to find a difference between the groups
(Fig. 1, Part A).
3.1.2. Number of Entrances (errors)
Two-way ANOVA revealed significant ACPD treatment F(2,42)=
5.6, Pb0.01, MK-801 treatment F(1,42)=7.83, Pb0.01, and interac-
tion F(2,42)=6.15, Pb0.01. Newman–Keuls post-hoc test found that
rats injected with MK-801 0.1 mg/kg alone showed higher Number of
Entrances compared to controls (Pb0.001). ACPD at both doses
(0.01 mg/kg, 0.1 mg/kg) blocked the deteriorating effect of MK-801
0.1 mg/kg on this parameter. Application of ACPD alone produced no
significant effect at either dose compared to the control group (Fig. 1,
Part B).
3.1.3. Maximum Time Avoided
This parameter was found to be affected by both ACPD F(2,42)=
5.72, Pb0.01, and MK-801 administration F(1,42)=6.89, Pb0.05.
However the interaction remained insignificant F(2,42)=2.35. New-
man–Keuls post-hoc test revealed that MK-801 0.1 mg/kg group
decreased Maximum Time Avoided compared to controls (Pb0.05)
and co-application of ACPD 0.1 mg/kg and MK-801 0.1 mg/kg
reversed this effect (Pb0.01). Administration of ACPD alone had no
Fig. 1. Effect of non-specific mGlu receptor agonist ACPD on Active Allothetic Place
Avoidance task performance. (A) Locomotor activity was not found to be affected by
MK-801 or ACPD administration. (B) Number of Entrances was increased in the MK-801
group (0.1 mg/kg), administration of both doses of ACPD (0.01 and 0.1 mg/kg) blocked
the effect of MK-801. Application of ACPD alone produced no significant effect at either
dose. (C) Maximum Time Avoided was decreased after application of MK-801 0.1 mg/
kg, which was reversed after co-application of higher (0.1 mg/kg), but not lower
(0.01 mg/kg) doses of ACPD. The injection of ACPD alone produced no significant effect
at either dose. Number of animals in each group indicated in parentheses. All values±
S.E.M.; *Pb0.05, ***Pb0.001 compared to control group; # Pb0.05, ## Pb0.01, ###
Pb0.001 compared to MK-801 0.1 mg/kg group.
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significant effect at either dose compared to control group (Fig. 1,
Part C).
3.2. Allosteric modulator of mGlu2/3 receptor: LY-487379
3.2.1. Locomotor activity
Two-way ANOVA conducted on locomotor activity yielded no
significant effect of LY-487379 or MK-801 (Fig. 2, Part A).
3.2.2. Number of Entrances (errors)
Two-way ANOVA confirmed that MK-801 affected entering into
punished region F(1,34)=23,17, Pb0.0001 but failed to find a
significant effect of LY-487379, or an interaction. A Newman–Keuls
post-hoc test revealed that the combination of LY-48739 0.1 mg/kg
(but not 10 mg/kg) and MK-801 0.1 mg/kg produced the same deficit
in performance as the MK-801 0.1 mg/kg only group. Both groups
were significantly worse than control rats. Moreover, co-application
of LY-48739 0.1 mg/kg and MK-801 0.1 mg/kg deteriorated perfor-
mance compared to LY-48739 0.1 mg/kg only group (Fig. 2, Part B).
3.2.3. Maximum Time Avoided
Similarly to Number of Entrances, ANOVA conducted on Maximum
Time Avoided found effect of MK-801 F(1,34)=21,37, Pb0.0001, but
not significant effect of LY-487379 or interaction. As Newman–Keuls
post-hoc test revealed, administration of LY-487379 alone at either
dose had no effect compared to controls, however, co-application of
LY-487379 0.1 mg/kg and MK-801 0.1 mg/kg significantly deteriorat-
ed performance below the level of LY-487379 0.1 mg/kg only group
(Pb0.01), or control group (Pb0.05) (Fig. 2, Part C).
3.3. Agonist of mGlu5 receptor: CHPG
3.3.1. Locomotor activity
Analysis of the paths taken during the sessions failed to detect a
significant CHPG factor, but revealed a significant effect of MK-801
F (1,42)=9.32, Pb0.01, as well as an interaction F(2,42)=4.18,
Pb0.01. Detailed analysis by Newman–Keuls post-hoc test specified
that combination of MK-801 0.1 mg/kg and CHPG 0.1 mg/kg treat-
ment induced mild hyperlocomotion compared to controls (Pb0.05)
or CHPG 0.01 mg/kg group (Pb0.01) (Fig. 3, Part A).
3.3.2. Number of Entrances (errors)
Both CHPG F(2,42)=3,37, Pb0.05, and MK-801 F(1,42)=9.89,
Pb0.01 affected entering into the punished region; moreover, there
was a significant interaction between the two factors F(2,42)=4.21,
Pb0.05. Newman–Keuls post-hoc test revealed that CHPG at either
dose combined with MK-801 0.1 mg/kg was able to reverse the
deteriorating effect of MK-801 0.1/mg/kg alone. In addition, there was
no difference between each CHPG+MK group and its corresponding
CHPG counterpart (Fig. 3, Part B).
3.3.3. Maximum Time Avoided
Atwo-wayANOVAperformedonMaximumTimeAvoidedshowed
a significant effect of MK-801 F(1,42)=5.46, Pb0.05, but not CHPG
F(2,42)=0.72, PN0.05, and a significant interaction F(2,42)=4.07,
Pb0.05. Closer examination by post-hoc test found that controls
differed from MK-801 group in Maximum Time Avoided Pb0.05
(Fig. 3, Part C).
3.4. Allosteric positive modulator of mGlu5 receptor: DFB
3.4.1. Locomotor activity
Application of DFB did not affect locomotion, since a two- way
ANOVA did not reveal an effect of DFB or MK-801 factors (Fig. 4,
Part A).
3.4.2. Number of Entrances (errors)
A two-way ANOVA failed to show a significant effect of DFB,
contrasting to the significant MK-801 0.1 mg/kg F(1,34)=10.83,
Pb0.01; interaction fell below the level of significance. Newman–
Keuls post-hoc tests revealed that both doses of DFB efficiently
blocked the deteriorating effect of MK-801 on spatial performance
(Fig. 4, Part B).
3.4.3. Maximum Time Avoided
A two-way ANOVA revealed that the administration of MK-801
F(1, 34)=5.34, Pb0.05, but not DFB, affected this parameter; with a
significant interaction F(2, 34)=3.97 Pb0.05. However, Newman–
Fig. 2. Effect of Glu2/3 positive allosteric modulator, LY-487379 on Active Allothetic
Place Avoidance task performance. (A) Locomotor activity was neither affected by MK-
801 0.1 mg/kg alone, nor in combination with LY-487379. (B) Number of Entrances
remained at the level of control rats after application of either dose of LY-48739.
However, combined application of LY-487379 0.1 mg/kg and MK-801 0.1 mg/kg
worsened performance compared to LY-487379 0.1 mg/kg alone group or control
group. C) Maximum Time Avoided dropped significantly after combination of LY-
487379 0.1 mg/kg and MK-801 0.1 mg/kg compared to LY-487379 0.1 mg/kg alone
group or control group. All values±S.E.M.; *Pb0.05, **Pb0.01 compared to control
group; §§ Pb0.01 compared to LY-487379 0.1 mg/kg group.
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Keuls post-hoc failed to find a significant effect between the particular
groups. (Fig. 4, Part C)
3.5. Positive allosteric modulator of mGlu5 receptor: CDPPB
3.5.1. Locomotor activity
CDPPB application did not interfere with locomotor activity.
Although there was a significant effect of MK-801 (F(1,36)=8.74,
Pb0.05, interaction remained insignificant and Newman–Keuls post-
hoc test failed to find significant difference between particular groups
(Fig. 5, Part A).
3.5.2. Number of Entrances (errors)
Atwo-wayANOVAfoundaneffectofCDPPBadministrationF(2,36)=
5.13, Pb0.05, MK-801 administration F(1,36)=6.67, Pb0.05, and a
significant interaction between the two factors F(2,36)=4.61, Pb0.05.
Subsequent post-hoc tests found that CDPPB blocked the deteriorating
effect of MK-801 in both doses (Pb0.01) (Fig. 5, Part B).
3.5.3. Maximum Time Avoided
The application of CDPPB F(2,36)=2.28, PN0.05, or MK-801 F
(1,36)=2.74, PN0.05 yielded no effect on Maximum Time Avoided. In
addition, the interaction between the two factors did not reach a level
of significance. (Fig. 5, Part C).
4. Discussion
The aim of this study was to assess the effect of mGlu receptor
agonists on cognitive coordination in rats in an animal model of
Fig. 3. Effect of orthosteric agonist of mGlu5 receptor, CHPG on Active Allothetic Place
Avoidance task performance. (A) Locomotor activity, was increased after combined
CHPG 0.01 mg/kg and MK-801 0.1 mg/kg administration compared to control or CHPG
0.01 mg/kg group. (B) Number of Entrances into punished region, which is increased
after application MK-801 0.1 mg/kg was blocked by co-administration with both doses
of CHPG. (C) Value of Maximum Time Avoided was decreased by MK-801, however,
was not fully restored by CHPG. All values±S.E.M.; *Pb0.05, **Pb0.01 compared to
control group; ## Pb0.01 compared to MK-801 0.1 mg/kg group; §§ Pb0.01 compared
to CHPG 0.01 mg/kg group.
Fig. 4. Effect of mGlu5 positive modulator, DFB on Active Allothetic Place Avoidance
task performance. (A) Locomotor activity was not affected by MK-801 or DFB
administration. (B) Number of Entrances into punished region, which was increased
by MK-801 administration, was blocked by either dose of DFB. (C) In Maximum Time
Avoided, in contrast to Number of Entrances, there were no significant differences
between groups. All values±S.E.M.; **Pb0.01 compared to control group; # Pb0.05
compared to MK-801 0.1 mg/kg group.
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schizophrenia-like behavior by an NMDA receptor antagonist. The
Active Allothetic Place Avoidance task is suitable for assessing the
executive function and attention of animals and was previously
validated for testing the effect of antipsychotics (risperidone and
haloperidol) in rats treated with MK-801 (Bubenikova-Valesova et al.,
2008b) In addition, the successful execution of this task requires that
the subjects differentiate between relevant and irrelevant stimuli,
similar to the concept that schizophrenic patients are often unable to
differentiate between relevant and irrelevant stimuli because their
information processing is impaired (Ellenbroek and Cools, 1990).
In our study we applied MK-801 at the dose of 0.1 mg/kg, i.p.,
which causes a cognitive deficit without an increase in locomotion
activity. This dose was selected on the basis of our previous results
(Vales et al., 2006). This setup allows a study of the effect of drugs on
cognition separately from the effect on hyperlocomotion.
The present study demonstrated that the administration of a non-
specific agonist of mGlu receptor (1S,3R-ACPD) alone at doses of
0.01 mg/kg and 0.1 mg/kg, i.p. did not have any effect on locomotor
activity or on cognitive parameters. In the literature we found that the
application of ACPD did not change behavior in the open-field test
(Zalewska and Wiśniewski, 1996). In another study, ACPD adminis-
tered alone had no effect on locomotion, recognition memory or
retention of memory but improved effectively motivated memory
(Zalewska and Wiśniewski, 1997). Another study revealed that post-
training intra-caudal infusion of ACPD impaired memory retention in
the inhibitory avoidance task and visible-platform water maze task,
but the same application did not affect the hidden-platform version of
the water maze task (Packard et al., 2001). From the above
mentioned, it can be concluded that although the effects of ACPD on
various behaviors are task-dependent, most studies show that
locomotion and higher cognitive functions are not disrupted by this
drug when it is injected alone.
Co-administration of both doses of ACPD to animals treated with
MK-801(0.1 mg/kg)significantly improvedthe performance of ratsin
the Active Allothetic Place Avoidance task when compared to MK-801
alone. Unfortunately we have not found any comparison data in the
literature.
Non-specific agonist ACPD activates both the mGluI receptor and
mGluII receptor groups, respectively. The mGluIII receptor group is
ACPD insensitive (Conn and Patel, 1994). Based on pharmacology we
can not say that the effect of ACPD is due to the mGluI or mGluII
receptor groups. This is why more specific compounds were used.
Whatcan be established basedonour data withACPDis thatthe effect
is not induced by ionotropic receptors (Conn and Patel, 1994).
In the second part of the study we focused on the effect of the
mGlu2/3 allosteric modulators on the performance of the Active
Allothetic Place Avoidance task. Application of an allosteric positive
modulator of mGlu2/3 receptor, LY487379, alone had no effect on
locomotion and cognition parameters (Number of errors and
Maximum Time Avoided). Our results are in accordance with studies
showing that administration of the mGlu2/3 receptor agonist induced
a delayed working memory deficit (Higgins et al. 2004). In another
study, the application of mGlu2/3 receptor agonist LY354740 induced
a working memory deficit, but had no effect on the acquisition of
passive avoidance (Schlumberger et al., 2009).
The agonist of mGlu2/3 receptor is the next promising target in the
pharmacotherapy of the cognitive symptoms of schizophrenia (Patil
et al., 2007). They are localized in various combinations of
presynaptic, postsynaptic, extrasynaptic and glial elements. Several
studies indicate that the activation of this group of receptors
“normalizes” glutamate release, presumably, via presynaptic recep-
tors localized on excitatory terminals (Cartmell and Schoepp, 2000).
Preclinical studies suggest that agonists of mGlu2/3 receptor diminish
the behavioral effects of NMDA antagonists such as stereotypy
(Cartmell et al., 1999) and working memory deficits (Moghaddam
and Adams, 1998). Recently, positive allosteric modulators of mGlu2/
3 receptor have been discovered, which may be more feasible for
long-term treatment than direct agonists (Johnson et al., 2003).
In our study, LY487379 did not ameliorate the cognitive impair-
ment induced by MK-801. Low doses of mGlu2/3 receptor agonist
(LY487379) in co-treatment with MK-801 increased the Number of
errors and decreased Maximum Time Avoided compared to controls
and LY487379 itself. At high doses the LY487379 had no effect. The
similar agonist, LY354740 did not modify the PCP-induced working
memory deficit. Moreover mGlu2/3 receptor agonists failed to
improve the deficit in pre-pulse inhibition induced by PCP (Schreiber
et al., 2000). In addition, the sub-chronic administration of LY354740
did notameliorateketamine-evokedbehaviorandneuronalactivityin
rats (Imre et al., 2006).
On the other hand, the agonists of mGlu2/3 receptor reduce the
behavioral effects of the NMDA antagonist phencyclidine, such as
stereotypic behaviors, hyperactivity and memory deficit (Cartmell
et al., 1999; Moghaddam and Adams, 1998). Similarly, LY379268
Fig. 5. Effect of mGlu5 positive modulator, CDPPB on Active Allothetic Place Avoidance
task performance. (A) Locomotor activity was not affected by MK-801 or CDPPB
administration. (B) Number of Entrances was impaired after application MK-801
0.1 mg/kg and was not restored after co-administration with CDPPB in both doses (0.5
and 5 mg/kg. (C) Deficit in Maximum Time Avoided induced by MK-801 was full
restored by CDPPB at dose 5 mg/kg. All values±S.E.M.; ** Pb0.01 compared to control
group; ## Pb0.01 compared to MK-801 0.1 mg/kg group.
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(mGlu2/3receptor agonist)is capable of preventing NMDAantagonist
induced hyperlocomotion, which is linked to a reduction of glutamate
exocytosis in the pre-frontal cortex (Imre et al. (2006). Finally the
mGlu2/3 allosteric positive modulator attenuated a deficit of social
novelty discrimination evoked by the neonatal administration of PCP
(Harich et al., 2007). It has been suggested that the mGlu2/3 receptor
agonists are more effective in models of positive symptoms (NMDA
antagonist induced hyperlocomotion, stereotypy) but not in models
of sensorimotor gating and cognitive impairment.
Our study is more focused on the agonist of mGlu5 receptor. We
used an orthosteric agonist, CHPG and two positive allosteric
modulators CDPPB and DFG both binding to the same side of mGlu5
receptor (Chen et al., 2007). Application of mGlu5 allosteric
modulators did not influence locomotor activity and cognition
parameters by itself. However, the application of CHPG decreased
the Number of errors in Active Allothetic Place Avoidance task in rats
treated with MK-801. However, both modulators blocked the
cognitive deficit induced by MK-801. The effect was more pronounced
after application of the more potent modulator CDPPB.
These results are in accordance with other studies, which showed
that application of an agonist of mGlu5 receptor (CDPPB) prevented
MK-801inducedexcessivefiringandreducedspontaneousburstingin
themedialpre-frontalcortexandnucleusaccumbens(Lecourtieretal.,
2007).TheOrthostericagonistaswellasallostericmodulatorofmGlu5
receptor, DFB, attenuated the locomotor hyperactivity, motor dis-
coordination,andcognitiveimpairmentinducedbyketamine(Chanet
al., 2008). However, antagonists of mGlu5 receptor induce a cognitive
deficit and potentiate the effects of NMDA receptor antagonists on
hyperlocomotion and disrupts pre-pulse inhibition (Kinney et al.,
2003), and working memory (Homayoun et al., 2004). In addition,
MPEP, a negative allosteric modulator of mGlu5 receptor, enhanced
locomotor activity increased by fencyclidine, NMDA antagonist, but
inhibited amphetamine-induced hyperactivity (Pietraszek et al.,
2005). The results are consistent with other findings suggesting that
mGlu receptor modulates the NMDA receptor function at molecular
and behavioral levels (Brody et al., 2004; Kinney et al., 2003; Marino
and Conn, 2002; Mannaioni et al., 2001; Ugolini et al., 1999).
Our findings show a difference in effect of the activation of mGlu5
and mGlu2/3 receptors in cognitive deficit induced by the NMDA
antagonist, MK-801. While activation of mGlu5 receptor leads to a
significant improvement of cognitive parameters without seriously
affecting locomotion, activation of mGlu2/3 receptor has no positive
effect on cognition parameters. This may be explained by their
different influences upon NMDA receptor function and glutamatergic
imbalance by mGlu5 vs. mGlu2/3 receptors or an interaction with
other neurotransmitter systems (a dopaminergic system or the
interaction of 5-HT2A with mGlu2/3 receptor) (Liu et al, 2008;
Marek et al, 2000)
Altogether, our data show that especially the mGlu5 receptor
agonist could be effective in the treatment of cognitive deficit in
patients with schizophrenia. However, the pro-cognitive effect of the
agonist of mGlu2/3 receptor was not clearly demonstrated in the
present study.
Acknowledgements
This work was supported by grant IGA MZD NR/9178-3 and by
AV0Z 50110509. We thank Misa Fialova for technical assistance and
Peter M. Luketic for language review and Abbott for unselfishly
providing the two drugs.
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