74 Short report
The selective metabotropic glutamate 2/3 receptor agonist
MGS0028 reverses psychomotor abnormalities and
recognition memory deficits in mice lacking the pituitary
adenylate cyclase-activating polypeptide
Yukio Agob, Naoki Hiramatsua,b, Toshihiro Ishihamab, Keisuke Hazamaa,c,
Atsuko Hayata-Takanoc,d, Yasuhiro Shibasakic, Norihito Shintanic,
Hitoshi Hashimotoc,d, Toshiyuki Kawasakie, Hirotaka Onoee,
Shigeyuki Chakih, Atsuro Nakazatog, Akemichi Babaf, Kazuhiro Takumab
and Toshio Matsudab,d
Previous studies suggest that metabotropic glutamate
2/3 receptors are involved in psychiatric disorders.
In this study, we examined the effects of the selective
metabotropic glutamate 2/3 (mGlu2/3) receptor agonist
MGS0028 on behavioral abnormalities in mice lacking the
pituitary adenylate cyclase-activating polypeptide (PACAP),
an experimental model of psychiatric disorders such as
schizophrenia and attention-deficit/hyperactivity disorder.
We found that PACAP-deficient mice showed impairments
in the novel object recognition test and these impairments
were improved by MGS0028 (0.1 mg/kg). Similarly,
MGS0028 improved hyperactivity and jumping behaviors,
but did not reverse increased immobility times in the
forced swim test in PACAP-deficient mice. These results
suggest that MGS0028 may be a potential, novel treatment
for psychiatric disorders. Behavioural Pharmacology
c 2013 Wolters Kluwer Health | Lippincott
24:74–77 Williams & Wilkins.
Introduction
The pituitary adenylate cyclase-activating polypeptide
(PACAP) is a multifunctional neuropeptide that acts as a
neurotransmitter and as a neuromodulator (Vaudry et al.,
2009). Hashimoto et al. (2007, 2010) showed that PACAP
gene single nucleotide polymorphisms are associated with
schizophrenia and major depressive disorders, suggesting
that PACAP may be a risk factor for psychiatric disorders.
They also reported that PACAP-deficient (Adcyap1 – / – )
mice show behavioral abnormalities, such as hyperlocomotion and jumping behavior in the novel open-field test
and prepulse inhibition deficits in a startle test, and
these changes were improved by the atypical antipsychotic drug risperidone and the attention-deficit/hyperactivity disorder drugs amphetamine and methylphenidate
(Hashimoto et al., 2001; Tanaka et al., 2006). Therefore,
Adcyap1 – / – mice are considered an animal model that
provides valid assessments of potential antipsychotic
drugs.
c 2013 Wolters Kluwer Health | Lippincott Williams & Wilkins
0955-8810 Behavioural Pharmacology 2013, 24:74–77
Keywords: cognition, hyperactivity, metabotropic glutamate 2/3 receptor,
mouse, pituitary adenylate cyclase-activating polypeptide, psychiatric
disorders
a
Japan Society for the Promotion of Science, Tokyo, bLaboratory of Medicinal
Pharmacology, cLaboratory of Molecular Neuropharmacology, Graduate School
of Pharmaceutical Sciences, dUnited Graduate School of Child Development,
Osaka University, Osaka, eFunctional Probe Research Laboratory, RIKEN,
Center for Molecular Imaging Science, Kobe MI R&D Center, fHyogo University
of Health Sciences, Hyogo, gStrategic Planning of Drug Discovery, R&D
Headquarters of Pharmaceutical Operation, Taisho Pharmaceutical Co. Ltd and
h
Molecular Function and Pharmacology Laboratories, Taisho Pharmaceutical
Co. Ltd, Saitama, Japan
Correspondence to Toshio Matsuda, PhD, Laboratory of Medicinal
Pharmacology, Graduate School of Pharmaceutical Sciences, Osaka University,
1-6 Yamada-oka, Suita, Osaka 565-0871, Japan
E-mail: matsuda@phs.osaka-u.ac.jp
Received 19 July 2012 Revised 2012 Accepted as revised 14 October 2012
A metabotropic glutamate (mGlu) 2/3 receptor agonist
and an mGlu2 allosteric potentiator each inhibit amphetamine-induced and phencyclidine-induced hyperactivity
(Galici et al., 2005; Johnson et al., 2005). Moreover,
mGlu2/3 receptor agonists ameliorate isolation rearinginduced hyperactivity and deficits of sensorimotor gating
and recognition memory (Jones et al., 2011; Ago et al.,
2012). These findings suggest that activation of mGlu2/3
receptors may have therapeutic benefits for patients with
psychiatric disorders such as schizophrenia. In this
respect, we have previously found that Adcyap1 – / – mice
show both behavioral abnormalities and cognitive impairments. In the present study, we examined the effects of
the potent and selective mGlu2/3 receptor agonist
MGS0028, which has a very high affinity for mGlu2
(EC50 = 0.57 nmol/l) and mGlu3 (EC50 = 2.07 nmol/l)
receptors (Nakazato et al., 2000; Ritzén et al., 2005), on
cognitive impairments, hyperactivity, jumping behavior,
and depression-like behavior in Adcyap1 – / – mice.
DOI: 10.1097/FBP.0b013e32835cf3e5
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Effect of mGluR2/3 agonist in PACAP-deficient mice Ago et al. 75
Methods
Subjects and drugs
All experiments were conducted in accordance with
protocols approved by the Animal Care and Use
Committee of the Graduate School of Pharmaceutical
Sciences, Osaka University. Homozygous PACAP-null
mutant mice were generated and characterized as
described previously (Hashimoto et al., 2001), and they
were backcrossed for more than 10 generations onto CD1
mice. All mice used were obtained from the intercross of
the heterozygous animals, and all experiments were
conducted on 8-week-old to 10-week-old male mice.
The wild-type control and Adcyap1 – / – mice were housed
separately under a standard 12 h light/dark cycle (lights
on at 8:00 a.m.), at a constant temperature of 22±11C,
with free access to food and water. MGS0028 (Taisho
Pharmaceutical Co., Saitama, Japan) was dissolved in saline
(0.9% NaCl) and adjusted to pH 7.4 with 0.5 mol/l NaOH.
The drug was injected at 10 ml/kg intraperitoneally.
Open-field test
Each mouse was placed in the center of an open cubic
transparent acrylic box with a black Plexiglas floor
(45 cm 45 cm 30 cm) and allowed to explore the
environment for 60 min. Their horizontal locomotor
activities and jumping behaviors were monitored using
an infrared photocell beam detection system (Acti-Track;
Panlab, Barcelona, Spain) (Ishihama et al., 2010).
Novel object recognition test
Each mouse was habituated by housing it in the test cage
(22 cm 38 cm 20 cm) for 3 days. In each training
session, two identical objects (yellow Lego blocks) were
placed in the cage, and the mouse was allowed to explore
the cage for 15 min. The retention sessions were
conducted 6 h after the training sessions. In each 5-min
retention session, the mouse was placed back in the test
cages with a familiar object that had been presented
in the training session and a novel object (wooden ball).
The behaviors of the mice were videotaped and evaluated
by observers blinded to the genotype and treatment
conditions. The objects were fixed to the floor and
cleaned thoroughly between trials to ensure the absence
of olfactory cues. Exploratory preferences were calculated
as the ratio of the time spent exploring the novel object
versus the total time spent exploring both the familiar
and the novel objects, and these ratios were used as the
dependent measure of recognition memory.
Forced swim test
The forced swim test was carried out as reported
previously (Ishihama et al., 2010). The total immobility
time was measured during the final 4 min of a 6-min
swimming session by observers blinded to the genotype
and treatment conditions.
Statistical analysis
All data were analyzed using two-way analysis of variance,
followed by the Tukey–Kramer test. Statistical analyses
were carried out using the software package Statview 5.0 J
for Apple Macintosh (SAS Institute, Cary, North Carolina,
USA). A value of P less than 0.05 was considered
statistically significant.
Fig. 1
Training session
Exploratory time (s)
(b)
Retention session
150
250
100
Exploratory preference (%)
(a)
200
100
150
100
50
50
0
0
Vehicle
MGS0028
(0.1 mg/kg)
75
∗
50
25
0
Vehicle
Wild type
MGS0028
(0.1 mg/kg)
Vehicle
MGS0028
(0.1 mg/kg)
Adcyap1−/−
Effects of MGS0028 on recognition memory in Adcyap1 – / – and wild-type mice in the novel object recognition test. After habituation, the wild-type
or Adcyap1 – / – mice were exposed to two identical objects in 15-min training sessions (a), and then the recognition memory was tested 6 h later with
one of the familiar objects replaced by a novel object in 5-min retention sessions (b); MGS0028 (0.1 mg/kg) or vehicle was administered 1 h before
the training session. The total exploratory time of the mouse in either the training or the retention session was measured, and the percentage of the
total exploration time spent exploring the novel object was calculated. Values are expressed as the mean ± SEM of 8–9 mice/group. *P < 0.05,
compared with vehicle-treated wild-type mice; wwP < 0.01, compared with vehicle-treated Adcyap1 – / – mice. PACAP, pituitary adenylate
cyclase-activating polypeptide.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
76 Behavioural Pharmacology 2013, Vol 24 No 1
Fig. 2
300
200
100
Depression-like behavior
∗∗
150
100
50
Wild type
kg
m
g/
0.
3
m
g/
kg
0.
3
m
g/
kg
0
0.
1
kg
m
g/
0.
3
0.
1
m
g/
kg
0
Ve
hi
cl
e
0
∗∗
200
m
g/
kg
100
(c)
0.
1
200
Jumping behavior
400
Ve
hi
cl
e
(b)
Immobility time (sec)
Horizontal activity
∗∗
Ve
hi
cl
e
300
Number of jumps
Distance traveled (m)
(a)
Adcyap1−/−
Effects of MGS0028 on hyperactivity, jumping behavior, and immobility in the forced swim test in Adcyap1 – / – mice. One hour after the injection of
MGS0028 (0.1, 0.3 mg/kg) or vehicle, the wild-type or Adcyap1 – / – mice were placed in the open-field apparatus (a, b) or in the acrylic cylinder for
the forced swim test (c); The total distance traveled (a) and the number of jumps (b) during the 60 min open-field session and the total duration
of immobility during the final 4 min of the 6 min forced-swim sessions (c) were measured. Values are expressed as the mean ± SEM of 10–11
mice/group (a, b) and 7 mice/group (c). **P < 0.01, compared with vehicle-treated wild-type mice; wwP < 0.01, compared with vehicle-treated
Adcyap1 – / – mice. PACAP, pituitary adenylate cyclase-activating polypeptide.
Results
Discussion
Figure 1 shows the effects of MGS0028 on novel object
recognition in Adcyap1 – / – and wild-type mice. The total
exploratory time in either the training or the retention
session did not differ between Adcyap1 – / – and wild-type
mice, and MGS0028 (0.1 mg/kg) did not affect exploratory behavior in either genotype (Fig. 1a and b, left).
In the retention session, the wild-type mice spent more
time exploring the novel object than the familiar object,
and this preference can be used as an index of object
recognition. The Adcyap1 – / – mice explored the novel
object to the same extent as the familiar target, and the
preference index in Adcyap1 – / – mice was significantly
lower than that in wild-type mice (Fig. 1b, right).
MGS0028 improved the impairment in recognition
memory in Adcyap1 – / – mice, whereas it did not affect
cognitive performance in wild-type mice (the significant
interaction between the genotype and treatment:
F1,31 = 5.10, P < 0.05). Figure 2 shows the effects of
MGS0028 on hyperactivity, jumping behavior, and immobility in Adcyap1 – / – mice. MGS0028 (0.1 and 0.3 mg/kg) did
not affect locomotor activity in wild-type mice. Adcyap1 – / –
mice showed greater locomotor activity in a novel
environment than wild-type mice, and this effect was
inhibited by MGS0028 (F2,58 = 3.24, P < 0.05) (Fig. 2a).
MGS0028 (0.1 and 0.3 mg/kg) also inhibited the jumping
behavior of Adcyap1 – / – mice (F2,58 = 10.67, P < 0.001)
(Fig. 2b). Adcyap1 – / – mice showed longer immobility
times in the forced swim test than wild-type mice, but
MGS0028 did not affect immobility times in either
genotype (Fig. 2c).
The present study examined the effects of the mGlu2/3
receptor agonist MGS0028 on abnormal behaviors in
Adcyap1 – / – mice. We first found that Adcyap1 – / – mice
showed impaired recognition memory in the novel object
recognition test. Consistent with this finding, Tajiri et al.
(2012) reported that Adcyap1 – / – mice showed impaired
spatial working memory in the Y-maze test. Taken
together with previous behavioral studies in Adcyap1 – / –
mice (Hashimoto et al., 2001, 2009; Tanaka et al., 2006),
this supported the use of Adcyap1 – / – mice to assess
potential antipsychotic drugs. The present study showed
that MGS0028 improved the impaired recognition
memory, hyperactivity, and jumping behavior, but not
immobility in the forced swim test, in Adcyap1 – / – mice.
In the novel object recognition test, the total time spent
exploring the objects was not altered in Adcyap1 – / – mice,
which are hyperactive in a novel environment. This may
be because we performed 3 days of habituation to the test
cage. Under these conditions, MGS0028 improved the
deficits of recognition memory in Adcyap1 – / – mice.
Similarly, Jones et al. (2011) found that LY379268, an
mGlu2/3 receptor agonist, improved recognition memory
deficits in isolation-reared rats. However, Horiguchi et al.
(2011) reported that LY379268 did not affect impaired
recognition in subchronic phencyclidine-treated rats. Moreover, MGS0028 improved hyperactivity and jumping behavior, but not immobility, in Adcyap1 – / – mice. This reversal
of hyperactivity is consistent with previous reports that
mGlu2/3 receptor agonists inhibit amphetamine-induced
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Effect of mGluR2/3 agonist in PACAP-deficient mice Ago et al. 77
and phencyclidine-induced hyperactivity [for a review,
see Chaki (2010)], and it is similar to the effects of the
atypical antipsychotic drug risperidone (Hashimoto et al.,
2007). In addition, the lack of effects on immobility in the
forced swim test is similar to the lack of effects of the typical
antipsychotic drug haloperidol (Hashimoto et al., 2009). As
such, each abnormal behavior of Adcyap1 – / – mice shows
different sensitivity to antipsychotic drugs, and it is likely
that these abnormal behaviors are mediated by different
mechanisms.
In clinical studies of the mGlu2/3 receptor agonist, Patil
et al. (2007) reported that LY2140023, the methionine
amide prodrug of the selective mGlu2/3 receptor agonist
LY404039, elicited a significant greater improvement in
both positive and negative symptoms in schizophrenia,
whereas Kinon et al. (2011) reported an inconclusive
result. After the submission of this paper, Eli Lilly and
Company unfortunately has reported negative results
from the H8Y-MC-HBBM study investigating LY2140023
for the treatment of patients with an acute exacerbation
of schizophrenia (http://newsroom.lilly.com/releasedetail.cfm?
releaseid = 690836). Therefore, it is not known whether
the mGlu2/3 receptor is actually a priority target for the
treatment of schizophrenia.
Conclusion
The present study showed that recognition memory is
impaired in Adcyap1 – / – mice, and MGS0028 improved
the impaired recognition, hyperactivity, and jumping
behavior in Adcyap1 – / – mice. Pozzi et al. (2011) have
recently reported that the mGlu2/3 receptor agonist
LY379268 reduced (R)-CPP-induced impairment in
attentional functioning and impulsivity. Taken together,
it is likely that the mGlu2/3 receptor might be a potential
target for the treatment of psychiatric disorders such
as attention-deficit/hyperactivity disorder.
Acknowledgements
This study was supported in part by the ‘Molecular
Imaging Research Program’ of the Ministry of Education,
Culture, Sports, Science and Technology of Japan. A part
of this work was also supported by Grants in Aid for
Scientific Research (B), Research Fellowships for Young
Scientists from the Japan Society for the Promotion of
Science (JSPS), and the Funding Program for Next
Generation World-Leading Researchers.
Conflicts of interest
There are no conflicts of interest.
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