The effect of mGlu5 receptor positive allosteric modulators on signaling molecules in brain slices

Article abstract of DOI:10.1016/j.ejphar.2006.02.003

Positive allosteric modulators of metabotropic glutamate receptor subtype 5 (mGlu5) have promising therapeutic potential. The effects of selective mGlu5 receptor positive allosteric modulators on signaling molecules in brain slices have not been previousl

Full text of DOI:10.1016/j.ejphar.2006.02.003

European Journal of Pharmacology 536 (2006) 262–268
www.elsevier.com/locate/ejphar
Short communication
The effect of mGlu5 receptor positive allosteric modulators
on signaling molecules in brain slices
a
a
a
a
Feng Liu a, , Guoming Zhang , Geoffrey Hornby , Dmytro Vasylyev , Mark Bowlby ,
⁎
Kaapjoo Park a,b, Adam Gilbert a,b, Karen Marquis a, Terrance H. Andree a
a
Wyeth Neuroscience Discovery Research, CN 8000, Princeton, NJ 08543, USA
b
Exploratory Medicinal Chemistry, Chemical and Screening Sciences, Wyeth Research, 401 North Middletown Road, Pearl River, NY 10965, USA
Received 12 January 2006; received in revised form 27 January 2006; accepted 7 February 2006
Abstract
Positive allosteric modulators of metabotropic glutamate receptor subtype 5 (mGlu5) have promising therapeutic potential. The effects of
selective mGlu5 receptor positive allosteric modulators on signaling molecules in brain slices have not been previously reported. The current study
demonstrated that the selective mGlu5 receptor positive allosteric modulator, N-{4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2yl)-methyl]
phenyl}-2-hydrobenzamide (CPPHA) potentiated the response to a subthreshold concentration of 3,5-dihydroxy-phenylglycine (DHPG) on
extracellular signal-regulated protein kinase (ERK) and cyclic-AMP responsive element-binding protein (CREB) activity, as well as N-methyl D-
aspartate (NMDA) receptor subunit NR1 phosphorylation in cortical and hippocampal slices. These results suggest that allosteric modulators of
mGlu5 receptor could have physiologically significant effects by potentiating the actions of glutamate.
© 2006 Published by Elsevier B.V.
Keywords: Metabotropic glutamate receptor subtype 5; Extracellular signal-regulated protein kinase; Cyclic-AMP responsive element-binding protein; N-methyl
D-aspartate receptor; N-{4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2yl)-methyl]phenyl}-2-hydrobenzamide
1. Introduction
affected by the endogenous ligand. A few potentiators of
mGlu5 receptor have been described, including DFB (3,3′-
difluorobenzaldazine), and more recently CPPHA (N-{4-
chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2yl)-methyl]
phenyl}-2-hydrobenzamide) (O'Brien et al., 2003, 2004). Both
compounds are devoid of direct agonist activity, but can act as
selective positive allosteric modulators of competitive agonists
of human and rat mGlu5 receptor expressed in Chinese hamster
ovary (CHO) cells. DFB potentiates threshold responses to
glutamate, quisqualate, and DHPG 3- to 6-fold, with EC50
values in the 2–5 μM range. At 10–100 μM, DFB shifts the
mGlu5 receptor agonist concentration response curves approx-
imately two fold to the left. CPPHA, a compound from a
different structural class, potentiates threshold Ca2+ responses
to glutamate 6- to 13-fold with EC50 values in the 150–400 nM
range, and at 10 μM shifts the mGlu5 receptor agonist concen-
tration response curves to glutamate, quisqualate, and DHPG 4-
to 9-fold to the left.
Positive allosteric modulators of metabotropic glutamate
(mGlu) receptors have recently emerged as novel pharmacolo-
gical targets offering an attractive alternative to the direct
activation of mGlu receptors by orthosteric competitive
agonists. These types of molecules have been discovered for
mGlu1, mGlu2 and more recently for mGlu5 receptors
(Knoflach et al., 2001; Johnson et al., 2003; O'Brien et al.,
2003, 2004). These positive allosteric modulators offer the
potential to increase the efficiency of glutamate transmission
during normal stimulation without the risk of inappropriate
stimulation of the system. Furthermore, such compounds are
more likely to achieve high receptor subtype selectivity by
targeting regions of the receptor that are different than these
⁎
Corresponding author. Wyeth Neuroscience Research, CN 8000, 865 Ridge
Road, Monmouth Junction, NJ 08852, USA. Tel.: +1 732 274 4017; fax: +1 732
274 4020.
In this report, we investigated the effect of DFB and CPPHA
on the signaling molecules ERK and CREB in hippocampal and
E-mail address: liuf2@wyeth.com (F. Liu).
0014-2999/$ - see front matter © 2006 Published by Elsevier B.V.
doi:10.1016/j.ejphar.2006.02.003

F. Liu et al. / European Journal of Pharmacology 536 (2006) 262–268
263
cortical slices as well as phosphorylation levels of the NR1
subunit of the NMDA glutamate receptor.
phospho-CREB Ser-133) antibody, 1:1000, phospho-NR1 Ser-
897, 1:500] overnight at 4 °C followed by incubation with
horseradish peroxidase-linked goat anti-rabbit IgG (1:10,000)
and developed using ECL. The blots then were incubated in
stripping buffer (62 mM Tris–HCl, pH 6.8, 2% SDS, and
100 mM β-mercaptoethanol). The stripped blots were incubated
with antibody-directed against total levels for the respective
protein (ERK, antibody, 1:1000; CREB antibody, 1:1000; NR1
antibody, 1:1000). Densitometric analysis of phospho-immu-
noreactivity and total immunoreactivity for each protein was
conducted using the Bio-Rad GS-710 Calibrated Imaging Den-
sitometer and quantified using Quantity One version 4.1.0.
Phosphorylated immunoreactivity was normalized to total im-
munoreactivity. Data were statistically analyzed by one-way
ANalysis Of VAriance (ANOVA) followed by Dunnett's mul-
tiple comparison tests using GraphPad Prism 4.0 software.
2. Materials and methods
2.1. Materials
All primary antibodies were purchased from Cell Signaling
Technology. DFB, DHPG and MPEP were obtained from To-
cris. CPPHA was synthesized by the route shown in Scheme 1.
Enhanced chemiluminescence (ECL) Western blotting detec-
tion reagents were obtained from Amersham Biosciences.
2.2. Hippocampal and cortical slice preparations
All experimental protocols were approved by the Wyeth
Animal Care and Use Committee and were conducted in accor-
dance with the internationally accepted principles in the care
and use of experimental animals. Hippocampal and cortical
slices were obtained from 6–8 weeks old male C57BL/6 mice.
Slices (300 μm) were placed in Kreb's bicarbonate buffer
containing in 125 mM NaCl, 4 mM KCl, 26 mM NaHCO3,
10 mM D-glucose, 1.5 mM CaCl2, 1.5 mM MgSO4 and
1.25 mM KH2PO4. The slices were preincubated in the buffer
for 30 min and then were exposed to different compounds of
interest for indicated times and snap frozen in liquid nitrogen.
The slices were sonicated in boiled homogenization buffer.
2.4. Neuronal cultures
Rat dissociated hippocampal cell cultures were prepared by
previously described methods (Brewer et al., 1993, Brewer and
Price, 1996). Briefly, hippocampal tissue was dissected from em-
bryonic day 20 Sprague–Dawley rats, cut into small pieces and
incubated for 30 min at 37 °C in solution equilibrated with 95% air/
5% CO2 and containing (in mM) 166 NaCl, 5.4 KCl, 26 NaHCO3,
1 NaH2PO4, 1.5 CaCl2, 1 MgSO4, 0.5 EDTA, 25 glucose, 1 L-
cysteine, and 15–20 U/ml papain. The cells were dispersed by
gentle trituration, and the dissociated suspension was collected and
spun at 1000 ×g for 5 min. The pellet was resuspended in neural
basal complete media (Gibco) and plated at 300,000 cells/ml on 8-
well poly-D-Lysine/laminin coated culture chambers.
2.3. Immunoblot analysis
Equivalent amounts of protein for each sample were resolved
in 4–12% SDS Polyacrylamide Gel Electrophoresis (SDS-
PAGE) gel and transferred to nitrocellulose membranes. The
membranes were blocked for 1 h in Tris-buffered saline (TBS)
containing Tween 20 and then incubated with the phosphor-
specific antibody of interest [phospho-ERK antibody, 1:1000;
2.5. Immunocytochemistry
Control cultures and cultures treated with either DHPG or
CPPHA or CPPHA plus DHPG were immediately placed in ice-
Scheme 1.

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F. Liu et al. / European Journal of Pharmacology 536 (2006) 262–268
cold 4% paraformaldehyde in PBS, pH 7.4, and fixed for 30 min
at 4 °C, followed by washing with TBST (TBS plus 0.1% Triton
X-100) for 5 min. The cells were blocked with 5% normal goat
serum in TBST for 60 min at 4 °C. The cells were incubated
with primary antibody [phospho-CREB, (1:100)] in 5% BSA in
TBSTovernight at 4 °C. Cells were washed with TBST and then

F. Liu et al. / European Journal of Pharmacology 536 (2006) 262–268
265
Fig. 1. CPPHA potentiates a subthreshold concentration of DHPG on ERK and CREB activation in cortical and hippocampal slices. (A, B) Cortical slices were treated
with DHPG (100 μM) for 5 min as the positive control. The slices were preincubated for 15 min with 20 μM of CPPHA or DFB followed by cotreatment of 20 μM
DHPG for 5 min. (C, D) Hippocampal slices were treated with DHPG (100 μM) for 5 min as the positive control. The slices were preincubated for 15 min with 20 μM
CPPHA in the presence or the absence of MPEP followed by cotreatment of 20 μM DHPG for 5 min. The levels of phosphorylation in ERK and CREB were assessed
via immunoblotting. A (Cortical) and C (Hippocampus), representative immunoblots for phosphorylated ERK (p-ERK), total ERK, phosphorylated CREB (p-CREB),
total CREB. B and D, p-ERK and p-CREB immunoreactivity were normalized to total ERK and total CREB immunoactivity, and followed by normalizing to untreated
⁎
control immunoactivity. Data were statistically analyzed by one-way ANOVA followed by Dunnett's multiple comparison tests. Denotes statistical significance
compared with control (pb0.05, n=4); #denotes statistical significance compared with 20 μM of DHPG (pb0.05, n=4). E, CPPHA potentiates a subthreshold
concentration of DHPG effects on ERK activity in primary hippocampal neuronal cultures. Representative immunostaining images were obtained from cultures that
were labeled using antibodies specific for phospho-ERK. Control, untreated cells; 20 μM DHPG for 5 min; 100 μM DHPG for 5 min; 20 μM CPPHA for 20 min;
20 μM CPPHA+20 μM DHPG, cells that were preincubated with 20 μM CPPHA for 20 min and then incubated with 20 μM DHPG for 5 min in the presence of 20 μM
CPPHA; 10 μM Forskolin for 5 min (positive control).
incubated with goat anti-rabbit Alexa Fluro@488 (Molecular
Probes). Cells were washed with TBS and the coverslips were
mounted with Prolong Antifade Mounting reagent (Molecular
Probes).
3. Results
3.1. The effect of DFB and CPPHA on phosphorylation of ERK
and CREB
2.6. Slice preparation for electrophysiology
The extracellular signal-regulated protein kinase (ERK)
and the transcription factor cyclic-AMP-responsive element-
binding protein (CREB) are perhaps the most intensively
studied molecules in the field of synaptic plasticity.
Phosphorylation of ERK1/2 on Thr202/Tyr204 leads to
activation of this kinase to mediate an array of various
signalling pathways that control neuronal plasticity (Thomas
and Huganir, 2004). Similarly, phosphorylation of CREB at
Ser133 leads to recruitment of other transcription machinery
of CREs to regulate gene transcription, which is an important
step in the induction of gene expression that is essential to
learning and memory (Carlezon et al., 2005). The effect of
DFB and CPPHA on a subthreshold concentration of DHPG
for the phosphorylation of ERK and CREB was assessed in
mouse hippocampal and cortical slices using phosphorylation
state-specific antibodies. Preincubation of cortical and hippo-
campal slices with CPPHA (20 μM) alone had no effect on
basal levels, but potentiated the DHPG (20 μM)-induced
increase in phosphorylation of ERK and CREB, approaching
NMDA-evoked current was recorded from visually IR-
camera identified CA1 pyramidal neurons in a standard whole-
cell recording mode. NMDA was delivered locally to the
recording site using a fast local perfusion technique (Vese-
lovsky et al., 1996). Electrodes were pulled from borosilicate
glass capillaries (TW150F; World Precision Instruments,
Sarasota, FL). Pipette resistances ranged between 2 and
5 MΩ when filled with the intracellular solution (in mM):
70 K-Gluconate, 70 CsCl, 1 CaCl2, 5 Mg-ATP, 5 EGTA,
10 HEPES, and 1 QX314, pH adjusted to 7.4. The extracellular
solution for voltage-clamp experiments was ACSF with
addition of 0.5 μM tetrodotoxin (Sigma) to suppress action
potentials. Data were collected using a MultiClamp 700A
amplifier and digitized at 1 kHz using DigiData 1322A and
pClamp9.0 software (all from Axon Instruments). Data were
analyzed using ClampFit9.0 (Axon Instruments). Experiments
were performed at 33 °C.

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F. Liu et al. / European Journal of Pharmacology 536 (2006) 262–268
levels seen with 100 μM DHPG alone. Treatment of slices
with DHPG (20 μM) alone did not increase ERK or CREB
phosphorylation. In contrast, DFB (20 μM) caused little
potentiation effect (Fig. 1A and B). The potentiating effects of
CPPHA with DHPG could be blocked by the mGlu5 receptor
selective antagonist MPEP (Fig.1C and D). In parallel with
the brain slices, we also analyzed the effect of CPPHA in rat
primary hippocampal cultures using an immunocytochemistry
approach. Consistent with brain slice observations, we found
that CPPHA can potentiate a subthreshold concentration
(20 μM) of DHPG on CREB phosphorylation in primary
hippocampal cultures (Fig. 1E).
Fig. 2. A, representative immunoblot for phosphorylated NR1-Ser897 (pNR1-S897), total NR1. B, pNR1-S897 immunoreactivity was normalized to total NR1
imunoactivity, followed by normalizing to untreated control immunoactivity. Data were statistically analyzed by one-way ANOVA followed by Dunnett's multiple
⁎
comparison tests. Denotes statistical significance compared with control (pb0.05, n=4); #denotes statistical significance compared with 20 μM DHPG (pb0.05,
n=4). (C, D), CPPHA enhances NMDA-evoked currents in CA1 pyramidal neurons. C, Representative recordings showing NMDA-evoked current in the presence of
10 μM CPPHA and its potentiation after co-application of 2 μM DHPG. Holding potential was −60 mV. Currents were elicited by a 1-s long pulses (60 s inter-pulse
interval) of 100 μM NMDA directly to the recording site. Nine out of 13 neurons showed response. D, Graph showing the time course of DHPG-induced potentiation
of NMDA-evoked current in the presence of CPPHA. Drug concentrations are the same as shown in C.

F. Liu et al. / European Journal of Pharmacology 536 (2006) 262–268
267
3.2. The effect of DFB and CPPHA on phosphorylation of
NMDA receptor subunit NR1 Ser-897
in rat hippocampal slices to test the effect of CPPHA on NMDA
receptor currents. We found that CPPHA can potentiate NMDA
evoked current in hippocampal slices, which is consistent with
data reported by O'Brien et al. (2003).
One of the major functions of the mGlu5 receptor is the direct
modulation of NMDA receptor activity which allows for the fine
tuning of glutamatergic transmission both in vitro and in vivo
(Awad et al., 2000; Mannaioni et al., 2001; Heidinger et al., 2002).
It has been shown in two independent brain samples from
schizophrenia patients, that there is a significant decrease in
phosphorylation levels at serine 897 of the NMDA receptor
(NR1) subunit (Emamian et al., 2004). Together with the report
that antipsychotics increase phosphorylation of NR1 at S897 in
vivo, these data suggest that insufficient phosphorylation at Ser-
897 may contribute to the neuronal pathology underlying schi-
zophrenia (Leveque et al., 2000). The effects of DFB and CPPHA
on NR1 Ser-897 phosphorylation were evaluated in slices. Similar
to studies with ERK and CREB, slices preincubated with CPPHA
(20 μM) alone did not alter the basal level of NR1, but potentiated
the DHPG (20 μM)-induced phosphorylation of NR1 Ser-897,
approaching levels seen with DHPG at 100 μM alone (Fig. 2A
and B). The effects were blocked by the mGlu5 receptor selective
antagonist MPEP, suggesting CPPHA's potentiation effect on
NR1-Ser897 is mediated by the mGlu5 receptor. DFB had no
effect on basal or DHPG-induced phosphorylation of NR1 Ser-
897. To further characterize the effect of CPPHA on NMDA
receptor activity, we also performed whole cell voltage-clamp
recordings from CA1 pyramidal cells in rat hippocampal slices on
NMDA receptor currents. Preincubation of slices with 10 μM of
CPPHA, which alone had no effect, potentiated a subthreshold
concentration of DHPG (2 μM) effect on NMDA-evoked currents
(Fig. 2C and D).
In conclusion, using a native system such as brain slices or
primary cultures, we have demonstrated that positive allosteric
modulators of mGlu5 receptor can alter major signaling mo-
lecules known to be important in modulating synaptic trans-
mission and neuronal plasticity. The ability to increase the
efficiency of glutamate transmission during normal stimulation
conditions is an advantage of positive allosteric modulators.
The result of this synaptic modulation may have useful phy-
siological implications in conditions where there are postulated
deficiencies in glutamatergic function such as schizophrenia or
mild cognitive impairment. Recently, another novel and more
potent mGlu5 receptor positive allosteric modulator CDPPB
has been reported to have antipsychotic-like effects in rat
behavioral models (Kinney et al., 2005). As more mGlu5
receptor positive allosteric modulators are identified, the cur-
rently described native systems can be employed to study the
functional characteristics of these new compounds.
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In brain slices, we found that CPPHA can potentiate the
effects of DHPG on ERK and CREB activity and on NMDA
NR1 Ser-897 phosphorylation. Also, in primary rat hippocam-
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phosphorylation. There were previous reports that activation of
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mGluR5 (Choe and Wang, 2001; Mao and Wang, 2003). Our
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positive allosteric modulator in native systems, suggesting that
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modulator can have physiological significance. DFB showed a
lesser potentiation than CPPHA on these signaling molecules.
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