1521-0103/350/2/375–386$25.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright ª 2014 by The American Society for Pharmacology and Experimental Therapeutics
http://dx.doi.org/10.1124/jpet.114.213959
J Pharmacol Exp Ther 350:375–386, August 2014
The [18F]FDG mPET Readout of a Brain Activation Model to
Evaluate the Metabotropic Glutamate Receptor 2 Positive
Allosteric Modulator JNJ-42153605
Tine Wyckhuys, Leonie wyffels, Xavier Langlois, Mark Schmidt, Sigrid Stroobants,
and Steven Staelens
Molecular Imaging Center Antwerp, University of Antwerp, Antwerp, Belgium (T.W., St.S.); Nuclear Medicine Department,
University Hospital, Antwerp, Belgium (L.w., Si.S.); and Department of Neuroscience, Janssen Pharmaceutica NV, Beerse,
Belgium (X.L., M.S.)
Received February 13, 2014; accepted June 2, 2014
Introduction
Glutamate is the most abundant neurotransmitter in the
central nervous system, and an imbalance in its transmission
is implicated in various neurologic and psychiatric disorders
(Pilc et al., 2008; Johnson et al., 2009; Lüscher and Huber,
2010; Chiechio and Nicoletti, 2012). Schizophrenia is a severe,
disabling chronic disorder affecting approximately 1% of the
This work was funded by Antwerp University, Belgium, through a full-time
associate professor position for St.S., a part-time full professor position for Si.S.,
and a postdoctoral position for T.W.; and by Antwerp University Hospital,
Belgium, through a part-time departmental position for Si.S. and a full-time
position for L.w. M.S. and X.L. are with Janssen Research and Development,
Beerse, Belgium.
Part of this work was presented as follows: Wyckhuys T, wyffels L, Langlois
X, Schmidt M, Stroobants S, and Staelens S (2013) Evaluation of mGluR2
positive allosteric modulator JNJ-42153605 in an animal model of glutamatergic dysfunction using [18F]FDG microPET. Society for Neuroscience Annual
Meeting; 2013 Nov 9–13; San Diego, CA.
dx.doi.org/10.1124/jpet.114.213959.
(20 mg/kg i.p.) or saline. Fifteen minutes later, [18F]FDG was
injected (37 MBq i.v.) followed by a mPET/computed tomography scan. The increase due to memantine is significant for all
relevant brain areas, whereas for ketamine this is not the case.
Standard uptake values (SUVs) of the LY404039 pretreated and
memantine-challenged group display a full reversal. Pretreatment
with JNJ-42153605 also dose-dependently decreases SUV with
a full reversal as well (for 10 mg/kg). Moreover, specificity of JNJ42153605 is reached at this dose. In conclusion, this mPET
experiment clearly indicates that subanesthetic doses of memantine induce significant increases of [18F]FDG SUVs in discrete
brain areas and that the novel mGluR2 PAM has the capacity to
dose-dependently and specifically reverse memantine-induced
brain activation.
population (Lindsley et al., 2006). Numerous studies have
generated support for the N-methyl-D-aspartate (NMDA)
receptor hypofunction hypothesis of schizophrenia (Olney
et al., 1999; Lindsley et al., 2006; Gunduz-Bruce, 2009) causing
excess glutamate excitotoxicity. Figure 1 illustrates this
interplay between the NMDA receptor (NMDAR), GABA,
and glutamate, where in nonpathologic conditions, NMDARs
on GABAergic neurons mediate glutamate release by excitatory neurons modulating metabotropic glutamate receptor
(mGluR)-5 activation on connecting neurons (Fig. 1A). In
addition to the aforementioned postsynaptic mGluRs on
connecting neurons, the concentration of extracellular glutamate is also regulated by excitatory amino acid or glutamate
transporters on neuroglia (Fig. 1A) and by presynaptic mGluRs
functioning to limit the release of intracellular glutamate (Fig.
1A). NMDARs are ionotropic receptors that are both ligandgated, requiring glutamate to bind with coactivation of D-serine
or glycine, as well as voltage-dependent through channel blocks
ABBREVIATIONS: BINA, potassium 39-([(2-cyclopentyl-6-7-dimethyl-1-oxo-2,3-dihydro-1H-inden-5-yl)oxy]methyl)biphenyl l-4-carboxylate; CT,
computed tomography; FBP, filtered backprojection; [18F]FDG, [18F]fluorodeoxyglucose; JNJ-42153605, 3-cylcopropylmethyl-7-(4-phenylpiperidin1-yl)-8-trifluoromethyl [1,2,4] triazolo[4,3-a]pyridine; LY404039, (2)-(1R,4S,5S,6S)-4-amino-2-sulfonylbicyclo[3.1.0]hexane-4,6-dicarboxylic acid;
LY487379, N-(4-(2-methoxyphenoxy)-phenyl-N-(2,2,2-trifluoroethyl-sulfonyl)-pyrid-3-ylmethylamine; mGluR, metabotropic glutamate receptor;
MK-801, (5S,10R)-(1)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine; NMDA, N-methyl-D-aspartate; NMDAR, NMDA receptor;
PAM, positive allosteric modulator; PCP, phencyclidine; PET, positron emission tomography; SUV, standard uptake value; THIIC, N-(4-((2(trifluoromethyl)-3-hydroxy-4-(isobutyryl)phenoxy)methyl)benzyl)-1-methyl-1H-imidazole-4-carboxamide; VOI, volume of interest.
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ABSTRACT
Using [18F]fluorodeoxyglucose m–positron emission tomography ([18F]FDG mPET), we compared subanesthetic doses of
memantine and ketamine on their potential to induce increases
in brain activation. We also studied the reversal effect of the wellknown metabotropic glutamate receptor (mGluR)-2/3 agonist
LY404039 [(2)-(1R,4S,5S,6S)-4-amino-2-sulfonylbicyclo[3.1.0]
hexane-4,6-dicarboxylic acid] and the novel mGluR2 positive
allosteric modulator (PAM) JNJ-42153605 [3-cylcopropylmethyl7-(4-phenylpiperidin-1-yl)-8-trifluoromethyl [1,2,4] triazolo[4,3-a]
pyridine]. First, rats (n 5 12) were subjected to LY404039
(10 mg/kg s.c.) or vehicle, 30 minutes prior to saline, ketamine
(30 mg/kg i.p.), or memantine (20 mg/kg i.p.). Second, rats (n 5
12) were subjected to 2.5 mg/kg or 10 mg/kg mGluR2 PAM JNJ42153605 or vehicle (s.c.), 30 minutes prior to memantine
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Fig. 1. Illustration of the interplay between NMDAR, mGluR, and energization. (A) In glutamatergic neurons such as neuron (2), glutamine is converted
through glutaminase into glutamate, which is transported by vesicular glutamate transporter (VGLUT) to be released in the synaptic cleft, where it is taken
up (among others): (i) postsynaptically by mGluR5 to relay the excitatory signal to a connecting neuron such as neuron (3) (ii) by mGluR2 to modulate the
glutamate release of neuron (2), and (iii) by excitatory amino acid or glutamate transporters on neuroglia to terminate the excitatory signal by converting
glutamate back to glutamine via glutamine synthetase. This process is largely mediated by GABAergic neurons such as: neuron (1), which take up glutamate
through their NMDARs to release GABA in the synaptic cleft, thereby inhibiting glutamatergic neurons such as neuron (2). The energy empowering excited
neurons such as neuron (2) comes mainly from the glycolysis and aerobic respiration of lactate, which is anaerobically generated in astrocytes that take up
glucose from the plasma in circulation via the glucose transporter 1 (GLUT1), and partly from direct glycolysis followed by aerobic respiration in the neuron
receiving plasma glucose via GLUT3. (B) Detail of the ionotropic NMDA receptor, which is (i) ligand-gated, requiring glutamate to bind with coactivation of
D-serine or glycine, and (ii) voltage-dependent through channel blocks by extracellular Zn2+ and Mg2+ ions. (C) Detail of the metabotropic mGluR2 receptor,
which is a G protein–coupled receptor with allosteric binding sites in the 7-transmembrane (TM) domain receptors and an extracellular N-terminal for the
binding of a ligand through the Venus fly trap mechanism, thereby causing a conformational change that activates the G protein of which the a subunit
causes a downstream cascade.
by extracellular Zn21 and Mg21 ions (Fig. 1B). When the NMDARs
function improperly, the inhibitory GABAergic tone is reduced,
causing excess glutamate to be released (Fig. 1A).
Therefore, neuropharmacology researchers are especially
interested in targeting glutamate receptors to modulate
the synaptic efficacy of glutamate (Kew and Kemp, 2005).
Metabotropic GluR2 PAM JNJ-42153605 Evaluation by mPET
which of these NMDA antagonists is best suited as an animal
model. Although memantine and ketamine are similarly potent
at human GluN1/GluN2A receptors in electrophysiological
assays (Gilling et al., 2009), there are small but clear differences
in the biophysical properties of these two uncompetitive NMDA
receptor antagonists. Ketamine was shown to have slower
kinetics than memantine (Parsons et al., 1995). Both the onset
and the offset kinetics for memantine were two times faster than
those of ketamine (kon 0.32 6 0,11 106 M21 s21, koff 0.53 6 0.10
second21 and kon 0.15 6 0.05 106 M21 s21, koff 0.22 6 0.05
second21, respectively). However, the Kd values (1.65 6 1.05 mM
and 1.47 6 0.68 mM for memantine and ketamine, respectively)
confirm that the two compounds have similar affinity for the
receptor despite the difference in kinetics. Memantine is also
slightly more voltage-dependent than ketamine, as it binds
to a deeper site and senses more of the electrical field (fraction
0.90 6 0.09 and 0.79 6 0.04, respectively). Finally, memantine
shows a 20% untrapping where ketamine has little or no
partial untrapping (Gilling et al., 2009).
A robust criterion for a suitable schizophrenia animal model
is a pronounced increase of glucose usage in delineated brain
structures as the NMDAR hypofunction causes hyperactive
glutamatergic neurons. The energy empowering these excited
glutamatergic neurons is mainly lactate, which is anaerobically
generated in astrocytes that take up glucose from the plasma in
circulation via the glucose transporter 1, and partly direct
aerobic glycolysis in the neuron receiving plasma glucose via
glucose transporter 3 (Fig. 1A). The radiotracer [18F]fluorodeoxyglucose ([18F]FDG) is a radiolabeled glucose analog and is
used to visualize changes in glucose consumption. [18F]FDG
becomes phosphorylated to [18F]FDG-6-PO4 and, due to the
molecule modifications in contrast to glucose, gets trapped once
transported within cells. The concentration of [18F]FDG within
cells of specific brain regions is considered an indirect measure
of neural activity (Bailey et al., 2004).
Therefore in the present study, we used in vivo [18F]FDG
molecular imaging positron emission tomography (PET) in
the rat, using a volume-of-interest (VOI)–based approach to
compare both memantine and ketamine: 1) on their potential
to induce increases in activation in discrete brain regions, and
2) on the reversal effects in these models induced by the wellcharacterized mGlu2/3 agonist LY404039 [(2)-(1R,4S,5S,6S)4-amino-2-sulfonylbicyclo[3.1.0]hexane-4,6-dicarboxylic acid]
(Patil et al., 2007; Rorick-Kehn et al., 2007). Second, we
determined the effect of the novel mGluR2 PAM JNJ42315605 (Cid et al., 2012) in the best schizophrenia model
of the latter two (memantine/ketamine). The major advantage
of using mPET for screening of novel antipsychotics in
comparison with other preclinical screening techniques, such
as autoradiography, microdialysis, and ex vivo tissue analysis, is its noninvasiveness, allowing longitudinal studies and
intra-animal comparisons with significantly lower numbers of
animals (Lancelot and Zimmer, 2010).
Materials and Methods
Animals
Twenty-four male Sprague-Dawley rats (Charles River Laboratories,
Lyon, France) weighing 275–350 g and aged 9–12 weeks were
treated in accordance with the European Ethics Committee (decree
86/609/CEE), and the study protocol was approved by the local Animal
Experimental Ethical Committee of the University of Antwerp,
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Recently, mGluR2/3 have proven to be interesting targets.
mGluR2/3 are preferentially expressed on presynaptic terminals (Fig. 1A); they negatively modulate glutamate levels and
are highly abundant in brain areas, such as the prefrontal
cortex and hippocampus, which are implicated in disorders
such as anxiety and schizophrenia (Cartmell and Schoepp,
2000; Vinson and Conn, 2012). Therefore, compounds known
to activate the mGluR2/3 are considered potential antipsychotics and anxiolytics. The mGluR2 receptor is a G protein–coupled
receptor with allosteric binding sites in the 7-transmembrane
domain and an extracellular N-terminal domain for binding a
ligand through the Venus fly trap mechanism, thereby causing
a conformational change in the 7-transmembrane domain,
resulting in activation of the G protein of which the a subunit
causes a downstream cascade (Fig. 1C). Previously, it was
shown that receptor agonists for the mGluR2/3 have robust
anxiolytic effects and can improve negative and positive
symptoms in schizophrenia in both animal models and
humans (Galici et al., 2005; Patil et al., 2007; Nikiforuk
et al., 2010; Wiero
nska et al., 2012).
In addition to mGluR2 and mGluR2/3 agonists, compounds
that can increase the effect of glutamate by binding to the
aforementioned allosteric sites (Conn et al., 2009), called
positive allosteric modulators (PAMs), are being investigated
(Johnson et al., 2003; Galici et al., 2006; Dhanya et al., 2011;
Fell et al., 2011; Lundström et al., 2011; Cid et al., 2012). The
use of PAMs compared with agonists has the advantage of
a higher selectivity and potentially overcoming the desensitization of G protein–coupled receptors observed after repeated
dosing of agonists (Bonnefous et al., 2005). In a recent study,
a potent and highly selective mGluR2 PAM JNJ-42153605 [3cylcopropylmethyl-7-(4-phenylpiperidin-1-yl)-8-trifluoromethyl
[1,2,4] triazolo[4,3-a]pyridine] has been identified (Cid et al.,
2012). This compound significantly influences rat sleep-wake
organization by decreasing rapid eye movement sleep, and it
reverses phencyclidine (PCP)-induced locomotor activity in
mice, denoting antipsychotic-like effects (Cid et al., 2012).
In developing new antipsychotics, the use of preclinical
screening methods using animal models of schizophrenia can
be informative for confirmation of pharmacologic effect and
dose response. In the search for suitable models, focus is on the
similarities of the animal model with the clinical behavioral
symptoms of schizophrenia and the increased activation in
discrete brain areas associated with the disease. In addition,
validity of these animal models is confirmed by determination
of the effects of known antipsychotics on the reversal of the
pathologic behavior and the increased brain activation. Acute
administration of PCP, MK-801 [(5S,10R)-(1)-5-methyl-10,11dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine], or subanesthetic doses of ketamine have been used as on-demand models
of schizophrenia, as both the behavior resembles the clinical
pathology (Javitt and Zukin, 1991; Andiné et al., 1999) as well
as the increased activation in distinct brain areas, such as the
prefrontal and cingulate cortices and hippocampus (Duncan
et al., 1998a). For example, reversal of ketamine-induced
effects was evaluated using both the typical antipsychotic
haloperidol and the atypical antipsychotic clozapine (Duncan
et al., 1998b) and confirms the model’s validity. Besides the
NMDAR antagonist ketamine model, administration of another NMDA receptor antagonist, memantine, has also been
suggested as an appropriate on-demand model of schizophrenia
(Dedeurwaerdere et al., 2011). However, controversy exists on
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Belgium (2011–67). The animals were kept in individually ventilated
cages under environmentally controlled conditions (12-hour normal
light/dark cycles, 20–23°C, and 50% relative humidity) with food and
water ad libitum.
Drugs
Experimental Procedures
In the first part, animals (n 5 12) were subjected to subcutaneous
injection of the test compound, i.e., LY404039 (10 mg/kg) or vehicle, 30
minutes prior to challenge with the NMDA receptor antagonist,
i.e., ketamine (30 mg/kg i.p.), memantine (20 mg/kg i.p.) or saline,
each of which, in turn, was administered 15 minutes prior to [18F]FDG
injection (37 MBq i.v. under short anesthesia) as schematically
illustrated by Fig. 2A. In the second part, animals (n 5 12) were
subjected to subcutaneous injection of JNJ-42153605 (2.5 mg/kg or
10 mg/kg) or vehicle, 30 minutes prior to challenge with memantine.
Memantine (20 mg/kg i.p.) injection is performed 15 minutes prior to
[18F]FDG injection (37 MBq i.v. under short anesthesia) as shown in
Fig. 2B. In both studies, [18F]FDG injection was followed by a 30minute awake uptake period to reach steady state. Anesthesia with
isoflurane (mixture with medical oxygen: 5% for induction, 1.5%
during the experiment) was started 25 minutes after tracer injection;
the animal was positioned onto the mPET scan bed and scanned (PET)
for 20 minutes, followed by a micro–computed tomography (CT) scan.
For these scans the animals were fasted for at least 12 hours, and
during the entire procedure their body temperature was controlled to
reduce variability. [18F]FDG was prepared using a cassette-based
GE Fastlab synthesis module (GE Healthcare, Diegem, Belgium).
[18F]Fluoride was produced by bombarding 18O-enriched water using
an 11 MeV proton beam in a Eclipse HP cyclotron (Siemens,
Knoxville, TN). The purified [18F]FDG was diluted with 0.9% NaCl
(Baxter, Eigenbrakel, Belgium) and sterile filtered through a 0.22 mM
filter. Quality control was performed according to European Pharmacopoeia 7.1, while radiochemical identity was confirmed by highpressure liquid chromatography (Dionex; ThermoFisher, Waltham,
MA) and radio–thin layer chromatography. Radiochemical purity
was also determined by high-pressure liquid chromatography
(Dionex; ThermoFisher), and radionuclidic identity and purity
were confirmed by g spectrum analysis (Multichannel analyzer;
Canberra, Meriden, CT).
Imaging
Imaging was performed on a Siemens Inveon PET-CT scanner
(Siemens Preclinical Solutions, Knoxville, TN). For quantitative
analysis, mPET images were reconstructed using the two-dimensional
filtered backprojection (FBP). Before image reconstruction, threedimensional sinograms were converted into two-dimensional
sinograms by Fourier rebinning. For the FBP reconstruction, a ramp
filter with cut-off at one-half the Nyquist criteria (maximum sampling
frequency) was set. Normalization, dead time, random, CT-based
Fig. 2. Study design with imaging protocol. (A) For comparison between
ketamine and memantine model exemplified by LY404039. (B) For
investigation of JNJ-42153605 in memantine model; rat brain [18F]FDG
scan normalization with (C) standardized MR space with predefined VOIs
together with a [18F]FDG template in the standard space (Schiffer rat MR
atlas available in PMOD 3.3) was used (from left to right: MR image, MR
and VOI definitions, [18F]FDG template, PET/MR, image and VOIs). (D)
Sample individual [18F]FDG PET/CT scan as obtained from our PET
camera after cropping out the brain (left to right: CT image, PET image,
and PET/CT image). (E) The resulting normalized images after applying
the elastic transformation to the individual PET and CT images. The
transformation was calculated by matching the individual PET from (D) to
the PET template of (C). As a result the VOIs in the standard space can be
used for quantifying [18F]FDG brain uptake in different regions. The
double-sided arrow indicates a registration step (E, elastic) with filled
arrow pointing to the reference image.
attenuation, and single scatter stimulation (Watson, 2000) scatter
corrections were applied. The reconstructed spatial resolution
approached 1.4 mm in the center of the field of view, and images
are displayed in transverse, coronal, and sagittal planes with
a matrix size of 128 128 159. The image pixel size in the
FBP-reconstructed images is 0.77 mm with a slice thickness of
0.79 mm.
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For ketamine, a concentration of 30 mg/kg was chosen, based on
literature (Duncan et al., 1998b; Dedeurwaerdere et al., 2011) as
higher concentrations (.40mg/kg) are reported to show loss of excitation
(Miyamoto et al., 2000). For memantine, a concentration of 20 mg/kg
was chosen based on the findings in literature (Dedeurwaerdere et al.,
2011).
Both memantine (memantine hydrochloride; Sigma-Aldrich,
St. Louis, MO) (20 mg/kg) and ketamine (Ketalar; Pfizer, New York,
NY) (30 mg/kg) were dissolved in saline (NaCl 0.9%; B. Braun
Medical, Melsungen, Germany). The mGluR2/3 agonist LY404039 (10
mg/kg; provided by Janssen Pharmaceutica NV, Beerse, Belgium) was
dissolved in H2O 1 NaOH (3.5 , pH , 9) as the LY-vehicle. The
mGluR2 PAM agonist JNJ-42153605 (provided by Janssen Pharmaceutica NV) was dissolved in 10% cyclodextrin 1 HCl (3.5 , pH ,9) as
the JNJ-vehicle.
Metabotropic GluR2 PAM JNJ-42153605 Evaluation by mPET
Data Analysis
Results
The Effect of Memantine/Ketamine. In the first part,
after both vehicle and memantine or ketamine administration, widespread increases in [18F]FDG uptake are observed
in neuroanatomically distinct regions in comparison with the
vehicle 1 saline controls. The mean (6 S.E.M.) percentage
changes in compared SUVs are indicated in Table 1.
Statistically significant increases in [18F]FDG SUVs are
observed between vehicle 1 memantine and vehicle 1 saline
treatment in all of the eight relevant brain areas (Fig. 3). The
percentage increase due to this memantine challenge in
comparison with vehicle 1 saline treatment is between 31.0%
(68.9%) for thalamus and 50.1% (612.1%) for frontal cortex
(Fig. 4, A and C; Table 1). No statistically significant increases
in [18F]FDG SUVs are observed in any of the eight relevant
brain areas between vehicle 1 ketamine and vehicle 1 saline
treatment (Fig. 3). The percentage increase due to ketamine
challenge in comparison with vehicle 1 saline treatment is
between 13.2% (66.9%) for thalamus and 22.4% (69.8%) for
frontal cortex (Fig. 4, A and B; Table 1). Memantine induces
on average a 16.6, 25.0, 24.8, 22.9, 24.0, 23.3, 18.0, and 17.0%
greater [18F]FDG uptake in comparison with ketamine, for
the caudate putamen, cingulate cortex, frontal cortex, medial
prefrontal cortex, motor cortex, parietal cortex, anterodorsal
hippocampus, and thalamus, respectively.
The Effect and Specificity of LY404039 Treatment.
Comparison between vehicle 1 memantine and LY404039 1
memantine SUVs reveals significant decreases for all eight
relevant brain areas due to treatment with LY404039,
similarly as for the decrease in [18F]FDG uptake between
vehicle 1 ketamine and LY404039 1 ketamine (Fig. 3).
Percentage decreases due to LY404039 pretreatment are
between 230.2% (63.9%) for caudate putamen and 238.5%
(63.4%) for cingulate cortex (Fig. 4, C and F; Table 2) in the
memantine-challenged group and are between 229.2% (65.8%)
for anterodorsal hippocampus and 237.9% (65.8%) for frontal
cortex (Fig. 4, B and E; Table 2) in the ketamine group. However,
the mGlu2/3 agonist LY404039 also causes a statistically
significant decrease in [18F]FDG uptake in all eight relevant
brain areas (LY404039 1 saline) relative to the basal uptake
values in vehicle 1 saline of between 232.5% (63.2%) for
anterodorsal hippocampus and 238.5% (64.4%) for frontal
cortex (Fig. 4, A and D; Table 2). There are no statistically
significant differences between the three decreases reported in
Table 2 in any of the eight relevant brain areas, indicating lack of
specificity.
Animals that received a pretreatment with LY404039
and a memantine challenge show a decrease between 20.3%
(67.1%) for motor cortex and 210.9% (65.2%) for thalamus
compared with vehicle 1 saline treatment, indicating a full
reversal (Fig. 4, A and F; Table 1). On the other hand, animals
TABLE 1
Overview of (pre)treatment effects: different LY404039 experimental protocols in comparison with the vehicle + saline
condition
Percentage increase or decrease of activity (6 S.E.M.).
LY404039
Treatment
Versus Vehicle of LY404039 and Saline
Vehicle + Memantine
Vehicle + Ketamine
LY404039 + Memantine
LY404039 + Ketamine
%
Caudate putamen
Cingulate cortex
Frontal cortex
Medial prefrontal cortex
Motor cortex
Parietal cortex
Hippocampus (AD)
Thalamus
AD, anterodorsal.
32.0
47.6
50.1
43.5
47.3
42.2
32.3
31.0
6
6
6
6
6
6
6
6
8.7
10.9
12.1
9.6
11.9
12.1
8.8
8.9
14.6
20.2
22.4
18.8
20.9
17.2
13.7
13.2
6
6
6
6
6
6
6
6
7.0
9.1
9.8
8.6
9.5
9.1
6.1
6.9
25.3
26.7
20.9
25.9
20.3
21.7
28.0
210.9
6
6
6
6
6
6
6
6
5.6
6.2
7.3
5.9
7.1
7.1
4.8
5.2
221.2
227.1
226.3
225.0
223.8
221.7
220.7
223.5
6
6
6
6
6
6
6
6
5.0
5.7
5.9
5.4
6.1
5.6
4.6
4.5
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Image Analysis. Each individual reconstructed PET image was
transformed into the space of a standard [18F]FDG PET template
(Schiffer et al., 2006) using brain normalization in PMOD v3.3 (PMOD
Technologies, Zurich, Switzerland). The normalized images were then
overlaid with a magnetic resonance imaging–based rat-brain VOI
template (Schiffer et al., 2006) available in the same software package
(Fig. 2, C–E) and by default already coregistered with the PET normal
database. Averaged activity concentrations for the different VOIs Ai
(kBq/ml) were extracted and the standard uptake values (SUVs) for
these VOIs was calculated as SUVi 5 Ai/ID mi, where i is the VOI
index, ID (kBq) is the injected activity, and m (g) is the weight of the
animal. The SUV values for eight clearly identifiable and relevant
brain areas were determined for the six experimental conditions, for
all animals. The rationale was for caudate putamen: coherent
intrinsic activity of the dorsal striatum increases and correlates with
positive symptoms, such as delusion and hallucination (Sorg et al.,
2013); cingulate cortex: receives input from the thalamus and is an
integral part of the limbic system, which makes it important in
cognitive function (Adams and David, 2007); frontal cortex and medial
prefrontal cortex: hyperfrontality (Soyka et al., 2005; WhitfieldGabrieli et al., 2009); motor cortex: motor abnormalities are
frequently part of the pathophysiology (Walker et al., 1994); parietal
cortex: involved in sensory integration; (anterodorsal) hippocampus:
increased hippocampal drive to the ventral tegmental area (Adams
et al., 2013), and thalamus: abnormality in thalamo-prefrontal
cortical circuitry (Lewis et al., 2001).
Statistical Analysis. Data were expressed as mean 6 S.E.M. To
determine the effect of challenges in comparison with saline, and for
treatments in comparison with vehicle we performed a repeated
measures analysis of variance, taking into account a Bonferroni
adjustment (SPSS20; SPSS, Chicago, IL). For statistical comparison
of the two treatment doses for JNJ-42153605, however, we performed
repeated measures analysis of variance, taking into account a Dunnett
correction (SPSS20). For determining the specificity of LY404039 and
JNJ-42153605, paired t tests were performed. Significance is reached
at P , 0.05.
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receiving LY404039 and ketamine challenge show a decrease
between 220.7% (64.6%) for anterodorsal hippocampus and
227.1% (65.7%) for cingulate cortex (Fig. 4, A and E; Table 1),
proving that the ketamine enhancement is not as high as with
memantine, resulting in an overshoot of the reversal.
The Effect and Specificity of JNJ-42153605 Treatment. As the second part of this study entails another
treatment (and thus also another vehicle), we first confirm
again that administration of memantine replicates the results
of the first part of the study, namely widespread significant
increases in [18F]FDG uptake in neuroanatomically distinct
regions in comparison with the corresponding vehicle 1 saline
controls (Figs. 4, G and H, and 5). The increases in this second
part of the study range similarly between 25.4 6 7.6% for
thalamus and 51.0 6 10.6% for frontal cortex (first columns of
Tables 1 and 3 are similar).
Administration of 2.5 mg/kg and 10 mg/kg JNJ-42153605 in
memantine-challenged animals causes a decrease in SUV in
comparison with memantine-challenged animals under vehicle pretreatment. The decrease in [18F]FDG uptake ranges
from 222.4% (64.5%) for the caudate putamen to 231.1%
(64.1%) for the cingulate cortex under the 10 mg/kg JNJ42153605 dose (Table 4) and is significant for all relevant
brain areas (Figs. 4, H and L, and 5). In the 2.5 mg/kg dose,
the decrease in SUV is only significant for cingulate cortex,
frontal cortex, medial prefrontal cortex, motor cortex, parietal
cortex, and anterodorsal hippocampus (Fig. 5) and ranges
from 25.2% (66.5%) for the caudate putamen to 211.2%
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 14, 2017
Fig. 3. Average SUV (6 S.E.M.) of [18F]FDG tracer uptake for eight relevant brain areas (caudate putamen, cingulate cortex, frontal cortex, medial
prefrontal cortex, motor cortex, parietal cortex, anterodorsal hippocampus, and thalamus) under saline, memantine, or ketamine with vehicle (white) or
LY404039 (dashed) pretreatment; error bars 6 S.E.M. *P , 0.05; **P , 0.01; ***P , 0.005.
Metabotropic GluR2 PAM JNJ-42153605 Evaluation by mPET
381
(66.3%) for the cingulate cortex (Fig. 4, H and J; Table 4).
Moreover, administration of 2.5 mg/kg and 10 mg/kg JNJ42153605 in saline-challenged animals induces a decrease in
SUV in comparison with saline-challenged animals under
vehicle pretreatment (Fig. 5). In the 10 mg/kg dose, the
decrease ranges from 215.8% (63.8%) for the caudate putamen to 224.3% (64.3%) for the frontal cortex (Fig. 4, G and
K; Table 4) and is significant for all relevant brain regions
(Fig. 5). For the 2.5 mg/kg dose, the decrease in SUV is only
significant for the motor cortex (Fig. 5), as it ranges from
0.2% (66.4%) for the caudate putamen to 26.4% (65.7%) for
the cingulate cortex (Fig. 4, G and I; Table 4). There is no
statistically significant difference between the SUVdecreasing effects induced by JNJ-42153605 in the 2.5 mg/kg
concentration for memantine-challenged versus salinechallenged animals (Fig. 6; Table 4). However, for the 10 mg/kg
concentration, there is a statistically significant difference
between the effect induced by JNJ-42153605 in the
memantine-challenged animals versus the saline-challenged
animals for the cingulate cortex, the motor cortex, the parietal
cortex, and the anterodorsal hippocampus (Fig. 6; Table 4),
showing the specific effect of JNJ-42153605 at this dose of
10 mg/kg.
Animals that received memantine as challenge and 2.5 mg/kg
of JNJ-42153605 as a treatment still show an increase of
SUV ranging from 11.4% (62.9%) for the thalamus to 28.4%
(63.9%) for the frontal cortex compared with vehicle 1 saline
(Fig. 4, G and J; Table 3). However, for the 10 mg/kg treatment
TABLE 2
Overview of (pre)treatment effects: LY404039 + saline, LY404039 + memantine and LY404039 +
ketamine treatment groups in comparison with vehicle + saline, vehicle + memantine, and vehicle +
ketamine, respectively
Percentage increase or decrease of activity (6 S.E.M.).
LY404039
Treatment
Versus Vehicle of LY404039 and Respective Challenge
LY404039 + Saline
LY404039 + Memantine
LY404039 + Ketamine
%
Caudate putamen
Cingulate cortex
Frontal cortex
Medial prefrontal cortex
Motor cortex
Parietal cortex
Hippocampus (AD)
AD, anterodorsal.
233.6
238.4
238.5
236.5
236.1
233.7
232.5
6
6
6
6
6
6
6
3.1
3.9
4.3
3.4
4.5
4.6
3.2
230.2
238.5
235.6
236.2
233.9
232.4
232.2
6
6
6
6
6
6
6
3.9
3.4
3.7
3.6
3.8
4.0
3.7
229.7
237.7
237.9
234.7
235.3
231.7
229.2
6
6
6
6
6
6
6
6.3
5.7
5.8
6.2
5.8
5.6
5.8
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 14, 2017
Fig. 4. Illustration of [18F]FDG mPET images, averaged over all animals, pretreated with (A) LY-vehicle + saline, (B) LY-vehicle + ketamine (30 mg/kg),
(C) LY-vehicle + memantine (20 mg/kg), (D) LY404039 (10 mg/kg) + saline, (E) LY404039 (10 mg/kg) + ketamine (30 mg/kg), (F) LY404039 (10 mg/kg) +
memantine (20 mg/kg), (G) JnJ-vehicle + saline, (H) JnJ-vehicle + memantine (20mg/kg), (I) JNJ-42153605 (2.5 mg/kg) + saline, (J) JNJ-42153605 (2.5
mg/kg) + memantine (20 mg/kg), and (K) JNJ-42153605 (10 mg/kg) + saline and (L) JNJ-42153605 (10 mg/kg) + memantine (20 mg/kg). SUV range: 0–8.
382
Wyckhuys et al.
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Fig. 5. Average SUV [18F]FDG for eight relevant brain areas (caudate putamen, cingulate cortex, frontal cortex, medial prefrontal cortex, motor cortex,
parietal cortex, anterodorsal [AD] hippocampus, and thalamus) under saline or memantine with vehicle (white) or JNJ-42153605 (2.5 mg/kg–dashed) or
JNJ-42153605 (10 mg/kg–black) pretreatment; error bars 6 S.E.M. *P , 0.05; **P , 0.01; ***P , 0.005.
dose of JNJ-42153605, this ranges from 28.9% (63.9%) for the
thalamus to 2.7% (65.0%) for the frontal cortex in memantinechallenged animals achieving full reversal (Fig. 4, G and L;
Table 3).
Discussion
First, this mPET experiment clearly indicates that both
memantine and ketamine induce increases in rat brain
[18F]FDG uptake. Moreover, our study reveals that memantine is more potent at inducing statistically significant
regional increases in SUV [18F]FDG in the rat brain than
ketamine. This finding strengthens the hypothesis that both
memantine and ketamine animal models are suitable
on-demand animal models for schizophrenia, but that the
memantine-challenged model necessitates lower numbers of
animals to reach statistically significant results in comparison with ketamine-challenged animals.
Metabotropic GluR2 PAM JNJ-42153605 Evaluation by mPET
383
TABLE 3
Overview of (pre)treatment effects: different JNJ-42153605 experimental conditions in comparison with
the vehicle + saline condition
Percentage increase or decrease of activity (6 S.E.M.).
JNJ-42153605
Versus Vehicle of JNJ-42153605 and Valine
Treatment
JNJ Vehicle + Memantine
2.5 mg JNJ + Memantine
10 mg JNJ + Memantine
%
Caudate putamen
Cingulate cortex
Frontal cortex
Medial prefrontal cortex
Motor cortex
Parietal cortex
Hippocampus (AD)
Thalamus
27.1
44.8
51.0
39.2
44.8
40.4
31.3
25.4
6
6
6
6
6
6
6
6
7.8
9.3
10.6
9.2
9.1
8.9
7.4
7.6
15.6
22.9
28.4
19.4
24.5
20.5
14.9
11.4
6
6
6
6
6
6
6
6
24.1
23.4
2.7
22.8
21.7
24.7
27.5
28.9
2.9
3.7
3.9
3.9
3.4
3.2
2.9
2.9
6
6
6
6
6
6
6
6
4.3
4.5
5.0
4.8
4.4
4.1
3.7
3.9
AD, anterodorsal.
moleculare of the hippocampus, etc. However, it is worth
highlighting the advantage of our approach: the use of
predefined and standardized VOI templates enables very fast
and robust analysis of many regions of interest, which is
a major advantage for the high-throughput screening of
pharmacological compounds and radiotracers using in vivo
functional imaging with mPET.
Normalizing excess glutamate levels by mGluR2/3 agonists
has led to identification of potential anxiolytics and antipsychotic drugs. Studies in animals have shown that mGluR2
agonists are able to improve cognitive impairment produced
by psychotomimetics and are active in several models related
to the positive symptoms of schizophrenia (Lorrain et al.,
2003; Patil et al., 2007; Pehrson and Moghaddam, 2010;
Seeman, 2013). Pharmacological reversal testing showed that
the mGlu2/3 receptor agonist LY404039 significantly attenuates both the memantine- and ketamine-induced [18F]FDG
uptake. LY404039 almost fully reverses the memantineinduced [18F]FDG increase, while for the ketamine-induced
increase, there is an overshoot in the reversal. These findings
again illustrate the superiority of the memantine model,
which was also highlighted in a previous 2DG autoradiography study with mice (Dedeurwaerdere et al., 2011). However,
the interpretation of the reversal effect of this mGlu2/3
receptor agonist becomes potentially biased due to a similar
decrease in [18F]FDG uptake in the LY404039 1 saline
condition as in the memantine and ketamine models. This
TABLE 4
Overview of (pre)treatment effects: JNJ-42153605 (2.5 mg/kg) + saline, and JNJ-42153605 (10 mg/kg) + saline treatment
groups in comparison with vehicle + saline and for JNJ-42153605 (2.5 mg/kg) + memantine, and JNJ-42153605 (10 mg/kg) +
memantine treatment groups in comparison with the vehicle + memantine
Percentage increase or decrease of activity (6 S.E.M.).
JNJ-42153605
Treatment
Versus Vehicle of JNJ-42153605 and Respective Challenge
JNJ (2.5 mg) + Saline
JNJ (2.5 mg) + Memantine
JNJ (10 mg) + Saline
JNJ (10 mg) + Memantine
%
Caudate putamen
Cingulate cortex
Frontal cortex
Medial prefrontal cortex
Motor cortex
Parietal cortex
Hippocampus (AD)
Thalamus
AD, anterodorsal.
0.2
25.5
26.4
23.7
24.9
24.4
23.6
22.8
6
6
6
6
6
6
6
6
6.4
5.9
5.7
6.2
5.8
5.8
5.5
6.0
25.2
211.2
210.6
210.3
210.2
210.3
29.1
27.4
6
6
6
6
6
6
6
6
6.5
6.3
6.4
6.2
6.1
6.1
6.0
6.5
215.8
223.0
224.3
220.5
223.1
222.0
217.9
218.8
6
6
6
6
6
6
6
6
3.8
3.7
4.3
3.8
4.0
3.9
3.4
3.5
222.4
231.1
229.3
227.8
229.8
229.9
227.8
225.3
6
6
6
6
6
6
6
6
4.5
4.1
4.8
4.4
4.4
4.2
3.8
4.2
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It has been previously shown that ketamine and memantine have equipotent binding affinities (Kd ∼ 1.5 mM) at the
human NMDA receptor (Gilling et al., 2009) and have nearly
indistinguishable synaptic pharmacodynamics (Emnett et al.,
2013). However, the pronounced differences in pharmacokinetics between memantine and ketamine, both in humans
and in rats, might explain the difference in resulting increase
in [18F]FDG SUV, as memantine has a half-life in humans of
around 100 hours (Periclou et al., 2006), whereas ketamine is
extremely rapidly eliminated, with a redistribution half-life of
around 15 minutes (Clements and Nimmo, 1981) making it
very difficult to achieve a long steady-state for ketamine. In
this [18F]FDG mPET study, the overall degree of memantineinduced [18F]FDG increases correlates with the literature on
memantine activation in rodent brain (Dedeurwaerdere et al.,
2011). Minor disparities between the findings in literature
and the current findings might be explained by differences in
animal species and/or route of administration, etc. In
addition, the difference in VOI determination can contribute
to minor disparities. Other investigators (Duncan et al.,
1998a; Dedeurwaerdere et al., 2011) use manually outlined
VOIs in comparison with our fully automated predefined
Schiffer rat brain VOI template available from the PMOD
v3.3 software, which results in larger regions as a whole,
which could eventually even out very regional changes such as
the [18F]FDG uptake increases in the dentate gyrus, CA3
stratum radiatum, presubiculum, and stratum lacunosum
384
Wyckhuys et al.
raises the question of whether LY404039 is specifically
reversing the ketamine- and memantine-induced [18F]FDG
increases. LY404039 may have a polypharmacology: in
addition to being an mGlu2/3 agonist (Fell et al., 2008), it
also affects serotonin turnover in a GluR2/3-mediated way
(Lowe et al., 2012), and it may inhibit the binding of ligands to
dopamine D2 and D3 receptors (Seeman and Guan, 2009;
Seeman, 2013) although this is controversial (Fell et al., 2009;
Zysk et al., 2011) and unlikely to alter FDG uptake. Therefore,
teasing out the specific pharmacology responsible for the
decrease in LY404039 1 saline condition and its effect on
memantine-induced changes in [18F]FDG uptake is very
complex. Our findings on the nonspecific effect of LY404039
are in contrast with a previous 2DG autoradiography
(Dedeurwaerdere et al., 2011), where no effects of the
compound on baseline 2DG uptake was observed. Differences
in imaging techniques used or species differences may be an
explanation.
Positive allosteric modulation (i.e., compounds that bind
a site other than the orthosteric site) of the mGluR2 provides
a novel way to regulate glutamatergic function. The use of
mGluR2 PAMs in comparison with agonists to improve the
functional effects of glutamate has several advantages, such
as the possibility of higher selectivity, the lower risk of
potential tolerance and desensitization, and improved safety,
as the mGluR2 PAM will only activate the receptor in the
presence of increased glutamate. A range of potent mGluR2
PAMs has been discovered; i.e., BINA [potassium 39-([(2cyclopentyl-6-7-dimethyl-1-oxo-2,3-dihydro-1H-inden-5-yl)
oxy]methyl)biphenyl l-4-carboxylate] (Jin et al., 2010),
LY487379 [N-(4-(2-methoxyphenoxy)-phenyl-N-(2,2,2-trifluoroethyl-sulfonyl)-pyrid-3-ylmethylamine] (Nikiforuk
et al., 2010), THIIC [N-(4-((2-(trifluoromethyl)-3-hydroxy-4(isobutyryl)phenoxy)methyl)benzyl)-1-methyl-1H-imidazole-4carboxamide] (Fell et al., 2011), and JNJ-42153605 (Cid
et al., 2012), among others. The mGluR2 PAM JNJ-42153605
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Fig. 6. Specific effect induced by JNJ-42153605, both 2.5 mg/kg (dashed) and 10 mg/kg (black) in saline (SAL)- or memantine (MEM)-challenged
animals; error bars 6 S.E.M. *P , 0.05.
Metabotropic GluR2 PAM JNJ-42153605 Evaluation by mPET
Acknowledgments
The authors thank their coworkers from the Molecular Imaging
Center Antwerp, University of Antwerp, Belgium: Joke Parthoens
and Philippe Joye for technical assistance and Lauren Kosten and
Stijn Servaes for the artwork for Fig. 1; and Hilde Lavreysen from the
Department of Neuroscience, Janssen Pharmaceutica NV, Beerse,
Belgium, for discussions of mGluR2.
Authorship Contributions
Participated in research design: Wyckhuys, wyffels, Langlois,
Schmidt, Stroobants, Staelens.
Conducted experiments: Wyckhuys, wyffels.
Performed data analysis: Wyckhuys, Staelens.
Wrote or contributed to the writing of the manuscript: Wyckhuys,
wyffels, Langlois, Schmidt, Stroobants, Staelens.
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showed the best overall profile among 28 structurally related
compounds being screened, with an optimal in vitro profile
combining potency, metabolic stability, and acceptable preliminary cardiovascular profile. Furthermore, it has acceptable pharmacokinetics, high selectivity for mGluR2, and
excellent mGluR2 PAM activity (Cid et al., 2012). At a dose of
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may indicate that JNJ-42153605 is specific for memantinepretreated animals.
A few limitations should be acknowledged. It should be
noted that [18F]FDG is not a specific tracer for the mGluR2/3
receptor and that we thus only indirectly show that JNJ42153605 has a higher specificity for the mGluR2/3 receptor
than LY404039. Also, we could not work with relative
[18F]FDG quantification versus a reference region given the
wide distribution of NMDAR and especially the downstream
effects, as the [18F]FDG readout is an integrated signal over
time, so that the net effect can be distant to the NMDA-rich
area of initial pharmacology. Finally, although we show
a statistically significant specific effect of JNJ-42153605 in
four out of eight relevant brain regions, it should be noted that
JNJ-42153605 also causes significant decreases in both
control and memantine-challenged animals in the other brain
regions, albeit not statistically different in terms of specificity.
In summary, this study shows that memantine, more
pronounced than ketamine, induces significant regional
activation in the rat brain, which can be effectively reversed
by a pharmacologic challenge with the mGlu2/3 receptor
agonist LY404039. Further, these experiments indicate that
the mGluR2 PAM JNJ-42153605 has the capacity to dosedependently reverse such memantine-induced brain activation with specific effects at the 10 mg/kg dose.
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