Molecular Psychiatry (2003) 8, 752–765
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ORIGINAL RESEARCH ARTICLE
Neuronal mechanism of nociceptin-induced modulation
of learning and memory: Involvement of
N-methyl-D-aspartate receptors
T Mamiya1,2, K Yamada1,3, Y Miyamoto1,4, N König5, Y Watanabe6, Y Noda1 and T Nabeshima1
1
Department of Neuropsychopharmacology and Hospital Pharmacy, Nagoya University Graduate School of Medicine, Nagoya,
Japan; 2Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, Meijo University, Nagoya, Japan;
3
Laboratory of Experimental Therapeutics, Department of Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Kanazawa
University, Kanazawa, Japan; 4Department of Molecular Genetics, National Institute for Longevity Sciences, Oobu, Japan;
5
EPHE Quantitative Cell Biology, Montpellier University, Montpellier Cedex, France; 6Department of Physiology, Kagawa
Medical University, Kagawa, Japan.
Nociceptin (also called orphanin FQ) is an endogenous heptadecapeptide that activates the
opioid receptor-like 1 (ORL1) receptor. Nociceptin system not only affects the nociception and
locomotor activity, but also regulates learning and memory in rodents. We have previously
reported that long-term potentiation and memory of ORL1 receptor knockout mice are
enhanced compared with those in wild-type mice. Here, we show the neuronal mechanism of
nociceptin-induced modulation of learning and memory. Retention of fear-conditioned
contextual memory was significantly enhanced in the ORL1 receptor knockout mice without
any changes in cued conditioned freezing. Inversely, in the wild-type mice retention of
contextual, but not cued, conditioning freezing behavior was suppressed by exogenous
nociceptin when it was administered into the cerebroventricle immediately after the training.
ORL1 receptor knockout mice exhibited a hyperfunction of N-methyl-D-aspartate (NMDA)
receptor, as evidenced by an increase in [3H]MK-801 binding, NMDA-evoked 45Ca2 þ uptake and
activation of Ca2 þ /calmodulin-dependent protein kinase II (CaMKII) activity and its phosphorylation as compared with those in wild-type mice. The NMDA-induced CaMKII activation in the
hippocampal slices of wild-type mice was significantly inhibited by exogenous nociceptin via a
pertussis toxin-sensitive pathway. However, the a-amino-3-hydroxy-5-methyl-4-isoxazole
propionic acid receptor GluR1 subunit at Ser831 and Ser845, and NMDA receptor subunit
NR2B at Thr286 were phosphorylated similarly after NMDA receptor stimulation in both type of
mice. The expressions of GluR1 and GluR2 also did not change, but the levels of polysialylated
form of neuronal cell adhesion molecule (N-CAM) were reduced in the ORL1 receptor knockout
as compared with wild-type mice. These results suggest that nociceptin system negatively
modulates learning and memory through the regulation of NMDA receptor function and the
expression of N-CAM.
Molecular Psychiatry (2003) 8, 752–765. doi:10.1038/sj.mp.4001313
Keywords: nociceptin/orphanin FQ; ORL1 receptor; Knockout mice; hippocampus; NMDA
Opioid receptors are negatively coupled to adenylate
cyclase through Gi/o proteins and mediate the
inhibition of forskolin-induced cAMP accumulation
by morphine and endogenous opioid peptides.
Opioid receptor agonists have receptor subtypespecific actions on not only the nociceptive thresholds but also synaptic transmission and long-term
potentiation (LTP) in the hippocampus. Thus, opioid
Correspondence: Professor T Nabeshima, Department of Neuropsychopharmacology and Hospital Pharmacy, Nagoya University
Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku,
Nagoya 466-8560, Japan.
E-mail: [email protected]
Received 6 April 2002; revised 15 August 2002 and 23 August
2002; accepted 8 December 2002
system modulates certain forms of learning and
memory.1
Nociceptin, an endogenous ligand for opioid
receptor-like 1 (ORL1) receptors, shares sequences
with other opioid peptides, particularly with dynorphin A (53%).2,3 This peptide is capable of inhibiting
Ca2 þ currents through voltage-gated Ca2 þ channels in
dissociated hippocampal neurons,4 inducing hyperpolarizing currents via inward-rectifier K þ channels
in the hippocampal CA3 cells5 or suppressing
synaptic transmission in the hippocampal CA1
slices.6 Owing to its structural similarity to classical
opioids (68% homology with m receptor, 67% with d
receptor, 66% with k receptor) and high density of
ORL1 receptors in the hippocampus,7 it is of interest
to explore whether nociceptin, like other opioids,
Nociceptin and memory
T Mamiya et al
modulates the synaptic plasticity and memory function in the hippocampus. Recent studies demonand
intrastrated
that
intrahippocampal8,9
cerebroventricular10,11 injections of nociceptin impair
learning and memory in the water maze and passive
avoidance tests, respectively. We have also reported
that LTP in the hippocampal CA1 region and memory
are facilitated in the ORL1 receptor knockout mice
compared to wild-type mice.11–13 The enhanced LTP
in the hippocampus of ORL1 receptor knockout mice
was blocked by AP-V, an N-methyl-D-aspartate
(NMDA) receptor antagonist.13 The overexpression
of NMDA receptor subunit NR2B in transgenic mice
has been reported to enhance the retention of memory
and the extinction of learning in the contextual fear
learning task.14 These findings suggest that nociceptin/ORL1 receptor system plays an important role in
modulating learning and memory related with NMDA
receptor function in the hippocampus. Furthermore,
a-amino-3-hydroxy-5-methyl-4-isoxazloe proprionic
acid (AMPA) receptor also regulates the LTP and
long-term depression (LTD) with the phosphorylation
and dephosphorylation of AMPA receptor GluR1
subunit.15 However, the cellular mechanisms of crosstalk between ORL1 and NMDA/AMPA receptors
remain to be addressed.
Here, we assessed the performance in the contextual
fear learning task in the ORL1 receptor knockout mice
and the wild-type mice treated with exogenous
nociceptin. Next, we investigated the alteration of
NMDA receptor function by nociceptin, as manifested
by [3H]MK-801 binding in the membrane preparations,
45
Ca2 þ uptake into the synaptosomes, Ca2 þ /calmodulin-dependent protein kinase II (CaMKII) activity and
its phosphorylation in the hippocampal slices after
NMDA receptor stimulation. Finally, we compared the
expression of GluR1 and GluR2 AMPA receptor
subunits with polysialylated form of neuronal cell
adhesion molecule (PSA-NCAM) by the immunohistochemical technique because PSA-NCAM is involved
in the neuronal plasticity associated with learning and
memory. Furthermore, we investigated the phosphorylation of NMDA and AMPA receptor subunits after
NMDA receptor stimulation in wild-type and the
ORL1 receptor knockout mice.
Materials and methods
We used male wild-type littermate and the male ORL1
receptor knockout mice aged 9–12 weeks.16 The
animals were housed in a controlled environment
(23711C, 5075% humidity) and were allowed food
and water ad libitum. The room lights were on
between 7:00 and 19:00. All experiments were
performed in accordance with the Guidelines for
Animal Experiments of the Nagoya University School
of Medicine. The procedures involving animals and
their care were conducted in conformity with the
international guidelines ‘Principles of Laboratory
Animal Care’ (NIH publication no. 85–23, revised
1985).
Fear conditioning learning
Fear conditioning learning test was performed as
described in previous reports.17,18 The conditioning
chamber was in a soundproof box (90 cm 65 cm 60 cm). With the surrounding noise measuring 75 dB,
noise within the box registered 68 dB. To provide
background white noise (72 dB), a single computer
fan was installed in one of the sides of the isolation
chamber. The conditioning chamber (25 cm 30 cm
47 cm) is made of transparent Plexiglas with grid
floor for footshock. The floor is removable, and after
each experimental subject, it was cleaned with 70%
ethanol. The floor grid is connected to a shockgenerator (NS-SG01; Neuroscience Inc., Tokyo, Japan), a
device that delivers scrambled shocks. The chamber
was surrounded by Scanet SV10-AQ (Melquest,
Toyama, Japan), which is an apparatus that can
measure automatically the immobility time using
the digital counters with infrared cell sensors placed
on the walls. We have checked that the freezing
behavior is parallel with the immobility in our study
(data not shown). The loud speaker used to deliver
the conditioned stimulus (CS) is connected to a power
supply with an adjustable current output that was
kept constant throughout the experiments. For the
cued and contextual conditioning experiments, each
mouse was placed in the conditioning chamber for
1.5 min before the onset of the discrete CS (lasted 30 s
at 2800 Hz and 85 dB of sound). In the last 2 s of CS,
they were exposed to the unconditioned stimuli (US)
(0.8 mA for 2 s of continuous foot shock). After the
CS/US pairing, a mouse was left in the conditioning
chamber for another 1 min and then placed back in
their home cages. For contextual freezing, each mouse
was placed in the experimental chamber in which the
mice previously received a footshock. In this study,
we measured the immobility at the 1st, 2nd or 7th day
after the training, respectively. In the cued conditioned freezing, each mouse was placed in a novel
context (new plastic cage with soft floor) for 2 min
(pre-CS test), and then exposed to the CS for 2 min
(CS test) 1, 2 and 7 days after the training, respectively. Nociceptin and [Nphe1]nociceptin(1–13)–NH2
were administered intracerebroventricularly in a
volume of 5 ml per mouse. This volume is often used
in many researches10–12 and we confirmed no behavioral differences among saline treated, sham-treated
and nontreated mice.
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[3H]MK-801 binding
Mice were killed by decapitation, and brains were
quickly removed on an ice-cold glass plate. The
forebrain (minus the cerebellum and brainstem) was
rapidly dissected out, frozen and stored in a deep
freezer at 801C until assayed. [3H]MK-801 binding
was measured as described previously.19,20 Briefly,
frozen samples were thawed at room temperature and
homogenized in 40 volumes of 50 mM Tris-acetate
buffer (pH 7.4) containing 1 mM EDTA using a
sonicator. All further procedures were performed at
41C. The homogenates were centrifuged at 40 000 g
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T Mamiya et al
754
for 30 min, and the resultant pellets were washed
three times with the same volume of 50 mM Trisacetate buffer (pH 7.4). The final pellets were
suspended in 30 volumes of 0.32 M sucrose, and the
suspensions were frozen at –801C for no longer than 1
week until use. On the day of the experiments, the
frozen suspensions were thawed at room temperature
and treated with 0.08% Triton X-100 at 41C (an
approximate protein concentration of 0.32 mg/ml) for
10 min with gentle stirring. The treatment was
terminated by centrifugation at 40 000 g for 30 min,
and the pellets were washed five times with 40
volumes of 50 mM Tris-acetate buffer (pH 7.4) followed by centrifugation at 40 000 g for 30 min. For
determination of [3H]MK-801 binding, an aliquot
(0.3 mg of protein) of the membrane preparations
was incubated in the presence or absence of glutamate
(10 mM), glycine (10 mM) and spermidine (1 mM), with
5 nM ( þ )[3-3H]MK-801 (22.5 Ci/mmol; NEN Life
Science Products, Boston, MA, USA) in a total
volume of 0.5 ml of 50 mM Tris-acetate buffer (pH
7.4) at 301C for 16 h. The incubation was terminated
by rapid filtration through a Whatman GF/B glass
filter under a constant vacuum. The filter was rinsed
with 3 ml of ice-cold 50 mM Tris-acetate buffer three
times within 10 s. Radioactivity retained on the filter
was measured by a liquid scintillation spectrophotometry, at a counting efficiency of 57–59%. Nonspecific binding was defined by 0.1 mM cold ( þ ) MK801 (Sigma, St Louis, MO, USA), and the specific
binding accounted for more than 60% of the total
binding found in the absence of cold ( þ ) MK-801.
Ca2 þ Uptake
Ca2 þ uptake assay was measured as described
previously.20 Mice were killed by decapitation, the
brains were quickly removed, and the forebrain was
dissected out on an ice-cold glass plate. The forebrains were homogenized in 20 volumes of ice-cold
0.32 M sucrose at 41C in a Teflon glass homogenizer.
All further procedures were performed at 41C. The
homogenates were centrifuged at 1000 g for 10 min.
The supernatants were collected and then diluted 1:1
with basal buffer of the following composition:
135 mM NaCl, 5 mM KCl, 1 mM CaCl2 and 10 mM
HEPES, pH adjusted to 7.4 with Tris base, and
centrifuged at 10 000 g for 15 min. The pellets were
resuspended in basal buffer and used for the 45Ca2 þ
uptake assay. The synaptosomes suspension (0.5 mg
of protein) was preincubated in a total volume of
450 ml of basal buffer, in the presence or absence of
( þ ) MK-801 (100 mM), at 371C for 10 min. The 45Ca2 þ
uptake assay was initiated by adding 50 ml of prewarmed basal buffer containing 1 mCi/ml 45CaCl2
(18.1 mCi/mg; NEN Life Science Products), in the
presence or absence of NMDA (100 mM), glycine
(10 mM) and spermidine (1 mM) or high K þ (45 mM;
isomolar replacement of NaCl with KCl). The reaction
was terminated after 5 min by adding 3 ml of ice-cold
basal buffer. The mixture was rapidly filtered under
vacuum over Whatman GF/B glass filters, and the
45
45
Molecular Psychiatry
filters were rinsed twice with 3 ml of basal buffer. The
radioactivity was determined by a liquid scintillation
spectrophotometry at a counting efficiency of 90%.
Ca2 þ uptake was defined by subtracting the uptake at
41C. The basal values mean the nonstimulated [45Ca]
uptake. The values of stimulated [45Ca] uptake were
expressed as the total [45Ca] uptake minus baseline
uptake (basal values).
CaMKII activity assay
CaMKII activity was determined according to the
method described by Occur and Schulman,21 and
Colbran et al.22 Hippocampi were dissected within
20 s and hippocampal slices (300 mm) were prepared
using a Mcllwain Tissue chopper. After stimulation of
NMDA receptor with NMDA (100 mM), glycine
(10 mM) and spermidine (1 mM) for 5 min, the slices
were sonicated in an ice-cold lysis buffer (35 mM
HEPES–NaOH (pH 8.0), 1 mM EGTA, 10 mM sodium
pyrophosphate, 0.4 mM sodium molybdate, 2 mg/ml
leupeptin, 2 mg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mM dithiothreitol (DTT))
at 41C. The homogenate was centrifuged at 100 000 g
for 30 min and the resultant supernatant was subjected to the assay of CaMKII activity using syntide-2
as a synthetic substrate for CaMKII which is a 15amino-acid peptide. The assay was carried out in the
mixture (final volume of 50 ml) containing 35 mM
HEPES–NaOH (pH 8.0), 10 mM MgCl2, 50 mM syntide2, 10 mM [g-32P] ATP (500–900 cpm/pmol) and either
1 mM EGTA or 0.4 mM CaCl2 and 10 mg/ml calmodulin. For determination of the total activity, 0.4 mM
CaCl2, and 10 mg/ml calmodulin were added to the
assay mixture; 1 mM EGTA was added to the basic
mixture for determination of Ca2 þ -independent activity. The mixtures were preincubated at 301C for 2 min,
and all reactions were initiated by the addition of
enzyme preparations. After incubation, aliquots of
10 ml were spotted onto P81 phosphocellulose paper.
The papers were washed four times in 75 mM H3PO4
and dried. Then, the radioactivity of samples was
quantified by liquid scintillation counting.
Western blot analysis of CaMKIIa and GluR1
phosphorylation
Hippocampal slices (300 mm) were prepared as described above. After stimulation of NMDA receptor
with NMDA (100 mM), glycine (10 mM) and spermidine (1 mM) in the buffer (135 mM NaCl, 5 mM KCl,
1 mM CaCl2, 10mM HEPES–NaOH and 10 mM glucose, pH 7.4), the slices were homogenized by the
sonication in an ice-cold lysis buffer (50 mM Tris–
HCl, 150 mM NaCl, 10 mM NaF, 10 mM EDTA, 1 mM
sodium orthovanadate, 2 mg/ml pepstatin, 2 mg/ml
leupeptin, 2 mg/ml aprotinin, 1 mM PMSF, and 1 mM
DTT, pH 7.4). Samples were boiled in Laemmli
sample buffer, separated on a 7.5% polyacrylamide
gel and subsequently transferred to PVDF membranes
(Millipore). The membranes were blocked with
Detector Block Kit (KPL) for 2 h at room temperature
and then probed with antiphospho-CaMKIIa subunit
Nociceptin and memory
T Mamiya et al
(monoclonal anti-mouse peptide antibodies at 0.2 mg/
ml, ABR), antiphospho-GluR1 at Ser831 (polyclonal
anti-rabbit peptide antibodies at 0.2 mg/ml, Upstate
Biotechnology) or antiphospho-GluR1 at Ser845 (polyclonal anti-rabbit peptide antibodies at 0.2 mg/ml,
Upstate Biotechnology) overnight at 41C. Membranes
were washed with TBST buffer (10 mM Tris–HCl,
150 mM NaCl, 0.1% Tween 20, pH 7.4) and subsequently incubated with a goat anti-rabbit horseradish
peroxidase-conjugated secondary antibody for 2 h at
room temperature. The immune complexes were
detected by chemiluminescence (ECL, Amersham)
and exposed to X-ray film. The band intensities
of film were analyzed by densitometry. To confirm
equal loading of each protein, membranes were
stripped with stripping buffer (62.5 mM Tris–HCl,
100 mM 2-mercaptoethanol, 2% SDS, pH 6.7) at 501C
for 5 min, and then incubated with anti-CaMKIIa
subunit antibody (monoclonal anti-mouse peptide
antibodies at 0.2 mg/ml, ABR) or anti-GluR1 (antirabbit immunoaffinity-purified peptide antibodies at
0.2 mg/ml, Upstate Biotechnology) and detected as
described above.
Immunoprecipitation of phosphorylated NR2B subunit
Subsequent to NMDA receptor stimulation, the
hippocampal slices were homogenized by sonication
in an ice-cold lysis buffer (50 mM Tris–HCl, 150 mM
NaCl, 10 mM NaF, 10 mM EDTA, 1 mM sodium
orthovanadate, 1% Nonidet P-40, 2 mg/ml pepstatin, 2 mg/ml leupeptin, 2 mg/ml aprotinin, 1 mM
PMSF and 1 mM DTT, pH 7.4). The lysate was
centrifuged at 10 000 g for 10 min. Protein
A–Sepharose (Amersham) was incubated with antiphosphoserine (polyclonal anti-rabbit peptide antibodies at 0.2 mg/ml, Zymed Laboratories Inc.) for 6 h
and then incubated with lysate (500 mg protein)
overnight. The immunoprecipitate was boiled in
Laemmli sample buffer, separated on a 7.5% polyacrylamide gel and subsequently transferred to PVDF
membranes (Millipore). Phosphorylated NR2B was
detected with anti-NR2B antibodies (polyclonal antirabbit peptide antibodies at 0.2 mg/ml, Zymed Laboratories Inc.).
Immunocytochemical detection of polysialylated form
of neuronal cell adhesion molecule (PSA-NCAM)
Coronal vibratome sections (75 mm) through the
forebrain at the hippocampal level were made from
five wild-type and five homozygous mutant brains
(perfusion fixed with 4% paraformaldehyde). The
floating sections were prepared for immunocytochemical analysis with a confocal laser scanning
microscope (TCS, Leica, Heidelberg) as described
previously.23 The antibodies directed against PSANCAM (monoclonal mouse IgM, diluted 1:2000) were
kindly donated by Dr T Seki, Juntendo University,
Tokyo. To visualize also the environment of the
PSA-NCAM positive cells, we used simultaneously
antibodies against two other proteins strongly
expressed in the hippocampus: the AMPA receptor
subunits GluR-1 (polyclonal rabbit IgG, 1:200;
Chemicon, Temecula, CA, USA) and GluR-2 (monoclonal mouse IgG, 1:60; Chemicon). Triple staining
was performed using the following secondary antibodies: (1) CY3-conjugated donkey anti-mouse IgM
(1:1000; Jackson Immuno Research, West Grove, PA,
USA); (2) CY5-conjugated donkey anti-rabbit IgG
(1:200; Jackson Immuno Research); (3) Fc-specific
FITC-conjugated goat anti-mouse IgG (1:200; Sigma,
St Louis, MO, USA). Serial ‘optical slices’ were
recorded at different depth levels, and the resulting
image stacks were processed to yield three-dimensional pictures that can be viewed with a standard
stereo-lorgnette.
755
Data analysis
All results are expressed as means7SEM for each
group. Statistical difference between groups was
assessed with Dunnett multiple comparisons test.
For the analysis of fear-conditioned memory test, we
used analysis of variance (ANOVA) with repeated
measures. Differences were considered statistically
significant at a level of Po0.05.
Results
Fear conditioning learning and memory in the ORL1
receptor knockout mice
We tested the ORL1 receptor knockout mice in the
contextual fear conditioning learning and memory.
Our previous reports show that there is no difference
in the pain responses to the electric, chemical and
thermal stimuli between the ORL1 receptor knockout
and wild-type mice.12,16 Wild-type and ORL1 receptor
knockout mice displayed comparable immobility
when exposed to an electric footshock (conditioning)
(Figure 1A-a, -d), demonstrating that the ORL1
receptor knockout mice have no performance deficits,
such as an inability to freeze. At 2 days after
the conditioning, the ORL1 receptor knockout
and wild-type mice displayed the same degree of
freezing response when they were exposed to the
same context of the conditioning chamber (Figure 1Ab). At 7 days after the conditioning, the immobility
time in wild-type mice was considerably decreased to
about 50% and this immobility level was maintained
at least up to the 14th day. On the other hand, the
ORL1 receptor knockout mice showed the immobility
time as much as on the 2nd day, and were significantly higher than that in wild-type mice (Figure 1Ac). To evaluate the specificity of the enhancement of
contextual memory, we tested the ORL1 receptor
knockout mice in the cued conditioned fear conditioning memory test, which does not require the
hippocampal function in the rodents. In this test, on
both the 2nd and 7th day, there was no difference in
immobility time between the ORL1 receptor knockout
and wild-type mice in the presence or absence of tone
(Figure 1A-e, -f).
In order to investigate the direct interaction
between nociceptin system and NMDA receptor, the
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Nociceptin and memory
T Mamiya et al
756
Knockout
+ (+) MK-801 0.03 mg/kg
Wild-type
+ (+) MK-801 0.03 mg/kg
A
B
Immobility time (%)
a
100
90
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60
50
40
30
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10
0
Wild-type
Knockout
ES
Tone
1
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d
Immobility time (%)
b
Conditioning
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30
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10
0
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3
Wild-type
Knockout
ES
Tone
3
1
2
Time (min)
c
2nd day
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a
7th day
**
1
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f
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Time (min)
4
Tone
1
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Time (min)
ES
Tone
1
e
Tone
Conditioning
4
2
3
7th day
**
**
**
1
2
3
Time (min)
Figure 1 Performance of wild-type and the ORL1 receptor knockout mice in the fear conditioning test (A) and the effects of
(+) MK-801 on the fear conditioning learning and memory (B). A solid line indicates the presence of CS (85 dB tone), while
the arrow indicates the US (ES; 2 s footshock). (Aa–c): Contextual test. (Ad–f): Cued test. (Aa) and (Ad) represent pooled data
(N ¼ 20) from two experiments. (Ab) and (Ae) represent the performance on the 2nd day after conditioning (N ¼ 10). (Ac) and
(Af) represent the performance on the 7th day after conditioning (N ¼ 10). (Ba, b): (+) MK-801 was injected subcutaneously
20 min before conditioning. **P o 0.05 vs wild-type mice.
NMDA receptor antagonist ( þ ) MK-801 was administered in both the mice (Figure 1b). The enhanced
memory retention in the ORL1 receptor knockout
mice was blocked by ( þ ) MK-801 (0.03 mg/kg), while
it did not affect the performance in wild-type mice
(Figure 1B-b).
Effect of nociceptin on the fear conditioning learning
and memory in wild-type mice
To further investigate a role of nociceptin in learning
and memory process, we examined the effect of
intracerebroventricular injection (i.c. v.) of nociceptin
in the fear conditioning learning and memory in wildtype mice. We chose the 2nd day to investigate the
effects of nociceptin, because the immobility level on
the 2nd day is approximately medial. As nociceptin
(0.01 and 1 nmol/5 ml i.c.v.) was injected immediately
after the 3-min conditioning, there was no difference
in immobility during the conditioning among the
Molecular Psychiatry
three groups (Figure 2a and d). Nociceptin had no
effects on contextual freezing response on the 1st day
(data not shown), but it induced a dose-dependent
impairment of retention of contextual memory on the
2nd day (Figure 2b and c). We have examined
whether the effects of nociceptin are mediated
through the ORL1 receptor on the contextual memory
by using [Nphe1]nociceptin(1-13)-NH2, a selective
ORL1 receptor antagonist. The pretreatment of this
antagonist inhibited the impairment of memory
retention induced by nociceptin (Figure 2a–c). In
contrast, nociceptin failed to affect the cued conditioning memory (Figure 2e and f).
NMDA receptor function in the hippocampus of the
ORL1 receptor knockout mice
The present findings with our previous ones that LTP
in the hippocampus are enhanced in the ORL1
receptor knockout mice13 suggest that NMDA receptor
Nociceptin and memory
T Mamiya et al
757
Noc 0.01 nmol 5 µl
Noc 1 nmol 5 µl
[Nphe1]-Noe 30 nmol 5 µl + Noe 1 nmol 5 µl
b
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Immobility time (%)
Conditioning
Immobility time (%)
Immobility time (%)
a
Control
1
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##
**
**
-
-
0.01
1
30 nmol
1
Nociceptin [nmol]
ES
Tone
1
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Time (min)
f
Test
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Immobility time (%)
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Immobility time (%)
Conditioning
d
Without tone
100
90
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40
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With tone
0
0.01
1
Nociceptin
(nmol)
Figure 2 Effects of nociceptin on the fear conditioning test in the wild-type mice. A solid line indicates the presence of CS
(85 dB tone), while the arrow indicates the US (ES: 2 s footshock). Nociceptin (0.01 and 1 nmol/5 ml) was injected into the
cerebral ventricle immediately after the conditioning. [Nphe1]nociceptin(1-13)-NH2 was administered 5 min before the
conditioning. (a–c): Contextual test (N ¼ 10). (c) Shows the average of the immobility time during 3-min test on the 2nd day.
(d–f): Cued test (N ¼ 10). (f) Shows the average of the immobility time during each 2-min test with or without tone. **Po0.05
vs nociceptin 0-treated group, ##Po0.05 vs. nociceptin 1 nmol-treated group.
in the hippocampus may have an important role in
the nociceptin-induced modulation of learning and
memory. To demonstrate the functional alterations of
NMDA receptor in the ORL1 receptor knockout mice,
we first performed the radioligand binding assay
using a noncompetitive NMDA receptor antagonist,
[3H]MK-801 (Figure 3a). The binding of [3H]MK-801
was determined in synaptic membranes treated with
Triton X-100 to deplete endogenous amino acids.19,20
There was no difference in the basal specific binding
of [3H]MK-801 between wild-type and the ORL1
receptor knockout mice. The specific binding of
[3H]MK-801 in both wild-type and the ORL1 receptor
knockout mice was significantly increased when the
assay was performed in the presence of 10 mM
glutamate plus 10 mM glycine, or glutamate, glycine
plus 1 mM spermidine. Under the stimulated conditions, the specific binding of [3H]MK-801 in the ORL1
receptor knockout mice was significantly higher than
that in wild-type mice (Figure 3a). Glycine or
spermidine alone did not change the [3H]MK-801
binding in either mice (data not shown).
We next measured 45Ca2 þ uptake into synaptosomes through NMDA receptor (Figure 3b). There was
no difference in the basal 45Ca2 þ uptake into
synaptosomes between wild-type and the ORL1
receptor knockout mice. When the assay was performed in the presence of 100 mM NMDA, NMDA plus
10 mM glycine, NMDA, glycine plus 1 mM spermidine, 45Ca2 þ uptake was increased in both the groups.
The 45Ca2 þ uptake in the ORL1 receptor knockout
mice was significantly higher than that in wild-type
mice under the stimulated conditions with NMDA,
glycine plus spermidine (Figure 3b). The NMDA
receptor agonist-stimulated 45Ca2 þ uptake in both
groups was completely antagonized by 100 mM MK801 (data not shown). In contrast, there was no
difference in high K þ (45 mM)-stimulated 45Ca2 þ
Molecular Psychiatry
Nociceptin and memory
T Mamiya et al
758
b
800
**
700
600
Wild-type
Stimulated [45Ca] uptake
(nmol/mg protein/5 min)
Specific [3H] MK-801
binding (fmol/mg protein)
a
Knockout
500
**
400
300
200
100
0
6
5
4
**
Wild-type
Knockout
3
2
1
0
Glutamate Glutamate/ Glutamate/
Glycine/
Glycine
Spermidime
NMDA
NMDA/ NMDA/
Glycine Glycine/
Spermidine
Figure 3 Specific [3H]MK-801 binding in forebrain membranes (a) and NMDA receptor-stimulated 45Ca2 þ uptake into
forebrain synaptosomes (b) in wild-type and the ORL1 receptor knockout mice. Each column represents the mean7SEM
(N ¼ 6). (a) Triton-treated forebrain membranes were incubated with 5 nM [3H]MK-801 at 301C for 16 h, in the presence or
absence of 10 mM glutamate, glutamate plus 10 mM glycine or plus glycine plus 1 mM spermidine. **Po0.05 vs wild-type
mice. (b) The forebrain synaptosomes were preincubated at 371C for 10 min, in the presence or absence of 100 mM (+) MK-801.
The assay was initiated by adding prewarmed buffer containing 1 mCi/ml 45CaCl2 for 5 min, in the presence of 100 mM (t)
NMDA, NMDA plus 10 mM glycine or NMDA plus glycine plus 1 mM spermidine. **Po0.01 vs wild-type mice.
uptake between wild-type (19.870.5 nmol/mg protein/5 min) and the ORL1 receptor knockout mice
(20.570.7 nmol/mg protein/5 min).
CaMKII activity
Ca2 þ influx initiates a wide array of biochemical
events associated with learning and memory in the
synapse.24,25 One potential target for immediate
activation by this Ca2 þ influx is CaMKII.26 Since
CaMKII has long been known to be essential for the
expression of both LTP and memory,27 we examined
CaMKII activity. When CaMKII was preincubated
under conditions promoting Ca2 þ -dependent autophosphorylation and subsequently assayed, the activity detected in the presence of excess of EGTA (Ca2 þ independent activity) increased in a time-dependent
manner and reached a maximum at about 1 min as
previously reported.28 Thus, we examined the CaMKII
activity after 1-min preincubation. The activity
assayed in the presence of Ca2 þ and CaM (total
activity) was not different between wild-type
(19.273.1 nmol/min/mg protein) and the ORL1 re-
Ca2+-independent
CaMkII activity (% of control)
200
180
##
160
Wild-type
Knockout
**
140
120
100
80
60
40
20
0
Basal
NMDA
NMDA/
Glycine/
Spermidine
(+) MK-801
NMDA/
Glycine/
Spermidine
+
(+) MK-801
Figure 4 Ca2 þ -independent CaMKII activity in wild-type and the ORL1 receptor knockout mice. Hippocampal slices were
incubated in the presence or absence of 100 mM NMDA plus 10 mM glycine plus 1 mM spermidine for 5 min and then CaMKII
activity was determined. ( þ ) MK-801 (300 mM) was pretreated for 2 min before the NMDA receptor stimulation. Ca2 þ independent CaMKII activity in wild-type and the ORL1 receptor knockout mice under basal condition was 1.1570.1 and
1.2670.1 nmol/min/mg protein, respectively. Each column represents the mean7SEM (N ¼ 8). **P o 0.05 vs basal, ##P
o0.05 vs wild-type mice.
Molecular Psychiatry
Nociceptin and memory
T Mamiya et al
Phosphorylation
the effects of exogenous nociceptin on NMDAinduced CaMKIIa phosphorylation. Hippocampal
slices from wild-type mice were preincubated with
pertussis toxin (5, 50 and 500 ng/ml for 16 h) and/or
nociceptin (10 mM) for 10 min before 20-min NMDA
receptor stimulation (Figure 6).29 In our experiment,
the inhibitory effects of pertussis toxin (50 ng/ml)
showed the maximal effects. Nociceptin inhibited
significantly the phosphorylation of CaMKIIa
induced by NMDA receptor stimulation (Figures 5b
and 6). Furthermore, the pretreatment of pertussis
toxin reversed the inhibitory effect of nociceptin on
the NMDA-induced phosphorylation of CaMKIIa.
These results suggest that nociceptin negatively
regulates the phosphorylation of CaMKIIa induced
by NMDA receptor stimulation through pertussis
toxin-sensitive G proteins.
CaMKIIa To support the data that CaMKII activity is
facilitated after NMDA receptor stimulation, we
examined the phosphorylation of CaMKIIa at Thr286
in the hippocampal slices induced by NMDA receptor
stimulation with 100 mM NMDA, 10 mM glycine plus
1 mM spermidine (Figure 5). Under our experimental
conditions, the phosphorylation of CaMKIIa at Thr286
was detected after 5-min stimulation and remained
for 30 min in the ORL1 receptor knockout mice. On
the
other
hand,
in
wild-type
mice
the
phosphorylation was observed after 20-min
stimulation of NMDA receptor. These results show
that the rapid phosphorylation of CaMKIIa at Thr286 is
induced in the ORL1 receptor knockout mice
compared with wild-type mice. We also examined
GluR1 and NR2B
One target for regulation by CaMKII may be the
AMPA-type glutamate receptor subunit GluR1.15,30,31
Thus, we examined the phosphorylation of GluR1 at
Ser831 and Ser845 after NMDA receptor stimulation in
wild-type and the ORL1 receptor knockout mice by
Western blotting with specific antibodies, respectively. Although NMDA receptor stimulation increased significantly the phosphorylation of GluR1
at Ser831, but not at Ser845, there was no difference in
GluR1 phosphorylation between the two genotypes
(Figure 7a and b).
Another target for regulation by CaMKII may be the
NMDA receptor itself, especially NR2B subunit at
Ser1303 32,33 Thus, we also compared the phosphoryla-
ceptor knockout (22.474.3 nmol/min/mg protein)
mice. Ca2 þ -independent CaMKII activity in wild-type
and the ORL1 receptor knockout mice under basal
condition was 1.1570.1 and 1.2670.1 nmol/min/mg
protein, respectively. Although Ca2 þ -independent
CaMKII activity was not affected by NMDA alone
(100 mM) in both the genotype mice, this activity was
significantly increased when the assay was performed
in the presence of 100 mM NMDA plus 10 mM glycine
plus 1 mM spermidine (Figure 4). The increased
activity in the ORL1 receptor knockout mice after
NMDA receptor stimulation was significantly enhanced compared to that in wild-type mice. Additionally, the increased activities were inhibited by
MK-801 in both the genotype mice.
a
Knockout
Wild-type
759
b
CaMKIIα-p
total CaMKIIα
5
0
10
20
30
0
5
10
20
30
Time after NMDA receptor stimulation (min)
Wild-type
Knockout
35
**
**
30
**
**
25
**
**
20
15
10
5
40
Change of
phosphorylation (%)
Change of
phosphorylation (%)
40
35
30
**
25
20
15
##
10
5
0
0
0
5
10
20
Time after NMDA receptor stimulation (min)
30
Noc (10 µmol)
-
-
+
NMDA receptor
stimulation
-
+
+
Figure 5 Time course of CaMKIIa phosphorylation induced by NMDA receptor stimulation in the ORL1 receptor knockout
and wild-type mice. Phosphorylation of CaMKIIa induced by the NMDA receptor stimulation (100 mM NMDA plus 10 mM
glycine plus 1 mM spermidine for 20 min) was detected by the antibody against phosphorylated Thr286 (CaMKIIaP). Blots
were stripped and reprobed with the antibody recognizing the CaMKIIa (total CaMKIIa) (top). Summary of phosphorylation
changes in Thr286 after the NMDA receptor stimulation (bottom). Each column represents the mean7SEM (N ¼ 8). **Po0.05
vs corresponding time 0.
Molecular Psychiatry
Nociceptin and memory
T Mamiya et al
760
Figure 6 CaMKIIa phosphorylation induced by NMDA receptor stimulation in the presence or absence of nociceptin or
pertussis toxin in wild-type mice. Phosphorylation of CaMKIIa induced by the NMDA receptor stimulation (100 mM NMDA
plus 10 mM glycine plus 1 mM spermidine for 20 min) was detected by the antibody against phosphorylated Thr286
(CaMKIIaP). Blots were stripped and reprobed with the antibody recognizing the CaMKIIa (total CaMKIIa) (top). Summary
of phosphorylation changes in Thr286 after the stimulation (bottom). Subsequent to the incubation with pertussis toxin
(50 ng/ml) for 16 h, nociceptin (10 mM) was added 10 min before the NMDA receptor stimulation.29 Each column represents
the mean7SEM (N ¼ 5). **Po0.05 vs control, ##P o 0.05 vs (NMDA/glycine/spermidine)-treated group, wwPo0.05 vs
(NMDA/glycine/spermidine) þ nociceptin-treated group.
-/-
+/+
+/+
-/-
-/-
Change of
phosphorylation (%)
10
0
5
NMDA receptor stimulation (min)
Wild-type
Knockout
30
25
*
20
*
*
*
15
10
5
+/+
+/+
-/-
-/-
+/+
-/-
0
5
10
NMDA receptor stimulation (min)
Change of
phosphorylation (%)
+/+
Wild-type
Knockout
30
25
20
15
10
5
0
0
0
5
10
Time after NMDA receptor
stimulation (min)
0
5
10
Time after NMDA receptor
stimulation (min)
c NR2b Serine1303 phosphorylation
+/+
+/+
-/-
-/-
+/+
-/-
0
5
10
NMDA receptor stimulation (min)
Change of
phosphorylation (%)
a GluR1 Serine831 phosphorylation b GluR1 Serine845 phosphorylation
Wild-type
Knockout
30
25
20
*
15
*
*
*
10
5
0
0
5
10
Time after NMDA receptor
stimulation (min)
Figure 7 Phosphorylation of GluR1 at Ser831 (a), Ser845 (b) and NR2B at Ser1303 (c) by NMDA receptor stimulation in wildtype ( þ / þ ) and the ORL1 receptor knockout (/) mice. Phosphorylation of NR2B subunit induced by the NMDA receptor
stimulation (100 mM NMDA plus 10 mM glycine plus 1 mM spermidine for 20 min) was detected by the antibody against
phosphorylated Ser831 and Ser845 on GluR1, and Ser1303 on NR2B (top). Summary of phosphorylation changes in Ser831 and
Ser845 on GluR1, and Ser1303 on NR2B subunit after NMDA receptor stimulation (bottom). Each column represents the
mean7SEM (N ¼ 6). **Po0.05 vs corresponding time 0.
Molecular Psychiatry
Nociceptin and memory
T Mamiya et al
tion of NMDA receptor subunit NR2B at Ser1303 after
NMDA receptor stimulation in both the genotype
mice at various points (Figure 7c). The NR2B at
Ser1303 was phosphorylated by NMDA receptor stimulation in both the genotype mice. No differences
were observed in the phosphorylation levels between
wild-type and the ORL1 receptor knockout mice.
Polysialylated form of neuronal cell adhesion molecule
(PSA-NCAM) and GluR 1/2 expression
While there was few if any PSA-NCAM detectable in
neocortical areas (data not shown), the protein was
present in the hippocampal complex, and particularly
in the dentate gyrus of both wild-type and the ORL1
receptor knockout mice (Figure 8). The density of
PSA-NCAM positive processes in the dentate polymorph layer, and the number of cell bodies within
this layer as well as in the granular layer, were
reduced in the ORL1 receptor knockout (Figure 8b) as
compared with wild-type mice (Figure 8a). Expressions of both GluR1 and GluR2 in the ORL1 receptor
knockout were not changeable compared to the wildtype mice.
761
Discussion
Previously, we have reported that LTP in the
hippocampus and memory is facilitated in the ORL1
receptor knockout mice compared to the wild-type
mice.11–13 Moreover, exogenous nociceptin has been
reported to impair learning and memory in intact
mice10,11 and in rats.8,9 Here we investigated the
mechanisms of nociceptin-induced modulation in
learning and memory.
First of all, we examined the contextual and cued
conditioning response to see whether the enhancement of learning and memory in the ORL1 receptor
knockout mice is dependent on the hippocampal
NMDA receptor function. The NMDA receptor in the
hippocampus plays an important role in the contextual, but not cued conditioning memory.14,34–36 In
the ORL1 receptor knockout mice, the retention of
contextual fear conditioning memory was prolonged
without any alterations of cued conditioning response. The enhancement of memory retention in
the ORL1 receptor knockout mice was observed
compared with wild-type mice on the 7th day when
the immobility time was approximately B50% in the
Figure 8 Three-dimensional representation of wild-type (a) and the ORL1 receptor knockout (b) dentate gyrus, to be viewed
with a stereo-lorgnette. Reconstruction using nine serial ‘optical sections’ taken at 1 mm intervals. Five mice were used
independently in each group. Coronal sections. Dorsal is up; median is to the left. Coded in red: PSA-NCAM; blue: GluR-1;
green: GluR-2. Note the reduction of red-coded processes and cells in the ORL1 receptor knockout dentate gyrus (b).
Molecular Psychiatry
Nociceptin and memory
T Mamiya et al
762
wild-type mice, but not on the 2nd day when the
immobility time was about B80% in wild-type mice.
The lack of significant difference between the genotypes on the 2nd day may be because of the ceiling
effect in this task. On the other hand, nociceptin
(1 nmol) induced an impairment of the contextual,
but not cued conditioning memory. This acute effect
of nociceptin on the contextual memory in wild-type
mice was examined on the 2nd day (Figure 2),
because the level of immobility time was relatively
small on the 7th day so that the inhibitory effects of
nociceptin may not be detected because of the floor
effects. These results confirmed our previous results
that exogenous nociceptin impaired not only the
acquisition but also the retention in the passive
avoidance test, and that the ORL1 receptor knockout
mice showed an enhancement of the memory retention in the passive avoidance test.11,13 Importantly, the
impairment by nociceptin was blocked by a selective
ORL1 receptor antagonist. Furthermore, the enhancement of retention memory on the 7th day in the ORL1
receptor knockout mice was blocked by an NMDA
receptor antagonist, ( þ ) MK-801. Collectively, our
findings suggest that (1) nociceptin system is involved in the regulation of memory retention and (2)
the effects of nociceptin in learning and memory are
mediated via NMDA receptor in the hippocampal
function. In the previous reports, basal nociceptin
contents have been reported to be 55.5–58.7 pg/ml
(614–648 fmol/rat) in the brain of rat.37 The dose of
nociceptin used in the present study (1 nmol/mouse)
is far higher than basal nociceptin contents in the
brain, but is comparable to antinociceptive doses.2,3
Thus, it is considered that nociceptin (0.01–1 nmol)
impairs cognitive function at pharmacological doses.
Previous studies demonstrated that enhanced LTP
in the hippocampus of the ORL1 receptor knockout
mice was blocked by NMDA receptor antagonist,
indicating a role of NMDA receptor. No difference
between genotypes in the paired-pulse facilitation
suggests that the ORL1 receptors modulate LTP
probably through the postsynaptic mechanisms,13
because paired-pulse facilitation is used as an index
of the efficacy of releasing neurotransmitters from
presynapse. Furthermore, nociceptin was reported to
function as an inhibitory modulator for synaptic
function in the hippocampus.6 Accordingly, we
examined the effects of nociceptin on the NMDA
receptor function and signal transduction through
NMDA receptor. NMDA receptor is a ligand and
voltage-gated Ca2 þ channel. Binding of glutamate
released from the presynaptic terminal, coupled with
strong depolarization of the postsynaptic membrane
produces an influx of Ca2 þ into the postsynaptic
compartment. This Ca2 þ influx initiates a wide array
of biochemical events in the synapse that can lead to
LTP or LTD.24,25 One potential target for immediate
activation by this Ca2 þ influx is CaMKII.26 CaMKII
has long been known to be essential for the expression
of both LTP and LTD.38,39 Therefore, we measured the
[3H]MK-801 binding, and NMDA receptor-mediated
Molecular Psychiatry
Ca2 þ uptake into synaptosomes and CaMKII activity
to assess the NMDA receptor function in the ORL1
receptor knockout mice. When NMDA receptor is
activated by adding glutamate, glycine (for activating
glycine binding site) plus spermidine (for polyamine
binding site) in the assay mixture, the specific
binding of [3H]MK-801 in the ORL1 receptor knockout
mice was significantly increased compared with that
in wild-type mice, whereas there was no significant
difference on the basal level between wild-type and
the ORL1 receptor knockout mice. Similarly, 45Ca2 þ
uptake into synaptosomes obtained from the ORL1
receptor knockout mice was significantly higher than
that in wild-type mice when 45Ca2 þ uptake was
activated by NMDA, glycine and spermidine. The
binding sites of ( þ ) MK-801 exist inside the NMDA
receptor-associated cation channels.19,40 The basal
bindings of [3H]MK-801 in the Triton X-100-treated
membranes represent the minimally activated state of
NMDA receptor.19 When NMDA receptor is activated
with glutamate, glycine and/or spermidine, the
associated channels become open, and thereby
[3H]MK-801 binding sites are increased.20 Accordingly, these results suggest that the function of NMDA
receptor in the ORL1 receptor knockout mice is
potentiated.
The reduction of CaMKII activity by pharmacological41 or genetic38 means impairs LTP and learning,
whereas injecting or overexpressing CaMKII increases
synaptic strength, which occludes and is occluded by
electrically induced LTP.39,42 Crucial to its function in
LTP and spatial learning,27,43 CaMKII at Thr286 undergoes rapid phosphorylation following NMDA receptor-mediated
calcium
influx.
Since
this
autophosphorylation renders the kinase calcium
independent and has been proposed as a form of
molecular memory,44 we examined the CaMKII activity. NMDA receptor stimulation with NMDA, glycine
and spermidine increased the CaMKII activity in both
the wild-type and the ORL1 receptor knockout mice,
the increased activity in the ORL1 receptor knockout
mice was significantly enhanced compared with that
in wild-type mice. The increased activities were
inhibited by ( þ ) MK-801 in both the genotype mice.
Furthermore, the rapid phosphorylation of CaMKIIa
at Thr286 was induced in the ORL1 receptor knockout
mice compared with wild-type mice. The phosphorylation was inhibited significantly by nociceptin, and
pertussis toxin reversed the inhibitory effect of
nociceptin on the NMDA-induced phosphorylation
of CaMKIIa. Taken together, it is suggested that (1)
NMDA receptor/CaMKII cascade is enhanced functionally in the ORL1 receptor knockout mice and
(2) nociceptin regulates negatively NMDA-induced
CaMKIIa phosphorylation through the pertussis toxin-sensitive G proteins.
One target for regulation by CaMKII at the postsynaptic site may be the AMPA-type glutamate
receptor subunit GluR1, although uncertainty about
the topology of receptor folding has cast some doubt
on the significance of direct phosphorylation of
45
Nociceptin and memory
T Mamiya et al
GluR1 in vivo.15,30,31 We could not observe any
difference of both GluR1 and GluR2 expression in
the hippocampus between wild-type and the ORL1
receptor knockout mice. By the activation of NMDA
receptor with 100 mM NMDA, 10 mM glycine plus
1 mM spermidine, in both the genotype mice, GluR1
at Ser831 was phosphorylated, but there was no
difference between the two genotypes. GluR1 at
Ser845 was not significantly phosphorylated under
our conditions. Another target for regulation by
CaMKII at postsynaptic site may be the NMDA
receptor itself, especially NR2B subunit at
Ser1303.32,33 After NMDA receptor stimulation, we
detected increased phosphorylation of NR2B at
Ser1303, but there was no significant difference
between wild-type and the ORL1 receptor knockout
mice. Accordingly, molecular targets of NMDA receptor/CaMKII cascade which are responsible for the
hyperfunction of NMDA receptor in the ORL1 receptor knockout mice remain to be determined.
As one of the possibilities, abnormalities of the
signal-transduction systems involving protein kinase
C (PKC) or mitogen-activated protein (MAP) kinases
may contribute to an enhancement of learning and
memory in the ORL1 receptor knockout mice. Previous studies have indicated that PKC modulates CA1
LTP and learning,45,46 and is activated by stimulation
of the ORL1 receptor.29 However, PKC activity was not
affected by the lack of the ORL1 receptor in the
nonstimulated condition (unpublished data). Alternatively, MAP kinase activity increases in the hippocampus during fear conditioning.47 We also failed to
detect any differences in the MAP kinase phosphorylation of ERK1 and ERK2 when the NMDA receptor
was stimulated by 100 mM NMDA, 10 mM glycine plus
1 mM spermidine (unpublished data). Further studies
are necessary to elucidate the involvement of PKC
and MAP kinases in the modulation of learning and
memory by nociceptin.
The polysialylated form of NCAM (PSA-NCAM) is
strongly expressed in the embryonic nervous system
during cell migration and neurite extension. In the
adult brain, PSA-NCAM is still present in a few
restricted areas, including the hippocampus.48 The
presence of PSA-NCAM in the adult hippocampus
has been associated with neural plasticity49 involved
in learning processes.50 In the hippocampus, PSANCAM may support migration of newly formed
neurons that will settle in the dentate granular layer.
Alternatively, it may be necessary for the movement
of cell processes linked to adult neurogenesis or to
plasticity of established cells. It seemed therefore
interesting to have a look at this protein in the
hippocampal complex of the ORL1 receptor knockout
as compared with the wild-type mice.
We found that PSA-NCAM expression was reduced
in the ORL1 receptor knockout mice. With regard to
this unexpected findings, we have previously reported that the lack of telencephalin, one of NCAMs
induces the facilitation of LTP in the hippocampus
and the enhancement of learning and memory with-
out affecting NMDA and non-NMDA receptor expression.51 These characteristics in the telencephalin
mutant mice are similar with those found in the
ORL1 receptor knockout mice. Regarding the interaction of NCAM and NMDA receptor, administration of
NMDA receptor antagonist induces the expression of
NCAM in the cortex and dentate gyrus.52,53 In
addition, Bouzioukh et al.54 have reported that the
chronic blockade of NMDA receptor activation decreases the NCAM expression. Thus, these results
suggest that NCAM expression is modulated by the
NMDA receptor. Further studies are necessary to
elucidate the role of PSA-NCAM in nociceptininduced modulation of learning and memory.
In conclusion, we found and confirmed the enhancement of memory retention with hyperfunction
of NMDA receptor in the ORL1 receptor knockout
mice compared with wild-type mice. Moreover,
exogenous nociceptin, which inhibited the activation
of CaMKII induced by NMDA receptor stimulation
through G proteins, impaired memory retention. In
the ORL1 receptor knockout mice, the expression of
PSA-NCAM was reduced. These results suggest that
nociceptin system negatively modulates learning and
memory through the regulation of NMDA receptor
function and the expression of PSA-NCAM.
763
Acknowledgements
We are very thankful to Drs Takeshima and Nishi at
Tohoku University for supplying the ORL1 receptor
knockout mice. This work was supported, in part, by
a Grant-in-Aid for Special Coordination Funds for
Promoting Science and Technology, Target-Oriented
Brain Science Research Program from the Ministry of
Education, Culture, Sports Science and Technology of
Japan, the Health Sciences Research Grants for
Research on Pharmaceutical and Medical Safety from
the Ministry of Health, Labor and Welfare of Japan,
and the Hibino Memorial Fund and the Kyousaidan
Foundation Fund.
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