The Journal of Neuroscience, July 23, 2003, 23(16):6529-6536
Previous Article | Next Article 
Locus-Specific Rescue of GluR
1 NMDA Receptors in Mutant Mice Identifies the Brain Regions Important for Morphine Tolerance and Dependence
Makoto Inoue,1
Masayoshi Mishina,2,3 and
Hiroshi Ueda1
1Division of Molecular Pharmacology and
Neuroscience, Nagasaki University Graduate School of Biomedical Sciences,
Nagasaki 852-8521, Japan, 2Department of Molecular
Neurobiology and Pharmacology, Graduate School of Medicine, University of
Tokyo, Tokyo 113-0033, Japan, and 3Solution-Oriented
Research for Science and Technology, Japan Science and Technology Corporation,
Tokyo 113-0033, Japan
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Abstract
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Tolerance and physical dependence caused by chronic treatment of narcotics
are good models to study basic neuronal plasticity. Activation of the NMDA
subtype of the glutamate receptor has been implicated as an anti-opioid system
in the development of morphine analgesic tolerance and dependence. The present
study examines the specific role of the 
1 subunit of the NMDA receptor
using mice lacking the gene encoding 1 subunit of the NMDA receptor
(GluR1-/- mice). GluR1-/- mice showed
significant enhancement and prolongation of morphine anti-nociception,
compared with wild-type GluR1+/+ mice.
GluR1-/- mice also showed a marked loss of the analgesic
tolerance after repeated morphine treatments. In C57BL/6J mice treated with
chronic morphine after tolerance paradigm, the GluR1 protein expression
significantly increased in periaqueductal gray matter (PAG), ventral tegmental
area (VTA) and nucleus accumbens (NAc), but not amygdala or hippocampus. The
rescue of GluR1 protein by electroporation into the PAG and VTA, but not
NAc of GluR1-/- mice significantly reversed morphine
analgesic tolerance liability. Similar attempts were also performed in the
naloxone-precipitated physical dependence paradigm. GluR1-/-
mice showed marked loss of typical withdrawal abstinence behaviors, and
significant enhancement of GluR1 protein expression was only observed in
NAc by chronic morphine treatments after dependence paradigm. The rescue of
GluR1 protein by electroporation into the NAc of
GluR1-/- mice significantly reversed the loss of abstinence
behaviors. These findings suggest that GluR1 has locus-specific roles in
the development of morphine analgesic tolerance and physical dependence.
Key words: NMDA receptor; 1 subunit; morphine tolerance and dependence; KO mice; locus-specific rescue; neuronal network
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Introduction
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The systemic administration of morphine is the most effective means of
alleviating severe pain across a wide range of conditions. However, its
clinical use has been limited by undesirable side effects such as tolerance.
Furthermore, narcotic dependence brings serious social problems, accompanied
by psychological and physical dependence. These undesirable symptoms are
considered related to neuronal plasticity in the CNS. Many investigators have
attempted to elucidate the mechanisms underlying the development of morphine
analgesic tolerance and dependence, and some aspects of molecular and cellular
mechanisms are now revealed (Ueda et al.,
1995
,
2001;
Roth and Willins, 1999;
Mamiya et al., 2001;
Nestler 2001;
Tsao et al., 2001;
Williams et al., 2001;
Bodnar and Hadjimarkou, 2002;
Kieffer and Evans, 2002).
However, the molecular mechanisms through the neuronal networks remain
unknown. Drug-induced adaptation of neuronal networks appears a good model to
study basic neuronal plasticity. Among several attempts (for review, see
Crawley and Corwin, 1994;
Harrison et al., 1998;
Mogil and Pasternak, 2001),
the idea of an anti-opioidergic system provides us with a guide for an
approach to understanding the complexity of the neuronal networks. The NMDA
receptor-dependent glutamatergic system is the most prominent candidate for an
anti-opioid system in the development of neuronal plasticity because the
administration of various kinds of NMDA receptor antagonists inhibits side
effects such as morphine analgesic tolerance and dependence (for review, see
Trujillo, 2000;
Mao, 2002). However, a
molecular-based approach has not been attempted. Native NMDA receptors
comprise GluR
(NR1) and GluR (NR2) subunits
(Mori and Mishina, 1995), and
multiple GluRs [GluR1-GluR4 (or NR2A-NR2D)] subunits convey
NMDA receptor heterogeneity to the properties of ion channels and differential
functions caused by different temporal and spatial expressions
(Mori and Mishina, 1995;
Colwell et al., 1998). We
studied the physical liability of morphine tolerance and dependence using mice
lacking the gene encoding the GluR1 subunit of the NMDA receptor
(GluR1-/-) (Sakimura et
al., 1995). Furthermore, we demonstrate that the locus-specific
expression by in vivo electroporation of GluR1 NMDA receptors
into mutant mice restores the morphine tolerance and dependence.
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Materials and Methods
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Mice. Male C57BL/6J mice, wild-type
(GluR1+/+) mice, and mutant
(GluR1-/-) mice lacking the NMDA receptor 1 subunit
gene weighing 20-22 gm were used. The
GluR1+/+ and GluR1-/-
mice with the highly homogenous genetic background of the C57BL/6J strain had
been developed previously (Sakimura et
al., 1995). These mice were kept in a room maintained at 22
± 2°C with ad libitum access to a standard laboratory diet
and tap water. All experiments were performed in compliance with the relevant
laws and institutional guidelines. All procedures were approved by the
Nagasaki University Animal Care Committee and complied with the
recommendations of the International Association for the Study of Pain
(Zimmermann, 1983).
Evaluation of morphine analgesic tolerance. Mice were given
morphine-HCl at 10 mg/kg, subcutaneously daily for 6 d, and the nociceptive
responses on the first and sixth days were compared. L-Arginine (50
mg/kg, i.p.) was injected 15 min before the administration of morphine,
according to previous report (Kolesnikov et
al., 1998). The Haffner tail-pinch, which is known to reflect the
supraspinal nociceptive response, was performed as described previously (Ueda
et al., 1997,
2000). In these experiments,
an arterial clip was placed at the base of an animal's tail, and the
nociceptive response in this test was evaluated by measuring the latency of
biting behavior to the clip, whose strength had been adjusted for nontreated
mice to show 0.3-1.0 sec latency. A cutoff time of 15 sec was used to avoid
tissue damage. Morphine analgesia was expressed as the area under the curve
(AUC). The tolerance index was represented as given by the following equation:
tolerance index (%) = (sixth AUC/first AUC) x 100. Individual
comparisons were made by the Student's t test.
Analysis of morphine withdrawal signs. Physical dependence was
induced by repeated subcutaneous injections of morphine-HCl, at intervals of 8
hr, for 4 d, according to the method of Maldonado et al.
(1996) with slight
modification (Ueda et al.,
2000). The morphine-HCl dose was progressively increased as
follows: first day, 20 and 40 mg/kg; second day, 60, 80, and 100 mg/kg; third
day, 100 mg/kg x 3; and fourth day, 100 mg/kg (only one injection in the
morning). The control mice were treated with saline under the same conditions.
Withdrawal signs were precipitated by the injection of naloxone-HCl (1 mg/kg,
i.p.) 2 hr after the last morphine-HCl administration. Mice were placed
individually in a clear plastic observation area (30 cm diameter, 50 cm
height) with a white floor 30 min before naloxone-HCl injection. The number of
withdrawal signs such as jumping, wet-dog shaking, paw tremors, and backward
locomotion were counted for 30 min after the naloxone challenge. Individual
comparisons were made by the Student's t test.
Western blot analysis. To confirm the effect of the in
vivo transfection of GluR1 cDNA, immunoblot analysis using 8%
polyacrylamide gel, SDS-PAGE was performed as described
(Yoshida and Ueda, 1999). For
this experiment, 40 µg [periaqueductal gray matter (PAG) and ventral
tegmental area (VTA)] or 20 µg proteins [nucleus accumbens (NAc),
hippocampus, and amygdala] were used per lane. To obtain equal transfer
efficiency, the saline control and morphine-treated samples for each brain
region of mice (n = 6) were applied onto the same gel, and immunoblot
transfers were performed using the same membrane. The membranes were incubated
overnight with anti-GluR1 (1:1000). After extensive washing, the
membrane was incubated with peroxidase-conjugated anti-rabbit IgG (1:20,000)
for 1 hr at room temperature. All visualization of immunoreactive bands was
performed by the Light Capture (AE-6960/C/FC; Atto, Tokyo, Japan) with an
enhanced chemiluminescent substrate for the detection of horseradish
peroxidase, Super Signaling Substrate (Pierce, Rockford, IL). Because this
Light Capture can repeatedly expose at intervals of 5 min and automatically
superimpose these signals to obtain high contrast, we can detect enhanced
signals with less background signals. Subsequently, the intensities of the
immunoreactive bands were analyzed by NIH imaging for Macintosh.
Plasmid DNA. The cDNA encoding GluR1 from pBKSA1
(Meguro et al., 1992) was
cloned into pAdTrack-cytomegalovirus (CMV). This shuttle vector is used for
production of green fluorescent protein (GFP) trackable virus containing
transgenes under the control of a CMV promoter (a gift from Dr. Bert
Vogelstein, Howard Hughes Medical Institute and Johns Hopkins Oncology
Center).
In vivo electroporation. GluR1 cDNA
(DGluR1pAdTrack-CMV) were purified with an AccuPrep plasmid extraction
kit (Bioneer Company, Daejeon, Korea) and used for gene transfer into specific
brain loci. A pair of stainless steel electrodes, 0.5 mm in length and 0.3 mm
in diameter, was stereotaxically inserted into both sides of the ventrolateral
PAG (anterior: -3.28 mm, lateral: ±0.3 mm, height: 2.5 mm from bregma),
NAc (anterior: 1.78 mm, lateral: ±0.90 mm, height: 3.75 mm from
bregma), or the VTA (anterior: -3.28 mm, lateral: ±0.65 mm, height: 4.0
mm from bregma) of sodium pentobarbital-anesthetized (50 mg/kg, i.p.) mice.
Electric pulses were generated with a Square Electro Porator (CUY21; TR Tech,
Tokyo, Japan) at 10 pulses/sec (10 Hz). The shape of the pulse was a square
wave; i.e., the voltage remained constant during the pulse duration. For the
best gene transfer, cDNA at dose of 1 µg, which diluted in PBS immediately
before use was electroporated into brain regions by the electric pulses (2
msec) in 40-55 V duration to get 10 mA current. There were no significant
gross behavioral changes as a result of the electroporation of either the mock
or recombinant cDNA into the any brain regions. Morphine was injected from day
4 after electroporation for tolerance and dependence experiments. The
tolerance experiments were preformed at day 9 after electroporation, whereas
the dependence experiments were performed at day 7 after electroporation. We
performed histochemical confirmation of fluorescence of all mice after the
behavioral experiments. We used the results with mice expressing fluorescence
on both sides of the PAG, VTA, or NAc for evaluation of the effects by in
vivo electroporation of GluR1 or mock genes. Individual comparisons
were made by the Student's t test (p < 0.05) between the
mock-expressed GluR1+/+ mice and the
mock-expressed GluR1-/- mice or the mock-expressed
GluR1-/- mice and the GluR1-expressed
GluR1-/- mice.
Histological analysis. To confirm the tissue damage after the
in vivo electroporation, histological analysis was performed with
hematoxylineosin. Nine days after the in vivo electroporation or sham
operation without electroporation, the mice were deeply anesthetized with 50
mg/kg pentobarbital intravenously and intracardially perfused with 0.1
M PBS followed by 4% paraformaldehyde fixative. Brain was dissected
and cut into 10-µm-thick slices and stained with hematoxylineosin.
Statistical analysis. Statistical evaluations were performed using
the Student's t test. In some experiments, statistical evaluations
were also performed using the Scheffe test for multiple comparisons, after
one-way or repeated measures ANOVA. Data were expressed as mean ± SEM.
Significance was established at *p < 0.05;
#p < 0.05.
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Results
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Attenuated morphine analgesic tolerance in GluR1-/-
mice
There were no significant differences in basal pain responses in the tail
pinch test between wild-type (GluR1+/+;
0.33 ± 0.02 sec) and GluR1 mutant (GluR1-/-;
0.31 ± 0.03 sec) mice (Fig.
1A). In this test, morphine at 10 mg/kg given
subcutaneously increased the latency to show biting responses to an artery
clip applied to the tail root (Fig.
1A). When the morphine analgesic activity was evaluated
by AUC of time course (Fig.
1B) or using the value in the peak time for morphine
analgesia (45 min after morphine administration)
(Fig. 1C), an enhanced
analgesia in mutant mice was observed in dose-dependent manner, compared with
one in GluR1+/+ mice. After daily
injections of morphine for 5 d, morphine analgesia in
GluR1+/+ mice was markedly attenuated
on day 6 (Fig. 1D),
whereas the analgesic tolerance was only limited in GluR1-/-
mice (Fig. 1E). When
the morphine analgesic activity was re-evaluated by AUC of time course
morphine analgesia, the AUC value for sixth day morphine analgesia in
GluR1+/+ mice (158.6 ± 12.0)
appears to be markedly lower than that for the first day (1086.3 ±
92.7), as shown in Figure
1F. The tolerance index calculated by the ratio was only
14.6% in GluR1+/+ mice, whereas that in
GluR1-/- mice was significantly increased to 68.8%
(Fig. 1F).
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Figure 1. Attenuation of morphine tolerance in GluR1-/- mice in the
tail-pinch test. A, Time course of analgesia in
GluR1-/- (1-/-) and
GluR1+/+
(1+/+) mice (n = 6) after 10
mg/kg given subcutaneously. B, Dose-dependent morphine analgesia in
1-/- or 1+/+ mice. A
cutoff time of 15 sec was used to avoid tissue damage. The analgesic effect
was represented as the AUC during 15-120 min. Maximal AUC is calculated to be
1575. C, D, Time course of morphine-induced (10 mg, s.c.) analgesia
in 1+/+ (C) or
1-/- (D) mice with (sixth) or without (first)
chronic morphine treatments (10 mg/kg, s.c.; 5 d), respectively. E,
Comparison of morphine analgesia in mutant mice pretreated with or without
chronic morphine treatments. Morphine (10 mg/kg, s.c.) analgesia was
represented as AUC. ED50 (95% confidence limits) of morphine
analgesia at first and sixth day in
1+/+ mice was 8.1 (2.4-26.8) and 28.0
(15.4-50.9) mg/kg subcutaneously, respectively, whereas the one at the first
and sixth day in 1-/- mice was 3.0 (1.2-7.7) and 5.1
(2.8-9.4) mg/kg subcutaneously, respectively. F, Morphine tolerance
index in 1+/+ and 1-/-
mice. The tolerance index was represented as given by the following equation:
tolerance index (%) = (sixth AUC/first AUC) x 100. Results were
represented as the mean ± SEM of the tail-pinch latency (sec) from six
separate animals. *p < 0.05 compared with
1+/+ mice.
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Enhancement of GluR1 protein expression in specific brain
regions after repeated morphine treatments
C57/BL6 mice were daily injected with morphine (10 mg/kg, s.c.) and used
for the immunoblot analysis of GluR1 protein expression. NAc, amygdala,
the hippocampus, PAG, and VTA were chosen as pivotal brain regions in the
development of morphine analgesic tolerance and neuronal plasticity after
chronic morphine treatments (Mitchell et
al., 2000; Williams et al.,
2001; Nestler,
2001). Compared with the expression in saline-treated mice,
significant increases were found in the preparations of NAc, PAG, and VTA, but
not in those of amygdala and hippocampus
(Fig. 2).
Nitric oxide action in the GluR1-/- mice with
morphine chronic treatments
NMDA-nitric oxide (NO) cascades are reported to be involved in the morphine
analgesic tolerance (Pasternak et al.,
1995; Kolesnikov et al.,
1998). We next assessed the effect of L-arginine, which
is a NO precursor and substrate for NOS, on morphine analgesic tolerance in
both GluR1+/+ and
GluR1-/- mice. After daily administration of morphine (10
mg/kg, s.c.) for 5 d in GluR1+/+ mice,
morphine analgesic activity evaluated by AUC of time course analgesia
significant decreased on day 4 and 5, as shown in
Figure 3A. The
concurrent daily administration of L-arginine (50 mg/kg, i.p.)
along with morphine did not affect the acute morphine analgesia (day 1),
although it accelerated the development of analgesic tolerance in
GluR1+/+ mice
(Fig. 3A). In
GluR1-/- mice, on the other hand, daily morphine
administration showed significant, but less marked analgesic tolerance than in
GluR1+/+ mice, as shown in
Figure 3B. However,
the concurrent daily administration of L-arginine (50 mg/kg, i.p.)
along with morphine markedly accelerated the development of analgesic
tolerance in GluR1-/- mice. L-Arginine maximally
decreased the morphine analgesia in GluR1-/- mice day 4 or 5
to the level with GluR1+/+ mice, which
had been similarly given (Fig.
3B).
Locus-specific reversal of morphine analgesic tolerance by gene
transfer of recombinant GluR1 into GluR1-/- mouse brain
regions
The recombinant GluR1 clone with the CMV promoter was expressed in
PAG, VTA, or NAc neurons using the electroporation technique. Although we
electroporated 0.3-2 µg GluR1 cDNA into the brain regions by the
electric pulses (2 msec) in 10-20 mA current, the best gene transfer was
observed when 1 µg of cDNA was electroporated into brain regions by the
electric pulses (2 msec) in 40-55 V duration to get 10 mA current. As shown in
Figure 4A, a marked
green fluorescence used as the genetic marker was specifically observed in
both sides of the ventrolateral PAG on day 9 after electroporation. The
fluorescence was also observed within the limits of 0.45 mm each toward to
rostrum and caudal regions from electroporation region (data not shown). On
the other hand, when tissues were stained with hematoxylin-eosin, the
electroporation did not damage the morphology of brain regions showing
fluorescence (Fig.
4B). The expression of the GluR1 protein in PAG was
also confirmed with immunoblot analysis on day 4 after electroporation, and
the expression lasted until at least day 9
(Fig. 4C). We started
the injection of morphine into mice from day 4 after electroporation, and we
performed the tolerance experiment on day 9. As shown in
Figure 4D, the
enhancement of acute morphine analgesia (first) and attenuation of morphine
analgesic tolerance (sixth) in mice deleting GluR1 gene was not affected
by electroporation of mock vector into any brain regions including PAG. The
electroporation of recombinant GluR1 into these regions of
GluR1-/- mice slightly reduced the acute analgesia. However,
the significant reversal of tolerance liability was observed when GluR1
was electroporated into PAG and VTA, but not into NAc
(Fig. 4D,E).
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Figure 4. Locus-specific rescue of morphine tolerance by gene transfer of recombinant
GluR1 into GluR1-/- mouse brain regions. A,
GFP fluorescence to confirm the accurate expression of the recombinant gene on
day 9 after electroporation. A marked green fluorescence used as the genetic
marker was specifically observed in both sides of the ventrolateral PAG.
B, Lack of morphological change in the ventrolateral PAG region by
electroporation on day 9. The hematoxylin-eosin staining of ventrolateral PAG
region expressing GFP fluorescence in A (+). The staining of
corresponding PAG region without electroporation (-). C, Western blot
of GluR1 (1) in the PAG from mock-transferred
1+/+ or 1-/- or
recombinant 1-transferred
1-/-[1+/+(m),
1-/- (m) or 1-/- (1),
respectively] mice at days 4 and 9. D, Comparison of morphine
analgesia in 1+/+ (m),
1-/- (m), or 1-/- (1) mice
with or without chronic morphine treatments. Morphine (10 mg/kg, s.c.)
analgesia was represented as AUC. E, Morphine tolerance index in
1 gene mutant mice with gene transfer into several brain regions.
Results were represented as the mean ± SEM of the tail-pinch latency
(in seconds) from six separate animals. The details are described in the
legend of Figure 1.
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Attenuated morphine dependence in GluR1-/- mice
We proceeded with the study of the physical dependence liability of
morphine in both GluR1-/- and
GluR1+/+ mice, because we confirmed the
classical signs of high-dose (20-100 mg/kg, s.c.) morphine-induced behaviors,
such as Straub's tail symptom and increased locomotive behaviors in these
mice. The intraperitoneal injection of naloxone (1 mg/kg) precipitated somatic
symptoms of withdrawal abstinence behaviors, such as jumping, wet-dog shaking,
paw tremors, and backward locomotion in the
GluR1+/+ mice chronically treated with
increasing doses of morphine, according to the morphine dependence paradigms.
All of these abstinence scores during 30 min after the naloxone challenge were
significantly attenuated in the GluR1-/- mice, but the
withdrawal abstinence was relatively less marked
(Fig. 5).
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Figure 5. Attenuated morphine dependence in GluR1-/- mice. Mice were
pretreated with morphine-HCl every 8 hr as follows: first day, 20 and 40
mg/kg; second day, 60, 80, and 100 mg/kg; third day, 100 mg/kg x 3; and
fourth day, 100 mg/kg. The number of jumps, wet-dog shakes, paw tremors, and
backward locomotions as withdrawal signs were counted for 30 min after the
naloxone (1 mg/kg, i.p.) challenge 2 hr after the final morphine-HCl
injection. The results were represented as the mean ± SEM of withdrawal
signs from seven separate animals. *p < 0.05, compared
with 1+/+ mice.
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Enhancement of GluR1 protein expression in NAc after chronic
morphine treatments using dependence paradigm
Compared with the GluR1 protein expression in saline-treated C57BL/6J
mice, a significant increase was found only in NAc, but not in PAG, VTA,
amygdala, or hippocampus, whereas there was a slight but significant decrease
in the expression in amygdala (Fig.
6).
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Figure 6. Enhanced GluR1 protein expression in NAc after repeated morphine
treatments using dependence paradigm. Mice were pretreated with morphine-HCl
following dependence paradigm. The brain was isolated 2 hr after the final
morphine injection for Western blot. The results were represented as the mean
± SEM of withdrawal signs from seven separate animals. The details are
described in the legend of Figure
2.
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Reversal of attenuated morphine dependence in
GluR1-/-
mice by the in vivo transfection of recombinant
GluR1 into NAc As shown in
Figure 7A, when
recombinant GluR1 was transfected in NAc, a marked green fluorescence
was specifically observed in NAc (core and shell) on day 7 after
electroporation. The in vivo electroporation of mock cDNA into NAc of
GluR1+/+ mice slightly affected some of
naloxone-precipitated withdrawal abstinence scores, compared with those in
background C57BL/6J mice, as shown in Figures
5 and
7. However, the relative
attenuation of these scores in GluR1-/- mice compared with
those in GluR1+/+ mice was not
significantly affected by mock cDNA electroporation. The electroporation of
GluR1 cDNA into NAc of GluR1-/- mice recovered the
decreased withdrawal abstinence behaviors. Significant recovery was observed
in wet-dog shaking, paw tremors, and backward locomotion, but the recovery of
jumping was only partial.
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Discussion
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Here, we used GluR1-/- mice and in vivo
electroporation technique to rescue the GluR1 protein expression to see
the involvement of this receptor subunit in the neuronal plasticity observed
in morphine tolerance and dependence. For the study of morphine tolerance, we
focused the analgesic activity, a major pharmacological action of
morphine.
The lack of alteration in pain threshold in GluR1-/- mice
(Fig. 1A) is
consistent with other reports, which used different nociception tests
(Minami et al., 1998;
Kiyama et al., 1998;
Kishimoto et al., 2001).
Similar lack of alteration in pain has been also reported with the
GluR4-/- mice (Minami et
al., 1998), although heterozygous mutant mice for the GluR2
subunit showed decrease in the pain threshold
(Wainai et al., 2001).
Therefore, at least GluR1 unlikely plays a major role in the
determination of basal pain threshold. However, because NMDA receptors mediate
a wide range of brain processes, including central sensitization during
persistent pain (Yaksh, 1993;
Woolf and Costigan, 1999), the
role of GluR1 subunit in the NMDA receptor-mediated central
sensitization during persistent pain remains to be determined.
The acute morphine analgesia was slightly potent in
GluR1-/- mice, compared with that in
GluR1+/+ mice
(Fig. 1A-C). These
findings suggest that opioidergic neuron could be connected to the
glutamatergic neurons activating postsynaptic GluR1 NMDA receptor
subunit through unknown inhibitory neuron. Thus, the inhibitory opioid actions
on nociception are partially counteracted by these glutamatergic neurons. This
view could be supported by the reports that NMDA receptor antagonists
potentiate the morphine analgesia (Plesan
et al., 1998; Lutfy et al.,
1999).
Since the first discovery that MK-801, an NMDA receptor antagonist inhibits
the tolerance (Trujillo and Akil,
1991), a number of reports have supported the involvement of NMDA
receptor in the development of morphine analgesic tolerance (for review, see
Trujillo, 2000;
Mao and Mayer, 2001;
Mao, 2002). In the present
study we first observed that GluR1 NMDA receptor subunit is involved in
the morphine analgesic tolerance by use of GluR1-/- mice
(Fig. 1C). As a result
of several attempts to examine biochemical changes (gene and protein
expression and protein phosphorylation) in the brain regions of mice with
chronic morphine treatments, we succeeded in the detection of significant
change in GluR1 protein expression. Among brain regions related to
morphine analgesia and tolerance (including associated tolerance)
(Mitchell et al., 2000;
Nestler, 2001;
Williams et al., 2001),
significant increase in the GluR1 protein expression after chronic
morphine treatments was observed in PAG, VTA, and NAc
(Fig. 2). These findings
suggest that GluR1 NMDA receptor subunit is specifically involved in the
development of morphine analgesic tolerance as an anti-opioid system to
counteract inhibitory morphine actions.
To identify the loci in the brain that are involved in the tolerance
liability of morphine, we attempted to rescue the protein expression in
specific brain regions of GluR1-/- mice. First we confirmed
whether the pathways downstream to NMDA receptor are intact in the
GluR1-/- mice, because the question that always comes up with
a mutant mice is whether there are general organizational changes in the
animals caused by the absence of the protein of interest during development.
The role of NMDA receptors in opioid tolerance is often placed in the context
of an NMDA-NO cascade (Pasternak et al.,
1995; Kolesnikov et al.,
1998). As L-arginine, the substrate for NOS,
accelerated the morphine analgesic tolerance in GluR1-/- mice
as well as in GluR1+/+ mice
(Fig. 3), the pathways
downstream from NMDA receptor were proven to be intact in
GluR1-/- mice.
We performed the in vivo electroporation of GluR1 cDNA to
rescue the protein expression in specific brain regions of
GluR1-/- mice. As immunoblot analyses revealed that the
protein expression lasted for >9 d after the electroporation into PAG
(Fig. 4A), we planned
the experimental designs to complete chronic morphine treatments and
behavioral tests within 9 d after the start of electroporation. Under these
conditions, we performed direct gene transfection into mutant mice to
elucidate the brain locus-specific rescue of morphine analgesia and tolerance.
As shown in Figure 4D,
the electroporation of recombinant GluR1 cDNA into PAG, VTA, or NAc
partially reversed the enhanced acute morphine analgesia in
GluR1-/- mice, suggesting that the anti-opioid system through
GluR1 activation regulates morphine analgesia in all three regions.
However, morphine analgesic tolerance in GluR1-/- mice was
significantly reversed by the electroporation into PAG and VTA, but not NAc.
Thus, it is evident that the GluR1 system in PAG and VTA plays a
critical role in the development of morphine analgesic tolerance. There are
reports that PAG neurons are related to the descending mechanisms of the
pain-inhibitory roles of opioids, whereas dopaminergic neurons in VTA are
related to ascending ones (Franklin,
1998; Millan,
2002).
There is a view that anti-reward systems involving anti-opioidergic
systems, called allostasis, can contribute to motivational changes associated
with chronic administration of drugs of abuse
(Koob and Le Moal, 2001). The
glutamate or NMDA receptor system and some anti-opioid peptidergic systems are
known to be representative of anti-opioid systems
(Harrison et al., 1998;
Trujillo, 2000;
Ueda et al., 2000;
Mao and Mayer, 2001). In the
present study, we observed that naloxone-precipitated withdrawal abstinence
behaviors were significantly attenuated in the GluR1-/- mice
(Fig. 5). These findings
suggest that GluR1 system is also involved in the development of
morphine dependence. To examine the validity of the speculation that the
enhanced GluR1 anti-opioid system is also related to the plasticity in
morphine dependence, C57BL/6J mice were treated with morphine by dependence
paradigm and used for the analysis of GluR1 protein expression. Among
these five brain loci, only NAc showed a significant increase in GluR1
protein levels, whereas amygdala showed a slight decrease
(Fig. 6). It should be noticed
that no significant change was observed in either PAG or VTA, although a
significant increase in GluR1 protein levels was observed in both brain
loci after chronic morphine treatments by tolerance paradigm
(Fig. 2). Such difference may
be explained by the shorter period of morphine pretreatments for dependence
paradigm (4 d), compared with that for tolerance one (6 d). But this
possibility unlikely explains the selective enhancement of GluR1 protein
expression in NAc. Alternatively another feedback mechanism to reset the gene
expression of GluR1 may work through unidentified plasticity in neuronal
networks. Significant, but weak decrease in GluR1 protein expression in
amygdala might be another target related to different behavioral changes such
as reverse tolerance (sensitization) caused by chronic morphine
treatments.
It is interesting to examine whether highly integrated behaviors observed
in morphine withdrawal abstinence could be recovered by locus-specific
expression of GluR1 in the NAc of GluR1-/- mice. In
GluR1-/- mice transfected with GluR1 cDNA, withdrawal
behaviors such as wet-dog shake, paw tremors, and backward locomotion were
significantly recovered, whereas withdrawal jumping behavior was only partial
(Fig. 7B). These
findings suggest that some of withdrawal symptoms are derived from plastic
changes in relatively narrow brain loci. Among these four withdrawal
behaviors, only jumping behavior shows some resistance to the locus-specific
rescue of GluR1 cDNA into NAc (Fig.
7B). This finding may be related to our previous finding
that the intrathecal injection of naloxone more effectively induces withdrawal
jumping in chronic morphine-treated mice than the supraspinal injection
(Ueda et al., 1987).
Because GluR1-/- mice still have some liability to
morphine analgesic tolerance and physical dependence, it is possible that
other anti-opioid systems are also involved in this plasticity. We have
previously proposed that nociceptin/orphanin FQ receptor (NOP) system plays an
important role in the development of morphine analgesic tolerance and physical
dependence by use of specific NOP antagonist and NOP-/- mice (Ueda
et al., 1997,
2000). In this study, the
inhibition of tolerance development is more clearly observed by the
intrathecal injection of NOP antagonist than by the supraspinal injection.
These findings suggest that spinal anti-opioid mechanisms through N/OFQ system
are more likely involved in the plasticity in morphine analgesic tolerance.
Other anti-opioids may be also involved in the development of morphine
tolerance and dependence. Specific cholecystokinin 2 (CCK2) receptor
antagonist blocked the expression of associative morphine analgesic tolerance
(Mitchell et al., 2000),
whereas CCK2 receptor-deficient mice showed greater naloxone-precipitated
withdrawal responses than in wild-type mice
(Pommier et al., 2002). On the
other hand, specific neuropeptide FF antagonist or its antibody increased
morphine analgesia, reversed morphine analgesic tolerance, and attenuated
naloxone-precipitated morphine withdrawal syndromes
(Malin et al., 1990;
Lake et al., 1992). In
addition to these mechanisms, there are reports that some opioid systems
regulate the development of morphine analgesic tolerance in experiments using
mutant mice for delta opioid receptor or preproenkephalin genes
(Zhu et al., 1999;
Nitsche et al., 2002). Thus,
other anti-opioid systems or opioid systems as well as NMDA system may be also
related to the residual mechanisms for the development of morphine tolerance
or dependence.
The present study using in vivo electroporation of specific gene
into mice deleting the gene provides a new strategy to identify the brain loci
involved in drug-induced behaviors of interest. This strategy seems to have
more advantages in the case with pharmacological actions after chronic drug
treatments. To study the brain loci of interest, the microinjection of
antagonists could be another approach. However, in the case with the present
study, long-term infusion of NMDA receptor antagonists should be applied, but
it might be difficult to avoid unexpected side effects of such compounds. In
addition, specific antagonist for GluR1 is not available. Currently the
use of spatial and temporal conditional mutant mice might be a good strategy.
However, available promoters still cover broad brain regions. Therefore, the
strategy of rescue of the receptor by gene transfer into specific loci in
mutant mice looks promising. Adenovirus infection used for this purpose has
some limitations in its efficiency of gene transfer, and higher titers of
adenovirus may cause toxic effects
(Nestler, 2000). Additionally,
the use of lentiviruses highly efficient for transfection into neurons has
limitations in experimental conditions to avoid infection in humans.
Furthermore, the size of the DNA needs to be appropriately small for
construction into such viral vectors. Thus, the direct gene transfection into
mutant mice by in vivo electroporation would be convenient and useful
strategy to elucidate the brain locus-specific recovery of morphine tolerance
and dependence.
In summary, we have found, by use of mutant mice deleting specific gene and
in vivo electroporation technique that GluR1 NMDA receptor in
PAG and VTA plays a critical role in the development of analgesic tolerance,
and the receptor in NAc plays a specific role in the development of physical
dependence to chronic morphine. This approach using the locus-specific rescue
of gene expression may further clarify the neuronal plasticity, which modifies
psychological dependence and sensitization after chronic morphine
treatments.
|
Footnotes
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|---|
Received Feb. 24, 2003;
revised May. 13, 2003;
accepted May. 20, 2003.
Parts of this study were supported by Special Coordination Funds of the
Science and Technology Agency of the Japanese Government, Human Frontier
Science Program and Grants-in-Aid from the Ministry of Education, Science,
Culture, and Sports of Japan. We thank Akira Yoshida, Toshiko Kawashima, and
Md. Harunor Rashid for technical help. We also thank Dr. Bert Vogelstein
(Howard Hughes Medical Institute and Johns Hopkins Oncology Center) for the
generous gift of pAdTrack-CMV vector.
Correspondence should be addressed to Dr. Hiroshi Ueda, Division of
Molecular Pharmacology and Neuroscience, Nagasaki University Graduate School
of Biomedical Sciences, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan. E-mail:
ueda{at}net.nagasaki-u.ac.jp.
Copyright © 2003 Society for Neuroscience
0270-6474/03/236529-08$15.00/0
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Top
Abstract
Introduction
