Previous Article | Next Article 
The Journal of Neuroscience, June 15, 2001, 21(12):4451-4459
Morphine-Induced Dependence and Sensitization Are Altered in Mice
Deficient in AMPA-Type Glutamate Receptor-A Subunits
Olga Y.
Vekovischeva1, 2,
Daniel
Zamanillo3,
Oxana
Echenko1,
Timo
Seppälä4,
Mikko
Uusi-Oukari1,
Aapo
Honkanen1,
Peter H.
Seeburg3,
Rolf
Sprengel3, and
Esa R.
Korpi1
1 Department of Pharmacology and Clinical Pharmacology,
University of Turku, FIN-20520 Turku, Finland,
2 International Graduate School in Neurosciences,
University of Tampere Medical School, FIN-33101 Tampere, Finland,
3 Department of Molecular Neurobiology, Max-Planck
Institute for Medical Research, D-69120 Heidelberg, Germany, and
4 Laboratory of Substance Abuse, National Public Health
Institute, FIN-00300 Helsinki, Finland
 |
ABSTRACT |
AMPA-type glutamate receptors have been suggested to be involved in
the neurobiological mechanisms of drug addiction. We have made use of
two mouse lines, which both have modulated AMPA receptor responses. The
first line is entirely deficient in glutamate receptor-A (GluR-A) subunits (A
/ knock-out line) and, in the second
one, the Q582 residue of GluR-A subunits is replaced by an arginine residue (R/R mutants), which reduces the calcium permeability and
channel conductance of the receptors containing this mutated subunit.
Mice of both lines are healthy, but they show slightly increased
locomotor activity. Acute morphine administration enhanced locomotor
activity of the GluR-A/ and GluR-A(R/R) mice, at least as much as
that of their wild-type littermates. Only in the GluR-A/ mice did
we observe reduced tolerance development in tail-flick antinociception
and less severe naloxone-precipitated withdrawal symptoms after
treatment with increasing morphine doses, without differences in plasma
and brain morphine levels when compared with wild type. Repeated daily
morphine administration sensitized the locomotor activity responses in
the GluR-A/ and GluR-A(R/R) mice only when given in the measuring
cages, whereas the wild-type mice showed slightly increased responses
also when the repeated treatment was given in their home cages. Normal
or even enhanced context-dependent sensitization was observed also with
repeated amphetamine administration in the GluR-A subunit-deficient
mice. The results indicate that AMPA receptors are involved in the
acute and chronic effects of morphine, including context-independent sensitization, and that the GluR-A subunit itself is important for
morphine tolerance and dependence.
Key words:
neurobiology of addiction; glutamate receptors; AMPA receptors; transgenic mouse lines; morphine; amphetamine
| |
INTRODUCTION |
Addiction to chemical substances
develops in a set of molecular events in the brain. First, all
addictive substances act on primary molecular targets, some of them
being already well characterized (Rodriguez de Fonseca and Navarro,
1998
; Self, 1998; Self and Nestler, 1998; Merlo Pich et al., 1999).
Opiates require specific G-protein-coupled receptors for their
efficacy, e.g., morphine is acting primarily on µ-opioid receptors
(Matthes et al., 1996). Second, the mesolimbic dopaminergic system has
been suggested to be a pathway, which is activated by most addictive
drugs (Bardo, 1998; Koob et al., 1998; Everitt et al., 1999; Leshner
and Koob, 1999). Third, repeated usage of addictive drugs, producing,
for example, behavioral sensitization, leads to multiple adaptive neuronal responses, some of these adaptations including permanent changes in synaptic structures (Robinson and Kolb, 1997, 1999). The
adaptive responses require altered gene expression (Koob et al., 1998;
Berke and Hyman, 2000; Nestler, 2000). Some of these adaptive
processes may thus serve as acquired molecular twitches, making a
subject prone to develop dependence to chemical substances on repeated usage.
One interesting candidate protein that has been shown to be upregulated
in the brains of animals addicted to morphine and other drugs of abuse
is a subunit of AMPA-type glutamate receptors, namely GluR-A subunit
(Fitzgerald et al., 1996). Drug-induced changes in GluR-A subunit in
various brain regions have been observed at both protein and mRNA
levels (Lu et al., 1997; Churchill et al., 1999; Lu and Wolf, 1999;
Jang et al., 2000), indicating that GluR-A subunit-containing AMPA
receptors might be involved in the activation of brain pathways
associated with a drug-sensitized state. Importantly, Carlezon et al.
(1997) have produced evidence that viral-mediated upregulation of
GluR-A subunit levels in the ventral tegmental area, the origin of
mesolimbic dopaminergic pathway, is enough to mimic the
morphine-sensitized state in rats. This suggests a causal relationship
between the effects of drugs of abuse and behavioral effects via GluR-A
subunit-containing AMPA receptors (Carlezon et al., 1997).
To provide additional genetic evidence of the involvement of AMPA
receptors in drug addiction, we investigated mice of two lines, both of
which have altered AMPA receptor responses. Both lines were generated
by gene-targeted modification of the mouse GRIA-1 gene. In
mice of the GluR-A/ line, the GluR-A subunit is not expressed at
all, whereas mice of GluR-A(R/R) line express the mutated version of
GluR-A with glutamine (Q582) replaced by an arginine (R). In principal
neurons of the hippocampus, most AMPA receptor channels are now formed
by subunits containing R582, and those channels are known to have very
low single-channel conductance (Burnashev et al., 1992; Swanson et al.,
1997). In interneurons, which lack the GluR-B subunits, we expect no
reduction of AMPA currents but a shift to less
Ca2+-permeable channels. We found that
these novel mouse models do show responses to morphine and
D-amphetamine, although in a different manner
from their wild-type littermate controls, which clarifies the role of
AMPA receptors in the effects of and in the neuronal adaptation to
drugs of abuse.
| |
MATERIALS AND METHODS |
Generation of GluR-A/ and GluR-A(R/R) mice.
GluR-A/ mice were generated as described previously (Zamanillo et
al., 1999). GluR-A(R/R) mice were created by targeted GluR-A
mutagenesis in R1 mouse embryonic stem (ES) cells (Nagy et al.,
1993). The targeting vector pFCII-R contained 10.7 kb of the murine
GluR-A gene (GRIA1) with parts of introns 10 and 11 and
mutated exon 11, numbered according to the GluR-B gene (Kohler
et al., 1994) (Fig. 1). The Q582 to R582
mutation (CAG to CGG) was introduced by PCR in an exon 11-containing
plasmid. An NsiI-BglII wild-type fragment from the construct was substituted by an NsiI-BglII
segment encoding the CAG to CGG mutation. The neo selection
marker ploxPneo3 (Single et al., 2000) was inserted into an
EcoNI site, 700 bp into intron 11. In addition, in
225 nucleotides upstream of exon 10, a 28 bp PflMI fragment was
substituted by a 34 bp loxP site. ES cells (R1) were electroporated
with the targeting vector and linearized at the unique
HindIII site in the polylinker, and five positive clones
were identified by PCR with primers P1 and P2 (Zamanillo et al., 1999)
and confirmed by Southern blotting with a 162 bp SacI
fragment of intron 10 as 5' outside probe. One positive ES cell
clone was transfected with a Cre recombinase expression vector (pMC-Cre) (Gu et al., 1993), and clones with two loxP sites and without
neo selection marker in the modified GluR-A allele were identified by PCR with primer pair int5' (GGT AAT ATG CTG TCA AGA TGC)
and loxP0 (ACT CGA GGG ACC TAA TAA CTT CG). The positive clones were
confirmed by using a second primer set int5' and P3 (Zamanillo et al.,
1999). This primer pair identified the mutated and wild-type allele (a
60 nucleotide shorter product) by PCR. Cre-mediated deletion of the
neo gene was confirmed by Southern blotting using the 162 bp
SacI fragment described above. Subclone 57.4 was injected
into C57BL/6 blastocysts, chimeric animals were backcrossed to C57BL/6,
and intercrosses produced 25% GluR-A(R/R) mice.
View larger version (42K):
[in this window]
[in a new window]
|
Figure 1.
Generation of GluR-A(R/R) mice. A,
Representation of the GluR-A subunit and of GluR-A+,
GluR-A(Rneo), GluR-A(R), and GluR-A gene fragments.
Black boxes represent putative membrane segments M1 to
M4. For the gene segment, open boxes represent exonic
sequences and the PGK-NEO gene (neo). loxP sites are
shown by triangles. Two filled circles in
GluR-Aneo indicate 5' and 3' ends of
the targeting construct. Positions of BamHI
(B) and HindIII
(H) restriction enzyme recognition sites
are given. The 260 bp genomic SacI fragment used as
probe for the genomic Southern blot is depicted as a
black bar. P1 and P2
arrows represent primers used for screening of the targeted
allele. ES cell clones were confirmed by using the primer set int5' and
P3. B, Southern blot analysis of
BamHI-digested genomic ES cell DNA derived from ES cell
colonies, which were injected into blastocysts. The GluR-A+ and
GluR-A(R) alleles are visualized as 7.2 kb, the targeted
GluR-A(Rneo) as 9.2 kb, and
the GluR-A allele as 5.7 kb fragments, respectively.
Positions of the molecular weight marker in kilobase pairs are
given on the left.
C, RT-PCR on total brain mRNA
derived from mice with genotypes as indicated using primers exon9 and
exon12EcoRI to amplify a 800 bp fragment. By
BbvI digestion (b), GluR-A and
GluR(R) mRNA derived fragments of 471, 120, 106, and 103 bp and of 577, 120, and 103 bp can be visualized by ethidium bromide staining in a 1%
agarose gel. Size markers in kilobases are given on the
left. D, Immunoblot analysis
demonstrating the expression of the GluR-A(R) subunit in brains of
GluR-A(R/R) mice. Membrane proteins of forebrain from genotypes as
indicated were separated in 8% SDS-polyacrylamide gel and visualized
by anti-GluR-A antibody. The molecular weight marker in kilodaltons is
given on the left.
|
|
Homozygous GluR-A(R/R) showed normal expression levels of the mutated
GluR-A(R) subunit in the forebrain, whereas the GluR-A/ animals
lacked the subunit protein (Fig. 1). However, similar to the
GluR-A/ animals (Zamanillo et al., 1999), their AMPA receptor-mediated currents in somata of CA1 pyramidal cells were at
least 10-fold reduced (T. Borchardt and R. Sprengel, unpublished observations).
The mouse lines were constructed and backcrossed three times with
C57BL/6 mice in Germany, and breeding pairs were transferred to Turku,
Finland. The experimental animals were produced by heterozygous mating
from the F5-7 generations. The genotype of the
animals was checked out before behavioral experiments by PCR analysis of tail-tip DNA (Brusa et al., 1995). Primer pair MH60 (CAC TCA CAG CAA
TGA AGC AGG AC) and P3 amplified a 215 bp fragment from the wild-type
allele and a 250 bp fragment from the GluR-A(R) allele. Primer
combination 1005 (AAT GCC TAG TAC TAT AGT GCA CG) and P3 was used to
get a 1500 bp fragment from the wild-type allele and a 275 bp fragment
from the GluR-A allele.
Animal care was in compliance with the institutional guidelines at the
animal facility of the Center for Molecular Biology (Heidelberg,
Germany). Transgenic manipulations were performed according to a
license (37-9185.81/35/97) of the Regierungspräsidium (Karlsruhe, Germany).
In Turku, adult mice were maintained in individual polypropylene cages
(20 × 10 × 15 cm) in a facility artificially illuminated from 7:00 A.M. to 7:00 P.M. with air conditioning (21 ± 1 oC) and relative humidity of 50-60%.
Tap water and rodent pellets (Special Diet Service; Witham, Essex, UK)
were available ad libitum. All experimental protocols were
approved by the Institutional Animal Care and Use Committee of the
University of Turku. In most experiments, both male (weight of 28-38
gm) and female (weight of 20-28 gm) animals were used from all lines
because, in preliminary experiments, we failed to detect any gender
differences in locomotor activity or nociception (see Results).
Gene expression analysis. Reverse transcription (RT)-PCR
amplification of GluR-A mRNA sequences was performed with primers exon9
(GTC CAG AAT AGA ACC TAC ATC G) and exon12EcoRI (GTG
AAT TCT GCT TCC AAT GTG CCA TAA GC) to amplify a 800 bp DNA fragment. The origin of PCR fragments was determined by BbvI
restriction enzyme digestion, which produced 471, 120, 106, and 103 bp
fragments for wild type, and 577, 120, and 103 bp fragments for mRNA
derived from the mutated GluR-A(R) allele. For quantitative analysis, pictures of ethidium bromide-stained gels were digitized by scanning (Gt 9600; Epson, Long Beach, CA), and the densiometric analysis was performed by Image Gauge version 3.0 (Fujifilm, Tokyo, Japan). It
revealed 42% GluR-A(R) mutant mRNA expression relative to total GluR-A
mRNA, instead of the expected 50%. The slightly reduced expression
level of the mutant allele is probably attributable to the
insertion of loxP sites in intron 10 and 11.
Immunoblotting. Isolated hippocampi were homogenized in 0.32 M sucrose and 5 mM HEPES,
pH 7.4, containing a cocktail of protease inhibitors (Complete;
Boehringer Mannheim, Mannheim, Germany), and centrifuged for 15 min at
3000 × g. The supernatant was subsequently centrifuged
for 30 min at 10,000 × g. The pellet was resuspended in 1% SDS, and the amount of total protein was determined (Pierce, Rockford, IL). For each sample, 20 µg of total membrane protein was
resolved on an 8% SDS-polyacrylamide gel, and the separated proteins
were transferred to nitrocellulose membranes. Blots were probed with
anti-GluR-A polyclonal antibody at 1 µg/ml (Chemicon, Temecula, CA),
followed by horseradish peroxidase-linked anti-rabbit secondary
antibodies. The enhanced chemiluminescence method (ECL; Amersham
Pharmacia Biotech, Buckingamshire, UK) was used to detect GluR-A. To verify that equal amounts of protein were loaded in each
lane, the lower part of the SDS-polyacrylamide gel was cut off before
semidry electroblotting and Coomassie blue-stained, and then the
image was densitometrically analyzed (NIH Image version 1.56; data not shown).
Determination of locomotor activity. The horizontal
locomotor activity (ambulation) of the mice was measured in animals
placed in transparent 25 × 42 × 19 cm plastic cages
equipped with computer-controlled photocells automatically monitoring
the movements (Photobeam Activity System; San Diego Instruments, San
Diego, CA). The cages were arranged on the shelves and illuminated by
artificial lights with an average intensity of 290 lux. Locomotor
activity was recorded for 5 min periods (time course) and then
calculated for the total test time (total locomotor activity, usually
for 120 min).
Determination of tail-flick latencies. The mouse was lightly
restrained, and its tail (2 cm from the base) was exposed to a focused
infrared heat beam (intensity set to 50; Ugo Basile, Comerio,
Italy). The animal could voluntarily terminate the noxious stimulation
by flicking its tail, and the latency to this reaction was then
recorded. An animal that failed to respond in 10 sec (cutoff time) was
removed from the apparatus and assigned the latency of 10 sec. On the
test day (the first and ninth day of chronic morphine treatment),
baseline tail-flick latencies were first measured twice for each
animal. Saline (10 ml/kg, s.c.) or morphine (5 mg/kg) was injected
after the baseline measurements, and the mice were retested 30 and 60 min after the injections in two trials for tail-flick latencies.
Tail-flick latencies were converted to percentage of the maximal
possible effect (MPE). The individual antinociceptive values (%MPE)
were calculated according to the formula:
[(Texp TBL) × 100]/(10 TBL), where
Texp is the tail-flick latency and
TBL the baseline latency.
Determination of withdrawal signs. Naloxone-precipitated
opioid withdrawal symptoms were determined using a cylinder test. The
observations were performed in six identical glass cylinders (diameter
of 20 cm; height of 40 cm) by video recording. The behavioral parameters (number of jumps, digging, stretches, "wet dog"-like shakes, forepaw tremor, forepaw treading, head twitch, writhing and the
presence of ptosis, piloerection, and diarrhea) were monitored from the
video recordings for 15-25 min after naloxone injection. The body
weights were measured before the naloxone injection, and 20 and 60 min
after it. One score point was given of each parameter, and the total
number of score points was calculated for each mouse (Malin et al.,
1992). Withdrawal-induced changes in the total scores of the
morphine-treated mice were compared with naloxone-induced behavioral
and somatic symptoms in saline-treated control mice.
Morphine treatment regimens. Acute sensitivity of each mouse
line to morphine-induced locomotion was assessed in locomotor cages
using animals that had been habituated to the measuring cage for 2 hr/d
on 3 preceding days. Animals were injected subcutaneously with
saline (10 ml/kg) and 3, 10, and 30 mg/kg morphine hydrochloride (European Pharmacopoeia), and the locomotor activity was
recorded immediately for 2 hr. A 1 d interval was kept between the
increasing doses.
Sensitization to repeated morphine was assessed in the following manner
to find out the dependency on the environmental context of the
treatment. First, all experimentally naïve animals were adapted
to locomotor boxes for 2 hr/d on 3 preceding days. Then, all animals
received 3 mg/kg subcutaneous morphine and, immediately after the
injection, their locomotor activities were monitored for 2 hr (test
1). This locomotor activity reflected the initial sensitivity to
a low morphine dose, which afterward was compared with the activity
induced by the same morphine dose after repeated morphine treatment.
Repeated morphine treatment was performed using the following regimen.
One day after test 1, the animals were randomly divided into paired
(P), unpaired (UP), and control (C) groups. Each group received
two injections per day with 10 hr intervals. For the P group, the first
injection between 9:00 and 10:00 A.M. (10 mg/kg morphine) was
given in locomotor cages, in which the mice stayed for 2 hr after the
injection, and after the second (saline) injection these animals
remained in their home cages. The UP group received the injections on
the opposite schedule. Morphine was given in home cage, whereas saline
injection was followed by 2 hr in the locomotor cages. The C group
received saline injections in both home cages and locomotor cages.
Finally, retests of 3 mg/kg morphine-induced activity were performed at 24 hr (test 2) and 5 d (test 3) after the last injection of day 6 as described for test 1.
Development of tolerance and dependence to morphine was assessed during
a 9 d regimen (Belozertseva et al., 1996). On day 1, the mice were
randomly divided into two treatment groups (morphine and saline
groups). Before the drug treatment, all mice were tested with a
tail-flick test for the sensitivity to analgesic action of morphine (5 mg/kg). Ten hours after this test, the morphine group received morphine
(10 mg/kg, s.c.), and the saline group received the equivalent volume
of saline (10 ml/kg). During the next 7 d, the morphine group
received twice daily at 9:00 A.M. and 6:00 P.M. increasing subcutaneous
doses of morphine according to the following schedule (in mg/kg): day
2, 20; day 3, 30; day 4, 40; day 5, 50; day 6, 60; day 7, 70; and day
8, 80. The control group received saline at the same schedule. Body
weights were determined every second day. The first injection of
morphine on day 9 was 5 mg/kg, and the tail-flick test was performed
after 30 and 60 min to assess the development of tolerance. Immediately after the test, the morphine group received additionally 95 mg/kg morphine to reach the cumulative dose of 100 mg/kg. The second injection on day 9 was omitted. On day 10 (24 hr after the last dose of
morphine), all groups were treated with naloxone hydrochloride (0.4 mg/kg, s.c.; Research Biochemicals, Natick, MA) to determine the
severity of morphine withdrawal symptoms as described above.
The development of morphine dependence was repeated with GluR-A/
mutants and their littermates by using a slightly modified procedure
(Matthes et al., 1996). During 6 d, the morphine groups of both
lines received subcutaneous doses of morphine twice daily as follows
(in mg/kg): day 1, 20; day 2, 40; day 3, 60; day 4, 80; day 5, 100; and
day 6, only a morning dose of morphine (100 mg/kg) was given. Control
groups were treated with saline under the same conditions. The
withdrawal symptoms were precipitated to all mice 2 hr after the last
morphine or saline injection by naloxone (1 mg/kg, s.c.) and evaluated
for a period of 25 min as described above.
Amphetamine and dizocilpine administrations. On 3 consecutive days, the mice were adapted to locomotor boxes for 2 hr/d.
For drug treatment, the mice of each line were divided into two groups, which received subcutaneously either saline (10 ml/kg) or
D-amphetamine sulfate (2 mg/kg; Sigma, St. Louis,
MO) daily. After the injection, half of the animals were immediately
tested in familiar locomotor boxes (paired groups), whereas the others
remained as unpaired controls being transferred to their home cages
after the injection. The treatment was repeated on the following 4 d. The test for sensitization to amphetamine-induced locomotor activity
was performed 5 d after the last injection. The
amphetamine-treated groups received amphetamine (0.5 mg/kg). The saline
groups of each line were divided into two groups that were treated with
either saline or amphetamine (0.5 mg/kg). GluR-A(R/R) mice were studied
only in the paired protocol.
Adaptation of the mice to locomotor boxes was performed for 1 hr on the
day before the experiment began and before each drug session. Acute
effect of (+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d]
cyclohepten-5,10-imine maleate (dizocilpine; MK-801) (Sigma) on
locomotor activity of knock-out mice and their littermates was tested
by Latin square design using the following intraperitoneal doses: 0.1, 0.3, 0.5, and 1.0 mg/kg.
Determination of morphine concentrations in plasma and
brain. Male GluR-A/ mice and their controls were injected with
10 mg/kg morphine. The mice (n = 7-8 per group) were
decapitated 30 or 90 min later, and brains were dissected and rapidly
frozen to dry ice. Trunk blood was collected into heparinized tubes and centrifuged. Brain tissues and plasma were stored at 20°C until assayed. Before analyses, brains were homogenized mechanically (Ultra-Turrax; Janke & Kunkel GmbH, Staufen, Germany) in 0.1 M perchloric acid (Merck, Darmstadt, Germany) and
added up to a total of 4 gm. Morphine in rat plasma and brain were
determined by an HPLC method using an electrochemical detection
(Zharkovsky et al., 1999).
Statistical analyses. Statistical analysis was conducted
using SAS-STAT software (release 6.11; SAS Institute, Cary, NC). Analysis of the descriptive statistics procedure by the SAS-STAT Univariate procedure demonstrated that some of the data were not distributed normally (Wilks-Shapiro's test). The distribution-free three-factorial ANOVA was conducted using a combination of the rank and general linear model (GLM) procedures. Repeated-measures design was applied when necessary. Briefly, data were ranked, and the
ranks were later subjected to ANOVA (GLM procedure for unbalanced
design with unequal group size). For conditioned sensitization experiment, the factors were as follows: 1, strain (knock-outs or their
wild-type littermates); 2, treatment (levels of paired, unpaired, or
control); and 3, test day (test 1 or test 2, test 1 or test 3). For
tolerance and dependence experiment, the factors were as follows: 1, strain; 2, treatment (morphine or saline); and 3, test day (before the
chronic administration or after it). For analysis of acute locomotor
effect by various drugs, two-factorial analyses were used, with the
following factors: 1, strain; and 2, dose. Duncan's or Dunnett's
tests were conducted for post hoc analyses of between-group
comparisons (only when ANOVA revealed significant main effects). Null
hypothesis was rejected at the p < 0.05 level.
| |
RESULTS |
GluR-A subunit-deficient mice have increased locomotor activity in
a novel environment
In agreement with Zamanillo et al. (1999), GluR-A/ mice were
slightly hyperactive when placed in a novel environment of locomotor
cages compared with their littermate, wild-type controls (F(1,79) = 53; p < 0.0001) (Fig. 2). When the measurement
was repeated daily, both knock-out and wild-type mice showed clear habituation, but a slight difference in ambulation still prevailed. The
mice with genetically impaired GluR-A subunits (R/R mutants) were also
slightly hyperactive compared with their littermates (F(1,64) = 25; p < 0.0001) (Fig. 2), and, also in these mice, habituation to repeated
exposure to novel cages was detected.
View larger version (32K):
[in this window]
[in a new window]
|
Figure 2.
Spontaneous horizontal locomotor activity in a
novel cage (first trial, A and C) and
habituation to repeated exposures (third trial, B and
D) to the same environment in drug and handling
naïve GluR-A subunit-deficient mouse lines and their wild-type
controls. The mouse line codes (with the number of animals) are shown.
Both mutant lines had higher activity than the wild-type controls,
which can be seen in E, depicting the total activity
scores. Repeated trials demonstrated that all of the lines habituated
to the test environment, but still on the third trial the mutants
exhibited higher activity than their controls. Points
and bars indicate means ± SEM.
*p < 0.05 for the difference from the
corresponding activity of the control line; #p < 0.05 for the difference between the trials within a line; Duncan's
test.
|
|
In both line pairs, the locomotor activity difference was present in
both sexes [the gender factor was not significant for the locomotor
activity counts: GluR-A/ mutants and their littermates, F(1,79) = 0.38, NS; GluR-A(R/R)
mutants and their littermates, F(1,64) = 2.2, NS]. In all following experiments, except for using only males
in the determination of morphine pharmacokinetics and the effects of
MK-801 (see below), approximately equal number of males and females
were used. No significant gender effects were detected in any of the
experiments (statistics not shown).
Morphine-induced locomotor activity is slightly enhanced in
GluR-A/ mice
Acute injection of morphine dose dependently increased locomotor
activity in habituated animals of all mouse lines (GluR-A/ and
controls, F(3,120) = 35, p < 0.0001; GluR-A(R/R) mutants and controls,
F(3,64) = 78, p < 0.0001) (Fig. 3). The responses of GluR-A/ mice to morphine (F(3,120) = 15; p < 0.001), but not those of GluR-A(R/R) mutants
(F(3,64) = 1.2; p > 0.05), were significantly bigger than those of the wild type. However,
the baseline values of the GluR-A/ mice were higher than those of
their controls (Dunnett's test; p < 0.05), and no
interaction between the line and treatment factors was found
(F(3,120) = 1.35; p > 0.05).
View larger version (32K):
[in this window]
[in a new window]
|
Figure 3.
Dose dependency of acute subcutaneous morphine
injection on horizontal locomotor activity of GluR-A/ and wild-type
control mice (A) and on that of GluR-A(R/R) and
wild-type control mice (B). Number of animals is
given in the figure, in which the bars indicate means ± SEM for total activities for 2 hr. All measurements were performed
after three habituation trials. *p < 0.05 for the
difference from the corresponding saline-treated
(Sal) group; Dunnett's test.
#p < 0.05 for the difference from the value for
the corresponding control line; Duncan's test.
|
|
Reduced morphine tolerance and withdrawal in GluR-A/ mice
To test the development of morphine tolerance, the mice were first
tested for the antinociceptive action of 5 mg/kg morphine using the
tail-flick latency test, a reproducible and robust test for morphine
efficacy that has been demonstrated to be affected by AMPA receptor
antagonists in chronic studies (Kest et al., 1997). The baseline
latencies were similar in all mouse lines (Fig.
4A), except for
slightly longer latencies of the GluR-A/ mice in the first trial.
The test dose of morphine was antinociceptive in all animals. Repeated
treatment with escalating doses of morphine produced significant
tolerance to the antinociceptive effect of morphine in all mouse lines,
except for the GluR-A/ mice, in which morphine still produced a
clear antinociception (F(1,84) = 4.17, p < 0.05 and F(1,84) = 4.15, p < 0.05 for the interaction between
GluR-A/ and control mice and drug treatment at 30 and 60 min tests,
respectively) (Fig. 4B,C).
View larger version (30K):
[in this window]
[in a new window]
|
Figure 4.
Tail-flick latencies in GluR-A subunit-deficient
mouse lines and their wild-type controls in drug-free
(A) and morphine-affected states
(B, C). A, Tail-flick
latencies of the initial baseline trial and the trials 30 and 60 min
after the injection of saline (Sal; 10 ml/kg, s.c.).
B, C, Percentage of the maximal possible
effect (%MPE) at 30 and 60 min after saline or morphine
(Mor; 5 mg/kg) injections in GluR-A/ and wild-type
control mice (B) and in GluR-A(R/R) and their
control mice (C) before the chronic treatment and
15 hr after the last injection of 8 d of chronic morphine
treatment with escalating doses (Chr). The
bars indicate means ± SEM. *p < 0.05 for the difference from the corresponding acute morphine value;
#p < 0.05 for the difference from the
corresponding control mouse line; Duncan's test.
|
|
Thereafter, the same animals received naloxone (0.4 mg/kg) to
precipitate opioid withdrawal symptoms, which were scored for their
severity and frequency. In saline-treated animals, naloxone did not
induce any symptoms. All morphine-treated animals showed some signs of
withdrawal (Fig. 5A), but in
GluR-A/ mice, the total scores were significantly lower than in
wild-type littermates (F(1,22) = 6.13;
p < 0.05). There was no difference between the GluR-A(R/R) mutants and their wild types
(F(1,24) = 0.63; NS). In another
dependence experiment using slightly different morphine and naloxone
doses, ANOVA detected an interaction between the treatment (saline vs
morphine) and mouse line (GluR-A/ mice vs littermates) in the
withdrawal scores (F(1,27) = 6.08;
p < 0.05). The GluR-A/ mice had a lower number of
naloxone-precipitated symptoms than their littermates after chronic
morphine, but not after saline, treatment (Fig. 5B). The
withdrawal scores for mutants and wild types were higher in this second
experiment than in the first one (Fig. 5A), but they were
less severe because no weight loss or diarrhea were detected in any of
the mice.
View larger version (27K):
[in this window]
[in a new window]
|
Figure 5.
Naloxone-precipitated withdrawal symptoms after
chronic morphine administration. A, GluR-A/ and
GluR-A(R/R) mutant mice and their wild-type controls were treated with
escalating morphine doses for 9 d, and withdrawal symptoms were
precipitated 24 hr after the last injection by 0.4 mg/kg naloxone. The
bars indicate mean ± SEM withdrawal scores
(n = 11-12) summed from the number of appearance
of the following symptoms within 15 min of the naloxone injection:
forepaw tremor, head twitch, writhe, diarrhea, lying, and forepaw
treading. #p < 0.05 for the difference from the
corresponding control mouse line; Duncan's test. Naloxone at this dose
after saline treatment failed to induce any of these symptoms in any of
the mouse lines (data not shown). B, GluR-A/ mice
and their wild-type controls were treated with escalating morphine
(Mor) doses for 6 d, and withdrawal symptoms were
precipitated 2 hr after the last injection by 1.0 mg/kg naloxone.
Saline controls (Sal) were treated similarly. The
bars indicate mean ± SEM withdrawal scores
(n = 5-9) summed from the number of appearance of
the following symptoms within 25 min of the naloxone injection: jumps,
digging, forepaw tremor, head twitch, writhe, and lying.
*p < 0.05 for the difference from the
corresponding saline value; Dunnett's test. #p < 0.05 for the difference from the corresponding control mouse line;
Duncan's test.
|
|
The blood and brain concentrations of morphine were determined with
HPLC in GluR-A/ mice and wild-type littermates after a single
morphine injection (10 mg/kg, s.c.). There were no differences in the
morphine levels in brain and blood plasma samples
(F(1,29) < 2.1, NS) between the
genotypes, suggesting similar absorption and elimination of brain
morphine (Table 1). This indicates that the differential behavioral responses to morphine were attributable to
factors other than pharmacokinetic ones.
View this table:
[in this window]
[in a new window]
|
Table 1.
Mean ± SEM concentrations of morphine in plasma
(ng/ml) and brain (ng/gm) of GluR-A/ mice and their wild-type (WT)
controls 30 and 90 min after subcutaneous injection of 10 mg/kg
morphine (n = 7-8)
|
|
Context-dependent sensitization to morphine is retained in the
GluR-A subunit-deficient mice
To find out whether the environment in which the repeated
treatment with a moderate dose of morphine was performed affected the
responses of the mouse lines, we first tested all mice with a low dose
of morphine (3 mg/kg, s.c.). Then they received five daily injections
of morphine (10 mg/kg) in either the home (unpaired) or locomotor
activity (paired) cages, before being retested with 3 mg/kg morphine. A
saline control group was also included. The alterations in ambulatory
activity at the retest (test 1 vs test 2) was dependent on mouse line
(GluR-A/ mutants and their controls, F(1,158) = 6.76, p < 0.05; GluR-A(R/R) mutants and their controls, F(1,128) = 4.23, p < 0.05) and the treatment group (GluR-A/ mutants and their controls,
F(2,158) = 4.36, p < 0.05; GluR-A(R/R) mutants and their controls,
F(2,128) = 5.31, p < 0.01). The results show that, in the GluR-A/ and GluR-A(R/R)
mutants, morphine-induced sensitization took place effectively only in
the P group, which had repeated morphine injections in the same
environment as the final testing was performed (Fig.
6). These effects became clearer 5 d
after the repeated dosing (Fig. 6, Test3). The littermate controls showed increased activity in response to morphine injection independent of the environment of repeated morphine injections when
compared with the corresponding saline group. There was a small, but
significant sensitization to morphine test dose in the unpaired control
groups only when both wild-type groups were combined
(p < 0.05 between test 1 and test 3).
View larger version (51K):
[in this window]
[in a new window]
|
Figure 6.
Effects of 6 d repeated morphine (10 mg/kg,
s.c.) treatment on horizontal locomotor activity of GluR-A/
(B) and GluR-A(R/R) (D)
mutant mice and their wild-type controls (A,
C, respectively). The bars indicate
means ± SEM (n = 10-14) for ambulatory
activity counts after a morphine test dose of 3 mg/kg (subcutaneous),
which normally induces no clear locomotor stimulation. The tests were
performed first with naïve but habituated animals
(Test1), then 24 hr (Test2), and then
5 d after the last morphine dose (Test3). In each
mouse line, the treatment groups consisted of the control
saline-treated group (injected twice per day with saline, once in home
cages and once in locomotor activity cages), the unpaired morphine
group (saline in activity cages and morphine in home cages), and the
paired morphine group (morphine in activity cages and saline in home
cages). *p < 0.05 for the difference from the
corresponding saline control group; #p < 0.05 for
the difference from the corresponding activity counts in test 1;
Dunnett's test.
|
|
Context-dependent sensitization to D-amphetamine is
enhanced in GluR-A/ mice
To test whether stimulants affect differently the locomotor
activity in GluR-A/ mice and their littermates, we used repeated administration of D-amphetamine in context-dependent and
context-independent conditions. ANOVA indicated significant mouse line
(F(1,790) = 6.81; p < 0.05) and treatment (F(3,79) = 12.21;
p < 0.05) effects (Fig.
7), with
D-amphetamine inducing a greater
environment-dependent sensitization of locomotor activity in
GluR-A/ mice than in wild-type littermates
(p < 0.05). No significant
D-amphetamine-induced context-independent
sensitization was observed in the GluR-A/ or littermate controls. A
tendency for increased context-dependent sensitization was also
observed in the GluR-A(R/R) mutants (tested only in the paired
protocol; data not shown), because the increase in amphetamine-induced
activity after repeated amphetamine administration was significant only
in the GluR-A(R/R) mutants (p < 0.05). These results suggest that the GluR-A mutant mice display strong
context-dependent sensitization to stimulants, as they do to
morphine.
View larger version (23K):
[in this window]
[in a new window]
|
Figure 7.
The effect of repeated D-amphetamine
administration on amphetamine-induced locomotor activity of GluR-A/
mutant mice and their wild-type (WT) controls.
The mice were injected with 2 mg/kg (subcutaneous) amphetamine or 10 ml/kg saline for 5 d in either locomotor activity cages
(Paired) or home cages (Unpaired). After
5 d, immediately before being transferred into locomotor activity
cages, the saline-treated animals received either saline injection
(Saline) or 0.5 mg/kg amphetamine
(Control), and the amphetamine-treated animals
received 0.5 mg/kg amphetamine. The bars indicate
mean ± SEM (n = 12-16) horizontal activity
counts for 2 hr sessions. *p < 0.05 for the
difference from the corresponding saline and control group;
#p < 0.05 for the difference from the
corresponding control mouse line; Duncan's test.
|
|
NMDA receptor-uncompetitive blockers induce a high locomotor activity
(Ginski and Witkin, 1994; Velardo et al., 1998; Sukhotina et al.,
1999). We wanted to find out whether the specific NMDA receptor-uncompetitive antagonist MK-801 affects differentially the
mutant mouse lines and their control lines. However, the dose-dependent increase of ambulatory activity by MK-801 was similar in both line
pairs (drug effects: GluR-A/ male mice and their wild types, F(4,75) = 21, p < 0.001; GluR-A(R/R) male mice and their wild types,
F(4,63) = 35, p < 0.0001; line effects: GluR-A/ male mice and their wild types,
F(1,75) = 2.93, NS; GluR-A(R/R) male mice and their wild types, F(1,63) = 0.75, NS) (Fig. 8), suggesting that the
NMDA receptor activity is not altered in GluR-A mutant mice.
View larger version (28K):
[in this window]
[in a new window]
|
Figure 8.
The effect of acute NMDA receptor blockade by
MK-801 on locomotor activity of GluR-A/ and GluR-A(R/R) mutant mice
and their wild-type controls. The mice received either saline (10 ml/kg, i.p.) or different doses of MK-801, after which they were
immediately transferred into locomotor cages for the determination of
horizontal activity counts for 2 hr sessions. The bars
indicate mean ± SEM for 11-20 animals in GluR-A/ and control
groups and for 10-13 animals in GluR-A(R/R) and their control groups.
*p < 0.05 for the difference from the
corresponding saline group; Dunnett's test. There were no significant
differences between the mutants and their controls.
|
|
| |
DISCUSSION |
The actions of and adaptation to drugs of abuse are complex
neurobiological processes that have key neurochemical components. The
present findings clarify the role of one suggested molecular factor,
the GluR-A subunit of the AMPA-type glutamate receptors in
morphine-dependent behaviors. Our results confirm the previous suggestions that GluR-A subunits are operative in the process of
sensitization to morphine (Fitzgerald et al., 1996; Carlezon et al.,
1997; Lu et al., 1997; Churchill et al., 1999; Lu and Wolf, 1999) and
extend those results by indicating that the context-dependent sensitization is not eliminated by the loss of those subunits, whereas
the context-independent sensitization is. Considering that
context-dependent sensitization may resemble a learning process, our
results are in agreement with the normal learning ability of these mice
in spatial tasks (Zamanillo et al., 1999) and with their normal
habituation during repeated locomotor activity (the present study).
They also signify that environmental factors have a strong influence on
the effects of drugs of abuse via molecular mechanisms and brain
pathways that are different from those operative in the direct drug
actions. The GluR-A subunit-deficient mice exhibited at least as high
locomotor responses to acute morphine administration as their
littermates but had reduced development of tolerance and reduced
emergence of withdrawal symptoms. This suggests that GluR-A
subunit-expressing neurons, perhaps the interneurons known to express
GluR-A but not GluR-B subunits (Geiger et al., 1995), are involved in
neurochemical pathways normally affected by morphine.
The higher baseline locomotor activity of the GluR-A/ mice is a
confounding factor in the present study with regard to the sensitization phenomenon. However, for the interpretation of the present results, this factor is not essential, because we had another
mouse line with impaired GluR-A subunit-mediated synaptic transmission.
The GluR-A subunits were engineered to express an arginine in their
channel-forming loops critical for ion selectivity, strongly reducing
the calcium permeability and single-channel conductance in the same way
as in RNA-edited, arginine-containing GluR-B subunits (Burnashev et
al., 1992; Swanson et al., 1997). Because GluR-A subunits are strongly
expressed in most interneurons with low GluR-B subunit expression
(Geiger et al., 1995), the GluR-A(R/R) mutants are expected to have a
major change in the cation selectivity of their AMPA responses in the
interneurons, because the mutant receptors are
Ca2+ impermeable. The GluR-A(R/R) mice,
demonstrated to express the GluR-A(R) protein in their brains (Fig.
1D), had only slightly elevated locomotor activities
at baseline but still showed elevated responses to morphine selectively
in the specific environmental context. Furthermore, because these mice
entirely lacked sensitization when the treatment was repeated in home
cages, the data suggest that the wild-type GluR-A subunit-containing
receptors are needed for morphine sensitization in the absence of
specific environmental cues but not when environmental cues are
present. This is in line with the demonstration that viral
vector-mediated overexpression of GluR-A subunits in the rat brain
(Carlezon et al., 1997) without environmental cueing sensitizes the
locomotor responses of the animals to morphine.
Although the NMDA receptors have been implicated in the development of
tolerance to morphine, we failed to see any clear differences in the
locomotor actions of NMDA receptor blockade by MK-801 between the
mutant and their respective control lines. As far as MK-801-induced locomotor activity is caused by blockade of basal function of the NMDA
receptors, our results suggest normal functioning of the NMDA component
of the glutamate system in GluR-A/ mice and fail to explain the
diminution of morphine tolerance by reduced NMDA receptor functioning.
Because in our study the tolerance and dependence to morphine were
lower in the absence of GluR-A subunits, but not when only AMPA
receptor functionality was compromised [GluR-A(R/R) mutants], it is
likely that the mutant GluR-A(R) subunit can normally mediate the
AMPA-receptor adaptation under chronic morphine, e.g., by increased
membrane targeting. Whatever the mechanism, this adaptation to chronic
morphine is apparently not coupled to GluR-A subunit-containing AMPA
receptor-mediated Ca2+ conductance in the
interneurons. It could involve enhanced excitability of these neurons
because of a putative increase in GluR-A subunit-containing AMPA
receptor channels (Fitzgerald et al., 1996; Carlezon et al., 1997; Lu
et al., 1997; Churchill et al., 1999; Lu and Wolf, 1999), the process
of which is deficient in GluR-A/ mice.
There is recent evidence for potentiation of µ-opioid agonist-induced
analgesia by AMPA receptor antagonists (Nishiyama et al., 1998) and for
an interaction between µ-opioid and AMPA receptor responses studied
electrophysiologically in isolated spinal dorsal horn neurons (Kolaj
and Randic, 1996). It is thus likely that these effects were deficient
in GluR-A/ animals when tolerance to morphine was assayed by the
tail-flick test, although acute morphine produced similar
antinociception in the knock-outs and their littermates. Detailed
molecular events remain unknown, and it is not clear whether mechanisms
regulating pathways important for morphine-induced hyperlocomotion are
homologous to the spinal dorsal horn nociceptive pathways and pathways
defining various withdrawal symptoms. However, it is known that both
µ-opioid receptors and GluR-A subunits are expressed widely in the
forebrain regions (Sato et al., 1993; Mansour et al., 1994), including
the nucleus accumbens that is implicated in locomotor stimulation by
various drugs of abuse. There is no evidence for direct molecular
interaction between µ-opioid and AMPA receptors, and therefore, we
think that the AMPA receptor-containing neurons may be targets of
neuronal-depressing action of morphine, to which these neurons then
adapt by changes in AMPA receptors upon repeated morphine
administration. The present animal model fails to assess the
significance of AMPA receptors in any given brain region for morphine
effects, because the expression of GluR-A subunit mutations was under
native promoters. Actually, the slight acute enhancement of morphine
effects in the GluR-A/ animals might be better explained by reduced
AMPA responses in the nucleus accumbens than the ventral tegmental
area. This would be consistent with the results of Kelz et al. (1999)
who showed that an increased level of GluR-A subunit in the nucleus
accumbens is associated with aversion to cocaine-paired environment.
There are presently no AMPA receptor subtype-selective antagonists, and
therefore, it is difficult to assess whether our findings are
attributable to direct inactivation or malfunction of GluR-A subunit-containing AMPA receptors or to indirect alterations in neuronal circuitry caused by the primary receptor alteration. It should
be also noted that the findings of the present study may be partly
affected by the possible alterations in neuronal development in GluR-A
subunit-deficient mice, because these subunits are expressed early in
the brain development (Monyer et al., 1991). However, several
nonselective AMPA antagonists have been studied and found to affect the
development and expression of behavioral sensitization to stimulants
and opioids in various rat and mouse models (Burns et al., 1994; Li et
al., 1997; McLemore et al., 1997; Mead and Stephens, 1998; Carlezon et
al., 1999). These results have established the involvement of the AMPA
receptors in behavioral sensitization and, therefore, our present
results on a new animal model are consistent with the previous
pharmacological studies. Additional studies are warranted to dissect
out the role of GluR-A subunit-containing AMPA receptors in the
acquisition and expression of primary rewarding effects and other
conditioned activity. The existence of context-dependent sensitization
of the GluR-A subunit-deficient mouse lines was not restricted to
opioids but could be observed also with amphetamine, and with this
stimulant, the sensitization was actually enhanced in GluR-A mutants
compared with the wild types. However, the same protocol with unpaired
amphetamine administration schedule failed to induce any sensitization
in the wild-type (or GluR-A/) animals, preventing any conclusion on
the mechanisms of context-independent sensitization to stimulants.
Enhanced context-dependent sensitization to stimulants indicates a
wider role for AMPA receptors in adaptation to the effects of drugs of
abuse, in keeping with the proposition that the loss of AMPA receptors
in the ventral striatum, the main site of stimulant action, should
enhance context-dependent effects of stimulants (Kelz et al.,
1999).
The AMPA receptors are known to be rapidly regulated in neuronal
membranes (Turrigiano, 2000) during neuronal activity and, therefore,
they would seem to be suitable substrates for neuronal plasticity and
learning and memory. Our results support the role of GluR-A
subunit-containing receptors in plasticity associated with the
development of tolerance and dependence but not in the development
and/or expression of context dependency of morphine-induced locomotor
stimulation. The Ca2+-dependent processes,
especially in GluR-B subunit-deficient interneurons, are apparently
only required for some of the morphine-induced adaptations.
| |
FOOTNOTES |
Received Dec. 13, 2000; revised March 15, 2001; accepted March 23, 2001.
This work was supported in part by the Academy of Finland, the National
Technology Agency Tekes, and the Sigrid Juselius Foundation (E.R.K.), the Finnish Cultural Foundation (A.H.), European Union Framework Program Grant QLG3-CT-1999-01022 (P.H.S.), and Deutsche Forschungsgemeinschaft Sonderforschungsbereich Grant 317 and the Volkswagen-Stiftung (R.S.). D.Z. was the recipient of a postdoctoral fellowship from Human Frontier Science Program. We thank F. Zimmermann for technical assistance, Hua Gu for plasmid pMC-Cre, and
Andras Nagy for providing embryonic stem cells R1 and plasmid
ploxPneo1. Cre was used under a noncommercial research license
agreement between DuPont Pharmaceutical Company and the
Max-Planck-Society.
Correspondence should be addressed to Dr. Esa R. Korpi, Department of
Pharmacology and Clinical Pharmacology, University of Turku, FIN-20520
Turku, Finland. E-mail: esa.korpi{at}utu.fi.
| |
REFERENCES |
-
Bardo MT
(1998)
Neuropharmacological mechanisms of drug reward: beyond dopamine in the nucleus accumbens.
Crit Rev Neurobiol
12:37-67[ISI][Medline].
-
Belozertseva I,
Zvartau E,
Bespalov A
(1996)
Behavioral effects of MK-801 in morphine-dependent and non-dependent mice.
Life Sci
58:L55-L61.
-
Berke JD,
Hyman SE
(2000)
Addiction, dopamine, and the molecular mechanisms of memory.
Neuron
25:515-532[ISI][Medline].
-
Brusa R,
Zimmermann F,
Koh DS,
Feldmeyer D,
Gass P,
Seeburg PH,
Sprengel R
(1995)
Early-onset epilepsy and postnatal lethality associated with an editing-deficient GluR-B allele in mice.
Science
270:1677-1680[Abstract/Free Full Text].
-
Burnashev N,
Monyer H,
Seeburg PH,
Sakmann B
(1992)
Divalent ion permeability of AMPA receptor channels is dominated by the edited form of a single subunit.
Neuron
8:189-198[ISI][Medline].
-
Burns LH,
Everitt BJ,
Kelley AE,
Robbins TW
(1994)
Glutamate-dopamine interactions in the ventral striatum: role in locomotor activity and responding with conditioned reinforcement.
Psychopharmacology (Berl)
115:516-528[Medline].
-
Carlezon Jr WA,
Boundy VA,
Haile CN,
Lane SB,
Kalb RG,
Neve RL,
Nestler EJ
(1997)
Sensitization to morphine induced by viral-mediated gene transfer.
Science
277:812-814[Abstract/Free Full Text].
-
Carlezon Jr WA,
Rasmussen K,
Nestler EJ
(1999)
AMPA antagonist LY293558 blocks the development, without blocking the expression, of behavioral sensitization to morphine.
Synapse
31:256-262[Medline].
-
Churchill L,
Swanson CJ,
Urbina M,
Kalivas PW
(1999)
Repeated cocaine alters glutamate receptor subunit levels in the nucleus accumbens and ventral tegmental area of rats that develop behavioral sensitization.
J Neurochem
72:2397-2403[ISI][Medline].
-
Everitt BJ,
Parkinson JA,
Olmstead MC,
Arroyo M,
Robledo P,
Robbins TW
(1999)
Associative processes in addiction and reward. The role of amygdala-ventral striatal subsystems.
Ann NY Acad Sci
877:412-438[ISI][Medline].
-
Fitzgerald LW,
Ortiz J,
Hamedani AG,
Nestler EJ
(1996)
Drugs of abuse and stress increase the expression of GluR1 and NMDAR1 glutamate receptor subunits in the rat ventral tegmental area: common adaptations among cross-sensitizing agents.
J Neurosci
16:274-282[Abstract/Free Full Text].
-
Geiger JR,
Melcher T,
Koh DS,
Sakmann B,
Seeburg PH,
Jonas P,
Monyer H
(1995)
Relative abundance of subunit mRNAs determines gating and Ca2+ permeability of AMPA receptors in principal neurons and interneurons in rat CNS.
Neuron
15:193-204[ISI][Medline].
-
Ginski MJ,
Witkin JM
(1994)
Sensitive and rapid behavioral differentiation of N-methyl-D-aspartate receptor antagonists.
Psychopharmacology (Berl)
114:573-582[Medline].
-
Gu H,
Zou YR,
Rajewsky K
(1993)
Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-mediated gene targeting.
Cell
73:1155-1164[ISI][Medline].
-
Jang CG,
Rockhold RW,
Ho IK
(2000)
An autoradiographic study of [3H]AMPA receptor binding and in situ hybridization of AMPA sensitive glutamate receptor A (GluR-A) subunits following morphine withdrawal in the rat brain.
Brain Res Bull
52:217-221[Medline].
-
Kelz MB,
Chen J,
Carlezon Jr WA,
Whisler K,
Gilden L,
Beckmann AM,
Steffen C,
Zhang YJ,
Marotti L,
Self DW,
Tkatch T,
Baranauskas G,
Surmeier DJ,
Neve RL,
Duman RS,
Picciotto MR,
Nestler EJ
(1999)
Expression of the transcription factor
FosB in the brain controls sensitivity to cocaine.
Nature
401:272-276[Medline]. -
Kest B,
McLemore G,
Kao B,
Inturrisi CE
(1997)
The competitive alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor antagonist LY293558 attenuates and reverses analgesic tolerance to morphine but not to delta or kappa opioids.
J Pharmacol Exp Ther
283:1249-1255[Abstract/Free Full Text].
-
Kohler M,
Kornau HC,
Seeburg PH
(1994)
The organization of the gene for the functionally dominant
-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor subunit GluR-B.
J Biol Chem
269:17367-17370[Abstract/Free Full Text]. -
Kolaj M,
Randic M
(1996)
µ-Opioid receptor-mediated reduction of -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-activated current in dorsal horn neurons.
Neurosci Lett
204:133-137[ISI][Medline].
-
Koob GF,
Sanna PP,
Bloom FE
(1998)
Neuroscience of addiction.
Neuron
21:467-476[ISI][Medline].
-
Leshner AI,
Koob GF
(1999)
Drugs of abuse and the brain.
Proc Assoc Am Physicians
111:99-108[ISI][Medline].
-
Li Y,
Vartanian AJ,
White FJ,
Xue CJ,
Wolf ME
(1997)
Effects of the AMPA receptor antagonist NBQX on the development and expression of behavioral sensitization to cocaine and amphetamine.
Psychopharmacology (Berl)
134:266-276[Medline].
-
Lu W,
Wolf ME
(1999)
Repeated amphetamine administration alters AMPA receptor subunit expression in rat nucleus accumbens and medial prefrontal cortex.
Synapse
32:119-131[Medline].
-
Lu W,
Chen H,
Xue CJ,
Wolf ME
(1997)
Repeated amphetamine administration alters the expression of mRNA for AMPA receptor subunits in rat nucleus accumbens and prefrontal cortex.
Synapse
26:269-280[ISI][Medline].
-
Malin DH,
Lake JR,
Newlin-Maultsby P,
Roberts LK,
Lanier JG,
Carter VA,
Cunningham JS,
Wilson OB
(1992)
Rodent model of nicotine abstinence syndrome.
Pharmacol Biochem Behav
43:779-784[ISI][Medline].
-
Mansour A,
Fox CA,
Thompson RC,
Akil H,
Watson SJ
(1994)
µ-Opioid receptor mRNA expression in the rat CNS: comparison to µ-receptor binding.
Brain Res
643:245-265[ISI][Medline].
-
Matthes HW,
Maldonado R,
Simonin F,
Valverde O,
Slowe S,
Kitchen I,
Befort K,
Dierich A,
Le Meur M,
Dolle P,
Tzavara E,
Hanoune J,
Roques BP,
Kieffer BL
(1996)
Loss of morphine-induced analgesia, reward effect and withdrawal symptoms in mice lacking the µ-opioid-receptor gene.
Nature
383:819-823[Medline].
-
McLemore GL,
Kest B,
Inturrisi CE
(1997)
The effects of LY293558, an AMPA receptor antagonist, on acute and chronic morphine dependence.
Brain Res
778:120-126[Medline].
-
Mead AN,
Stephens DN
(1998)
AMPA-receptors are involved in the expression of amphetamine-induced behavioural sensitisation, but not in the expression of amphetamine-induced conditioned activity in mice.
Neuropharmacology
37:1131-1138[Medline].
-
Merlo Pich E,
Chiamulera C,
Carboni L
(1999)
Molecular mechanisms of the positive reinforcing effect of nicotine.
Behav Pharmacol
10:587-596[Medline].
-
Monyer H,
Seeburg PH,
Wisden W
(1991)
Glutamate-operated channels: developmentally early and mature forms arise by alternative splicing.
Neuron
6:799-810[ISI][Medline].
-
Nagy A,
Rossant J,
Nagy R,
Abramow-Newerly W,
Roder JC
(1993)
Derivation of completely cell culture-derived mice from early-passage embryonic stem cells.
Proc Natl Acad Sci USA
90:8424-8428[Abstract/Free Full Text].
-
Nestler EJ
(2000)
Genes and addiction.
Nat Genet
26:277-281[ISI][Medline].
-
Nishiyama T,
Yaksh TL,
Weber E
(1998)
Effects of intrathecal NMDA and non-NMDA antagonists on acute thermal nociception and their interaction with morphine.
Anesthesiology
89:715-722[ISI][Medline].
-
Robinson TE,
Kolb B
(1997)
Persistent structural modifications in nucleus accumbens and prefrontal cortex neurons produced by previous experience with amphetamine.
J Neurosci
17:8491-8497[Abstract/Free Full Text].
-
Robinson TE,
Kolb B
(1999)
Morphine alters the structure of neurons in the nucleus accumbens and neocortex of rats.
Synapse
33:160-162[ISI][Medline].
-
Rodriguez de Fonseca F,
Navarro M
(1998)
Role of the limbic system in dependence on drugs.
Ann Med
30:397-405[ISI][Medline].
-
Sato K,
Kiyama H,
Tohyama M
(1993)
The differential expression patterns of messenger RNAs encoding non-N-methyl-D-aspartate glutamate receptor subunits (GluR1-4) in the rat brain.
Neuroscience
52:515-539[ISI][Medline].
-
Self DW
(1998)
Neural substrates of drug craving and relapse in drug addiction.
Ann Med
30:379-389[ISI][Medline]
TOP
ABSTRACT
INTRODUCTION
