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Vol. 295, Issue 3, 1031-1042, December 2000
-Opioid Agonists
on Locomotor Activity in Mice
Departments of Neuroscience (A.K., S.-O.Ö.) and Clinical Neuroscience (J.S., L.T.), Karolinska Institutet, Stockholm, Sweden
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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The 
-opioid agonists U50488H, bremazocine, and BRL52537, and
the µ-opioid agonist morphine were compared in their ability to
modify spontaneous motor activity in male NMRI mice. Higher, analgesic
doses of the -agonists reduced rearing, motility, and locomotion in
nonhabituated mice. These effects, as well as the analgesic action of
U50488H, were blocked by the selective -opioid antagonists
nor-binaltorphimine and DIPPA. In contrast, lower, subanalgesic doses
(1.25 and 2.5 mg/kg for U50488H; 0.15 and 0.075 mg/kg for bremazocine,
and 0.1 mg/kg for BRL52537) time dependently increased motor activity.
The stimulatory effects of U50488H and bremazocine were not observed in
habituated animals and were reduced by dopamine depletion.
Surprisingly, the stimulatory effects of U50488H and bremazocine were
not blocked by nor-binaltorphimine and DIPPA but they were completely
eliminated by naloxone (0.1 mg/kg). The effects of morphine were
dose-dependent; an initial limited suppression was followed by
increased motility and locomotion (but not rearing) with a peak effect
at 20 mg/kg both in habituated and nonhabituated mice. The selective
µ-opioid antagonist 
-funaltrexamine blocked morphine-induced motor
stimulation and analgesia but failed to affect the analgesic and motor
stimulatory effects of U50488H. The results indicate that -opioid
agonists interact with different functional subtypes of opioid
receptors. A stimulatory, naloxone-sensitive but
nor-binaltorphimine- and DIPPA-insensitive subtype of opioid receptor appears to operate only when the dopamine system is tonically active in nonhabituated animals. At higher doses, -agonists produce analgesia and motor suppression, effects mediated by a "classic" (inhibitory) -opioid receptor.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The
pharmacology of opiates is complex in a number of aspects. Species
differences are very obvious. In mice, morphine induces excitation and
the typical Straub tail elevation, behaviors that are not observed in
the rat, which mainly reacts by inhibition. Another aspect of
complexity is the functional multiplicity and heterogeneity of opioid
receptors elaborated by Martin (1984)
. The distinction of the
morphine-type (µ-) and -receptor could be confirmed by cloning
(Dhawan et al., 1996). A third aspect of complexity becomes apparent in
the study of the electrophysiological actions of opiates. Depending on
the type of cell used and dose applied, both µ- and - opioids can
induce hyperpolarization or depolarization (Smart and Lambert,
1996) and either inhibit or stimulate neuronal cells.
In rodents it is generally observed that µ- and 
-agonists increase
locomotion, whereas -agonists decrease locomotion (Mansour et al.,
1995) in addition to inducing ataxia and sedation (Jackson and Cooper,
1988). However, in preweanling (Duke et al., 1997) and
monoamine-depleted rats (Hughes et al., 1998) -opioid agonists have
been reported to markedly increase locomotor activity. In Syrian
hamsters -agonists elicit biphasic effects inducing hyperactivity at
lower and hypoactivity at higher doses (Schnur and Walker, 1990).
Opioid-induced effects on motor activity have been mainly related to
interactions with mesolimbic and nigrostriatal dopamine (DA)-ergic
neurotransmission, although some authors also claim the importance of
substantia nigra and its non-DA projections in the motor effects of
µ- and -opoioid agonists (Matsumoto et al., 1988). µ- and
-Opioid receptor agonists have been shown to increase extracellular
dopamine levels in the nucleus accumbens and striatum (Spanagel, 1995),
whereas -opioid agonists exhibit the opposite action (Pan, 1998),
effects that are blocked by the unselective opioid antagonist naloxone.
Conversely, the selective -opioid antagonist nor-binaltorphimine
(nor-BNI) dose dependently increased DA release (Spanagel, 1995).
However, there are also conflicting results, showing that acute
administration (Spanagel, 1995) or repeated injections of the
-opioid agonists U-69593 and U50488H (Heidbreder et al., 1998) fail
to modify basal DA overflow in the nucleus accumbens and the caudate.
It has also been reported that U50488H markedly increased DA release in
the nucleus accumbens after local infusion (Donzanti et al., 1989) and
potentiated both basal and depolarization-evoked dopamine release from
a human neuroblastoma cell line (Keren et al., 1999). The reasons for
these contradictory results have not been clarified.
The existence of multiple -opioid receptors has been proposed since
the early studies of Attali et al. (1982). At least two -opioid
receptor subtypes have been defined on the basis of in vitro binding
studies, classified as 1, a receptor subtype that preferentially
binds arylacetamide -opioids such as U50488H (Lahti et al., 1982)
and 2 (3), a subtype that binds benzomorphan -opioids such as
bremazocine (Clark et al., 1989; Horan et al., 1993). The acceptance of
the existence of 1 and 2 subtypes has been limited due to the
lack of functional evidence providing support for the data obtained in
radioligand binding studies. Alternatively, the subtypes of the
-receptor could, most probably, correspond to different affinity
states of the same receptor, depending on its coupling with G proteins
(Richardson et al., 1992). Cloning data have so far only provided
support for the existence of a single -opioid receptor.
The aim of the present work was to study the influence of low
(subanalgesic) and analgesic doses of the -opioid agonists U50488H,
bremazocine, and BRL52537 on spontaneous motor activity in mice and to
evaluate the sensitivity of these effects to opioid receptor blockade
produced by the nonselective opioid antagonist naloxone, the selective
µ-opioid antagonist -funaltrexamine (-FNA), and the
-selective antagonists nor-BNI and DIPPA.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Animals. Experiments were carried out in male NMRI mice (18-22 g; B&K Universal, AB, Sollentuna, Sweden). Animals were kept under standard laboratory conditions with unlimited access to food and water. Animals were housed eight per cage in a light-controlled room (12-h light/dark cycle, lights on at 6:00 AM) at 21°C and 60% humidity. The experiments were approved by the local Ethics Board of Animal Experimentation.
Locomotor Activity System. Mice were individually tested in a dimly lit, sound-controlled area ventilated by fans. They were removed from their home cages and placed in the middle of an activity monitor (standard transparent A3 macrolon cage with 50 ml of wooden shavings on the floor) and the data-collecting system was immediately activated. In experiments with nonhabituated animals, mice were treated with the drug just before placement in the activity box and different parameters of motor activity were recorded during six 10-min intervals. In experiments with habituated animals, mice were habituated to the test cages for 30 min and thereafter the injection was made and the activity was recorded as described above.
Motor activity was measured in eight animals simultaneously by means of a multicage red and infrared-sensitive motion detection system (Ögren et al., 1979). The system is fully computerized and uses
beams of red and infrared lights in combination with vertical and
horizontal photocell arrays (4-cm distance between cells) to detect
movements of animals. Rearing was measured by counting the number of
times an animal stands on its hind legs and interferes with any of the
six invisible infrared beams passing horizontally through the cages.
The height of these photocells was adapted to the size of the animal.
Motility was measured as all movements of a distance of 4 cm or more
detected by 48 vertical photocells, and represents a measurement of
general activity. Locomotion was measured by counting the number of
times an animal had covered eight horizontal photocells and moved from
one side of the test cage to the other side (a distance of at least 32 cm).
Dose-dependent effects of drugs on rearing, motility and locomotion in
mice were analyzed by two-way ANOVA for repeated measures, the factors
being treatment (drug versus saline/control) and time (six 10-min
recordings). This was followed by a post hoc Newman-Keuls test for
every time point separately. The whole study was designed as a
between-subjects (independent groups) experiment (i.e., each animal was
used only once).
Analgesic Activity. Analgesic activity of U50488H and morphine was measured using the hot-plate (57°C) test. First, the basal nociceptive threshold was measured as the latency to paw-licking or lifting of the back limbs or jumping (whichever came first). Thereafter, the drug was injected and the nociceptive reaction was measured 30 min after treatment. The cut-off time for nonresponders was set at 30 s. The results (latencies and percentage of analgesia) were analyzed using ANOVA followed by a post hoc Newman-Keuls test.
Drugs.
Naloxone HCl (Endo Laboratories, Wilmington,
PA), U50488H
[trans-(±)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide, methanesulfonate hydrate; Upjohn, Kalamazoo, MI], BRL52537
[(±)-1-(3,4-dichlorophenyl)acetyl-2-(1-pyrrolidinyl) methylpiperidine; Tocris, Bristol, UK), morphine HCl (Sigma, St. Louis,
MO), and bremazocine HCl (Research Biochemicals International, Natick,
MA) were dissolved in saline and injected s.c. in a volume of 5 ml/kg
just before the start of the experiment. The tyrosine hydroxylase
inhibitor H44-68 (
-methyl-p-tyrosine methyl ester; AstraZeneca, Sodertalje, Sweden) was dissolved in saline and injected i.p. 2 h before an experiment. Nor-BNI 2HCl
[17,17'- (dicyclopropylmethyl)-6,6',7,7'-6,6'-imino-7,7'-bimorphinan-3,4',14,14'- tetrol], -FNA HCl
[(E)-4-[[(5,6)-17-cyclopropylmethyl)-4,5-epoxy-3,14-dihydroxymorphinan-6-yl]amino]-4-oxo-2-butenoic acid methyl ester], and DIPPA
[2-(3,4-dichlorophenyl)-N-methyl-N-[(1S)-1-(3-isothiocyanatophenyl)-2-(1-pyrrolidinyl)ethyl]acetamide] (all from Tocris) were dissolved in water [with addition of
2-hydroxypropyl--cyclodextrin (Research Biochemicals
International) 1:1 w/w to dissolved compound] and injected s.c.
48 h before an experiment. All doses of drugs refer to the salts.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Effects of Morphine, U50488H, Bremazocine, and BRL52537 in Nonhabituated Animals
Saline Control. In animals treated with saline (n = 16) there was a gradual decrease of motor activity over time and an almost complete elimination of rearing, motility, and locomotion after 1 h of habituation to the activity cages.
Morphine Effects (Fig. 1).
There
was a significant main effect of morphine treatment on motility
(P < .01) and locomotion (P < .05); a
significant time effect for rearing, motility, and locomotion
(P < .0001, P < .001, and
P < .001, respectively); and a significant dose × time interaction for all parameters [F(20,175) = 4.6, P < .001; 3.5, P < .001; and
2.4, P < .01, respectively]. The post hoc
Newman-Keuls test, analyzed for each time point separately, revealed
that there was a significant (P < .01) lower level of
rearing 10 and 20 min after treatment with morphine at the doses of 20 and 40 mg/kg, and higher (with respect to saline-treated group)
motility 30, 40, 50, and 60 min after treatment with 20 mg/kg, 40 to 60 min after treatment with 40 mg/kg, and 60 min after treatment with 10 mg/kg, and significantly higher locomotion 30 to 60 min after treatment
with morphine at the dose of 20 mg/kg. Taken together, the data show
that morphine in a time- and dose-dependent manner increases motility
and locomotion but not rearing. The peak effect of morphine to enhance
motor activity in mice was found at the 20-mg/kg dose.
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U50488H Effects (Fig. 1). There was a significant effect of U50488H treatment on rearing (P < .01), motility (P < .0001), and locomotion (P < .0001); a significant time effect for rearing, motility, and locomotion (P < .0001, P < .0001, and P < .0001, respectively); and a significant dose × time interaction for all parameters [F(20,175) = 3.3, P < .001; 5.0, P < .0001; and 4.6, P < .001, respectively]. The post hoc Newman-Keuls test, made for each time point separately, revealed that there was a significant (P < .01) increase (with respect to saline-treated group) in rearing and motility after 30 min of treatment with 1.25 mg/kg and a significant increase in rearing, motility, and locomotion after 40, 50, and 60 min of treatment with U50488H at the doses of 1.25 and 2.5 mg/kg. The lowest (1.25 mg/kg) dose of U50488H produced a monophasic stimulatory influence on motor activity, whereas the higher (2.5 mg/kg) dose exhibited a biphasic influence with inhibition of motor activity at 10 and 20 min and stimulation of activity at 40, 50, and 60 min post injection. Treatment with the 5-mg/kg dose tended to exhibit effects similar to those with 2.5 mg/kg, but the motor stimulation did not reach statistical significance. However, there was a significant inhibition of all parameters of motor activity after treatment with 5 or 10 mg/kg U50488H at 10 and 20 min after injection. In summary, U50488H displayed a dose-related influence on the locomotor activity of mice with inhibition at higher doses and stimulation at lower doses (preceded by slight initial suppression). The threshold dose range for the shift from stimulation to inhibition was between 2.5 and 5.0 mg/kg.
Bremazocine Effects (Fig. 2).
There was a significant effect of bremazocine treatment on motility
(P < .0001) and locomotion (P < .0001); a significant time effect for rearing, motility, and locomotion
(all P < .0001); and a significant dose × time
interaction for all parameters [F(20,175) = 4.9, P < .001; 12.5, P < .0001; and 11.6, P < .001, respectively]. The post hoc test (made for
each time point separately) revealed that there was a significant
(P < .01) increase (compared with saline) in motility
and locomotion at 40 min and in all parameters at 50 and 60 min with
0.075 mg/kg, and a significant increase after treatment with 0.15 mg/kg
in motility at 50 min and in all parameters at 50 and 60 min. The
effect was biphasic with an inhibition of motor activity at 10 and 20 min and stimulation of activity at 40, 50, and 60 min. At higher (0.312 mg/kg and higher) doses there was a significant inhibition of all
parameters of motor activity at 10-, 20-, and 30-min time measurements.
Experiments were also performed with 1.25-, 2.5-, and 5.0-mg/kg doses
of bremazocine (data not shown). These doses produced a complete
reduction of all parameters of motor activity at all time points of
measurement. In summary, similar to the experiments with U50488H,
bremazocine exhibited a biphasic influence on motor activity with an
initial inhibition followed by a stimulation at lower doses and an
inhibition at doses of 0.312 mg/kg and higher.
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BRL52537 Effects (Fig. 2).
There was a significant effect of
BRL52537 treatment on rearing (P < .001), motility
(P < .0001), and locomotion (P < .0001); a significant time effect for rearing, motility, and locomotion (all P < .0001); and a significant dose × time
interaction for all parameters [F(15,140) = 3.9, P < .001; 6.8, P < .0001; and 4.1, P < .001, respectively]. The post hoc test (made for
each time point separately) revealed that there was an increase (with respect to the saline-treated group) in rearing 40 to 60 min after treatment with 0.1 mg/kg and an increase in motility and locomotion at
30 to 60 min. Treatment with a dose of 0.01 mg/kg produced a
significant increase in rearing at 30 min, in motility at 30 to 50 min,
and in locomotion at 40 min. There was a significant (P < .01) decrease in all parameters (compared with saline control) at
all time points after treatment with 1.0 mg/kg. Thus, the highest dose
used (1.0 mg/kg) inhibited all parameters of motor activity, whereas
lower doses had a stimulating effect, i.e., the same biphasic pattern
that was observed with the other -agonists.
Effects of Morphine, U50488H, and Bremazocine in Habituated Animals (Fig. 3)
Animals habituated to the motor activity cages for 30 min were
tested in analogy with the experiments using nonhabituated animals.
ANOVA analysis indicated a significant main stimulatory effect of
morphine (10 and 20 mg/kg) treatment on motility (both P < .01) and locomotion (20 mg/kg, P < .05) but failed to reveal a significant influence of the two lower
doses of U50488H and bremazocine. The effects of higher doses of
-agonists were inconclusive because the activity was strongly
reduced by the habituation and could not be further inhibited
(floor-effect).
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Effect of Pretreatment with Dopamine Depletor H44-68 on the Motor Effects of U50488H (Fig. 4)
Mice were pretreated with H44-68 (100 mg/kg i.p.) 2 h before
injection of U50488H (1.25 and 10 mg/kg) or saline. Locomotor activity
was evaluated in nonhabituated animals. There was a significant treatment effect on motility and locomotion (P < .05 and P < .01, respectively) and a significant
treatment × time interaction for rearing
[F(10,65) = 9.2, P < .01] and
locomotion [F(10,65) = 2.3, P < .05]. The time effect was also significant (P < .0001) for all parameters. The post hoc comparison revealed a
significant (P < .01, against saline) decrease in
rearing at 20 min in both U50488H-treated groups but no differences
between high- and low-dose treatment groups. A significant decrease in
locomotion was found in both U50488H-treated groups at 20 and 30 min.
Again, there were no significant differences between the high- and
low-dose U50488H groups.
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Effects of Motor Stimulatory Doses of U50488H and Bremazocine in Nor-BNI-, DIPPA-, or Naloxone-Pretreated Nonhabituated Animals
Effects of Antagonists Alone. Nor-BNI (6 mg/kg) and DIPPA (4 mg/kg) were injected s.c. 48 h before an experiment. Naloxone (0.1 mg/kg) was injected s.c. just before an experiment. ANOVA analysis (data not shown) failed to reveal a significant influence of either compound on motor activity (compared with saline-treated animals) or a treatment × time interaction.
U50488H and Bremazocine Effects in Nor-BNI-Pretreated Animals (Fig.
5, Left).
There were significant
treatment effects on rearing, motility, and locomotion
(P < .05, P < .05, and
P < .05, respectively) and significant treatment × time interactions for all parameters [F(10,205) = 21.3, P < .0001; 86.2, P < .0001; and
129.6, P < .0001, respectively). The time effect was
significant (P < .0001) for all parameters. The post
hoc comparison revealed a significant (P < .01, comparison with saline-treated group) increase in rearing at 20, 30, 40, 50, and 60 min in the U50488H (1.25 mg/kg)-treated group and at 50 and 60 min in the bremazocine (0.075 mg/kg)-treated group. There was
also a significant increase in motility and locomotion at 30, 40, 50, and 60 min in the U50488H (1.25 mg/kg)-treated group and at 40, 50, and
60 min in the bremazocine (0.075 mg/kg)-treated group. However, at 20 min bremazocine produced a significant inhibition of motility and
locomotion. Taken together, the results show that nor-BNI treatment
failed to influence the locomotor stimulation produced by low doses of
U50488H and bremazocine.
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U50488H Effects in DIPPA-Pretreated Animals (Fig. 5, Middle).
There were significant pretreatment (DIPPA versus vehicle) effects on
rearing and significant treatment (U50488H versus saline) effects on
rearing, motility, and locomotion (P < .01, P < .001, and P < .001, respectively). The time effect was significant (P < .001) for all parameters. Treatment × time interactions were significant for motility and locomotion [F(15,100) = 3.4, P < .01; and F(15,100) = 1.8, P < .05, respectively]. The post hoc comparison
revealed a significant (P < .01, comparison with
vehicle-saline-treated group) increase in motility and locomotion at
40, 50, and 60 min in the U50488H (1.25 mg/kg)-treated groups (both
pretreated with vehicle and DIPPA) in comparison with the
vehicle-saline and DIPPA-saline groups. Rearing also increased in the
U50488H (1.25 mg/kg)-treated group, pretreated with vehicle, but not in
the group pretreated with DIPPA (4 mg/kg). In summary, DIPPA
pretreatment reduced the stimulation of rearing produced by the
-agonist, but failed to influence stimulation of motility and locomotion.
U50488H and Bremazocine Effects in Naloxone-Pretreated Animals
(Fig. 5, Right).
ANOVA revealed a significant treatment effect
only for locomotion (P < .05). However, time effect
and treatment × time interactions were significant
[F(15,100) = 12.6, 8.9, and 11.7; all
P < .0001) for all parameters. Post hoc comparison
revealed a significant (P < .01) inhibition of
rearing, motility, and locomotion in bremazocine-treated animals at 20 min. However, there was a significant increase in rearing and motility
in bremazocine-treated mice at 60 min. With respect to the group Sal + U50488H (1.25 mg/kg), there was a significant inhibition
(P < .01) of all parameters of activity starting from 30 min after -agonist injection in the group naloxone + U50488H (1.25 mg/kg). However, no differences in activity between groups "naloxone + vehicle" and "naloxone + U50488H" were found. It
was concluded that naloxone (0.1 mg/kg) completely abolished the motor stimulation produced by the -agonists. Similar results were obtained with 1.0 mg/kg naloxone (data not shown). The lack of effect at 60 min
might be explained by the short lasting effect of naloxone.
Effects of a High (Motor-Inhibitory) Dose of U50488H in Nor-BNI- and DIPPA-Pretreated Animals
U50488H Effects in Nor-BNI-Treated Animals (Fig.
6).
There was a significant
treatment effect on rearing, motility, and locomotion
(P < .01, P < .01, and
P < .01, respectively) and a significant
treatment × time interaction for all parameters [F(10,100) = 2.3, P < .05; 4.3, P < .01; and 4.4, P < .01, respectively]. The time effect was significant (P < .0001) for all parameters. The post hoc comparison revealed a
significant (P < .01, against saline) decrease in
rearing at 20, 30, and 40 min in the U50488-treated group pretreated
with vehicle and a significant increase in rearing at 50 and 60 min in
the nor-BNI-pretreated and U50488H (10 mg/kg)-treated group. The same
differences (i.e., decrease in activity at 20, 30, and 40 min in the
vehicle + U50488H and increase in activity at 50 and 60 min in the
nor-BNI + U50488H group) were found for motility and locomotion. Taken
together, the results show that nor-BNI pretreatment eliminated the
motor inhibitory effect of U50488H and reverted the effect of the drug
toward moderate motor stimulation.
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U50488H Effects in DIPPA-Treated Animals (Fig. 6). There were significant treatment effects on rearing, motility, and locomotion (P < .01, P < .001, and P < .001, respectively) and significant treatment × time interaction for all parameters [F(15,120) = 6.6, P < .01; 17.7, P < .001; and 17.1, P < .001, respectively]. The time effect was significant (P < .0001) for all parameters. In the group pretreated with vehicle, U50488H significantly (P < .01, comparison with vehicle-saline-treated group) inhibited rearing, motility, and locomotion at 20, 30, and 40 min. The post hoc comparison revealed a significant increase in rearing, motility, and locomotion at 40, 50, and 60 min in the U50488H (10 mg/kg)-treated group, pretreated with DIPPA. In summary, DIPPA pretreatment changed the effect profile of U50488H with no action in the first 30 min after administration and reversal of the inhibitory to a stimulatory action, 40 to 60 min after injection.
Effect of Nor-BNI Pretreatment on the Analgesic Activity of U50488H in the Hot-Plate Test (Table 1)
Nor-BNI (6 mg/kg) was injected s.c. to a separate group of
mice 48 h before the injection of U50488H similarly to that
described for experiments on motor activity. An overall repeated
measurement analysis of response latencies was performed with drug
treatment (nor-BNI-treated versus vehicle-treated mice) and U50488H
dose levels (four levels: 2.5, 5.0, 10, and 20 mg/kg) as between-group factors and time (basal versus 30-min values) as repeated measurement factor. ANOVA revealed that at 30 min the main effect of nor-BNI treatment (P < .0001), U50488H unit dose
(P < .01), and dose × treatment interaction
[F(3,24) = 6.20, P < .01] were
significant. The time effect was not significant, although the
time × dose × treatment interaction was significant
(P < .05). In vehicle-pretreated animals 10 and 20 mg/kg U50488H produced a significant increase in nociceptive latencies
at 30 min (P < .01). In nor-BNI-pretreated animals
neither dose of U50488H produced a significant analgesic effect.
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Effect of DIPPA Pretreatment on the Analgesic Activity of U50488H in the Hot-Plate Test (Table 2)
DIPPA (0.5, 1.0, 2.0, and 4.0 mg/kg) or its vehicle were injected
s.c. 48 h before the experiment. After testing the basal nociceptive reaction in the hot-plate (57°C) mice were treated with
U50488H (10 mg/kg) and nociceptive scores were measured 30 min after
-agonist injection. Two-way ANOVA analysis of the nociceptive latencies (time factor: basal versus 30-min measurement; treatment factor: four doses of DIPPA and saline) revealed significant time effect (P < .001), treatment effect (P < .01), and time × treatment interaction
[F(3,24) = 8.8, P < .001]. In
vehicle-pretreated animals 10 mg/kg U50488H produced significant
analgesia (P < .01). Treatment with DIPPA at the dose
of 4 mg/kg eliminated the analgesic effect of U50488H. In groups
treated with DIPPA at the doses 0.5, 1.0, and 2.0 mg/kg, the analgesic
effect of U50488H was less pronounced but still significant
(P < .05).
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Effect of -FNA Pretreatment on Analgesic and Stimulatory Effects
of U50488H and Morphine
Analgesia (Table 3).
Animals
were pretreated with either -FNA (20 mg/kg s.c.) or its vehicle and
48 h later the analgesic activity of morphine (5 and 10 mg/kg
s.c.) and U50488H (5 and 10 mg/kg s.c.) was evaluated. The basal
nociceptive reaction was measured before either U50488H or morphine
treatment and nociceptive scores were remeasured 30 min after opioid
agonist injection. An overall ANOVA analysis of the nociceptive
latencies (time factor: basal versus 30-min measurement; treatment
factor: saline, two doses of morphine and two doses of U50488H;
pretreatment factor: -FNA versus vehicle) revealed significant time
effect (P < .0001), treatment effect (P < .01), and pretreatment effect (P < .05) with significant (P < .01) interactions
between factors. In vehicle-pretreated animals both doses of morphine
and U50488H produced significant analgesia (P < .01).
In -FNA-treated groups only U50488H (both doses) produced
significant increase in nociceptive latencies. In summary, pretreatment
with -FNA eliminated the analgesic effect of morphine but failed to
affect analgesia produced by U50488H.
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Stimulatory Effect (Fig. 7).
Mice were pretreated with either vehicle or -FNA and the effects of
morphine (20 mg/kg) or U50488H (1.25 mg/kg) on locomotor activity were
evaluated 48 h after pretreatment. In morphine-treated groups
two-way repeated measurement ANOVA (group factor: -FNA + morphine,
vehicle + morphine, vehicle + saline; time factor: six 10-min
intervals) revealed significant treatment effect on rearing and
motility (P < .05 and P < .01, respectively) and significant treatment × time interaction for
all parameters (P < .001, P < .05, and P < .01, respectively). The time effect was highly
significant (P < .0001) for all parameters. The post
hoc comparison revealed a significant (P < .01, compared with saline-treated group) decrease in rearing at 20 and 30 min in both morphine-treated groups with no differences between
-FNA- and vehicle-treated groups. A significant increase in motility
at 30, 40, 50, and 60 min and a significant increase in locomotion at
50 and 60 min was found in the morphine-treated group pretreated with
vehicle.
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-FNA- and vehicle-treated groups. In summary, treatment with
-FNA reduced the analgesic and motor stimulatory effects of morphine
but not those of U50488H.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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With the development of transgenic technology, the mouse has
become a species of interest for behavioral studies. Extrapolation from
rat to mouse is, however, not trivial. For example, in the opioid field
mice and rats differ in the amount and distribution of opioid receptor
subtypes and in their way of responding to opioid drugs. Rats and mice
respond to µ- and -opioid agonists in an opposite way, giving
sedation in rats but stimulation in mice. However, the present study
indicates that -opioid agonists seem to give similar effects in rats
and mice at higher doses and opposite effects at lower doses. It was
unexpected that low subanalgesic doses of the -opioid agonists
U50488H, BRL52537, and bremazocine can stimulate spontaneous motor
activity in NMRI male mice, whereas higher, analgesic doses of the
drugs reduce locomotor activity. The stimulatory effect was present
only in animals not habituated to the new test environment and it was not blocked by -opioid antagonists, but it was significantly reduced
in naloxone-treated animals. On the contrary, both nor-BNI and DIPPA
blocked the inhibitory effect of high doses of U50488H on locomotor
activity and reversed it to moderate stimulation. Finally, it was found
that H44-68, which has been shown to block d-amphetamine-induced motor stimulation in mice via dopamine
depletion (Ögren and Ross, 1977), totally eliminated the motor
stimulatory effects of U50488H without altering motor inhibitory effects.
Binding studies have shown that U50488H is selective for -opioid
receptors with little affinity for µ- and -receptors (Clark et
al., 1983). Bremazocine is less selective with affinity for -, µ-,
and 
-receptors (Tseng and Collins, 1991). Nevertheless, both U50488H
and bremazocine are considered prototype -opioid agonists (Dhawan et
al., 1996). U50488H has been shown to bind to the 1 subtype of
receptors, whereas bremazocine interacts with all subtypes of
-opioid receptors (i.e., also binds to 2/3 receptors) (Nock et
al., 1990, Horan et al., 1993). BRL52537 was shown to be a highly
selective -opioid agonist without preferable binding to any subtype
of -receptors (Vecchietti et al., 1991). Both nor-BNI and DIPPA are
highly selective -opioid receptor antagonists in vivo and they exert
long-lasting antagonistic effects, which may persist for at least 1 month (Horan et al., 1992; Jones and Holtzman, 1992; Broadber et al.,
1994; Chang et al., 1994). However, their preferences for subtypes of
-opioid receptors or species differences are not known.
The type of receptor activated by low doses of -agonists and
resulting in motor stimulation is not easily defined. It could be
argued that it is not a -receptor because nor-BNI is inactive. However, DIPPA is partially active, giving evidence for a limited -opioid receptor involvement. The observation that naloxone at a low
(preferentially µ-selective) dose blocks the stimulatory effects of
U50488H and bremazocine further supports an opioid receptor
involvement. The failure of -FNA to influence the stimulation produced by -agonists excludes the possibility of µ-opioid
receptor involvement. This paradoxical finding remains to be studied further.
The observation that -agonists stimulate motor activity
only in nonhabituated animals with intact DA-ergic neurons
indicates that -opioids elicit the stimulatory effects probably by
lowering the threshold for activation of dopaminergic neurons by
sensory stimulation. In fact, depletion of dopamine by H44-68
totally eliminated the stimulatory effects of U50488H, indicating the requirement of an intact dopaminergic system for the stimulatory effects of -opioid agonists. Differences in the activation of DA
systems may also give some explanation for the discrepancies in the
data obtained in microdialysis studies (see the Introduction).
On the basis of electrophysiological studies it was previously
suggested that -opioid agonists elicit either stimulatory or
inhibitory influence on neurons, depending on the dose applied (Crain
and Shen, 1990; Keren et al., 1999). Studies in primary dorsal root
ganglion cultures showed that neuronal stimulation occurred at
nanomolar concentrations of -opioids, whereas inhibition occurred at
micromolar concentrations. This led to the proposal that there might be
separate stimulatory and inhibitory -opioid receptor subtypes linked
to different effector systems (Crain and Shen, 1990). Stimulatory
effects are blocked by cholera toxin treatment, whereas inhibitory
effects are blocked by pertussis toxin treatment (Shen and Crain,
1990), indicating receptor coupling through Gs or
Gi proteins, respectively. Because the natural concentration of the endogenous -opioid ligand dynorphin is in the picomolar range
(You et al., 1994) it is likely that the physiological effects of
-opioids are stimulatory.
Although it is difficult to extrapolate electrophysiological data
obtained in vitro to behavioral observations, the analogy with the
present results is intriguing, and suggests that two functional
subtypes of -opioid receptors exist. Subtype A (probably inhibitory)
has low affinity for agonists, mediates antinociceptive and motor
inhibitory effects of -opioids, and is blocked by the -opioid
antagonist nor-BNI. Subtype B (presumably stimulatory) has high
affinity for agonists and contributes negligibly to the analgesic
effects of -opioids, but it mediates the motor stimulatory effects
of -opioids. This receptor is insensitive to antagonism by nor-BNI.
Subtype A receptors may be linked to Gi or Go
proteins, whereas subtype B receptors may be associated with Gs
proteins. These two "subtypes" may in fact relate to two different
conformations of a single receptor activating two different second
messenger pathways (Pauwels and Wurch, 1998). It is important to note
that the proposed functional subtypes of receptors do not relate to the
1 and 2 terminology suggested on the basis of binding studies. Both U50488H (1-selective) and bremazocine (-unselective)
inhibited motor activity at higher doses while stimulating this
activity at lower doses (although the time pattern of the stimulatory
effects of the two drugs was slightly different). The inhibitory
effects of -agonists are readily inhibited at the "inhibitory"
subtype of -opioid receptors leaving effects at the stimulatory
subtypes, which are less sensitive to antagonist blockade.
In summary, -opioid agonists U50488H, BRL52537, and bremazocine
exhibit a bimodal dose-dependent effect on spontaneous motor activity
of mice. It is not possible to conclude at this stage whether this
effect can be generalized to other species than mice. The two opposite
-opioid receptor-mediated effects (i.e., stimulation at low doses
and inhibition at higher) are most likely linked to different
functional subtypes of -opioid receptors or high- and low-affinity
states of the same receptor. The stimulatory effect of -opioids was
observed only in nonhabituated animals and could be blocked by DA
depletion, which probably indicates that tonic activity in DA-ergic
neuronal systems is required for the stimulatory effect. The tonic
activity of the endogenous -opioid (=dynorphin) systems in systems
related to reward, memory, neuronal survival, etc., is probably low.
Consequently, the effects of low, subanalgesic doses of -opioid
agonists should be explored further.
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Acknowledgments |
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The support from The Marcus and Amalia Wallenberg Foundation and Swedish Medical Research Council is gratefully acknowledged.
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Footnotes |
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Accepted for publication August 15, 2000.
Received for publication March 28, 2000.
Send reprint requests to: Dr. Alexander Kuzmin, Department of Neuroscience, Karolinska Institutet, S-171 77 Stockholm, Sweden. E-mail: Alexander.Kuzmin{at}fyfa.ki.se
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Abbreviations |
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DA, dopamine;
nor-BNI, nor-binaltorphimine 2HCl
[17,17'-(dicyclopropylmethyl)-6,6',7,7'-6,6'-imino-7,7'-bimorphinan-3,4',14,14'-tetrol];
-FNA, -funaltrexamine HCl
[(E)-4-[[(5,6)-17-cyclopropylmethyl)-4,5-epoxy-3,14-dihydroxymorphinan-6-yl]amino]-4-oxo-2-butenoic acid methyl ester];
DIPPA, 2-(3,4-dichlorophenyl)-N-methyl-N-[(1S)-1-(3-isothiocyanatophenyl)-2-(1-pyrrolidinyl)ethyl]acetamide;
U50488H, trans-(±)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide,
methanesulfonate hydrate;
BRL52537, (±)-1-(3,4-dichlorophenyl)acetyl-2-(1-pyrrolidinyl) methylpiperidine;
H44-68, -methyl-p-tyrosine methyl ester.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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