University of Ljubljana, School of Medicine, Institute of
Pathophysiology, Ljubljana, Slovenia
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Introduction |
The
ergoline derivative LEK-8829
[9,10-didehydro-N-methyl-(2-propynyl)-6-methyl-8-aminomethylergoline
bimaleinate] is an antagonist on dopamine D2
receptors and on serotonin (5-HT)2 and
5-HT1A receptors and was designed as a potential
atypical antipsychotic drug (Krisch et al., 1994
, 1996). In vitro
experiments have shown that LEK-8829 stimulates adenylate cyclase
(Panlabs) and binds to dopamine D1 receptors with
moderate affinity (Krisch et al., 1994). On the other hand, the
drug has been tested in vivo for its effects on dopamine
D1 receptors only in dopamine-depleted animals,
using the rats with unilateral lesions of striatonigral neurons with 6-hydroxydopamine (6-OHDA). In this turning model, LEK-8829 induced a
dose-dependent contralateral turning and the expression of
c-fos mRNA in dopamine-depleted striatum that were both
blocked by SCH-23390 [(R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride], a dopamine D1 receptor
antagonist, but not by haloperidol (dopamine D2
receptor antagonist) or pindolol (5-HT1A receptor
antagonist). It was therefore proposed that LEK-8829 may be an agonist
on dopamine D1 receptors (
ivin et al.,
1996). The interaction of LEK-8829 and bromocriptine has also been
analyzed in a 6-OHDA model (ivin et al., 1998). It was found
that contralateral turning that was initiated with bromocriptine was
not inhibited by the treatment with either LEK-8829 or SCH-23390,
whereas the combined treatment with both drugs inhibited the turning.
It was concluded that LEK-8829 has a dual action on the dopamine
receptors in dopamine-depleted striatum, as an agonist on dopamine
D1 receptors and as an antagonist on dopamine
D2 receptors (ivin et al., 1998).
Drugs with a D2 and 5HT2
receptor-blocking and D1 receptor-stimulating
profile are promising for the treatment of the negative symptoms of
schizophrenia (Kahn and Davis, 1995; Okubo et al., 1997; Lidow et al.,
1998). Because the dopamine depletion does not seem to be involved in
the pathogenesis of schizophrenia, it is appropriate to study the
effects of such drugs in animals with normosensitive dopamine
receptors. The rats with unilateral striatal lesion with excitotoxic
glutamate analogs (kainic, ibotenic, or quinolinic acid) may be used
for this purpose. In the striatum injected with an excitotoxin, a
degeneration and death of intrinsic neurons occur, whereas the afferent
neurons and fibers on passage are relatively spared (McGeer and McGeer,
1976; Christensen-Nylander et al., 1986). Striatal degeneration on the
lesioned side leads to the functional imbalance of the basal ganglia
(Fields et al., 1978; Mayer et al., 1990), which results in the turning
behavior in response to directly or indirectly acting dopamine
agonists. In this model, the effects of dopamine drugs are
preferentially mediated by the dopamine-innervated dopamine receptors
of the intact striatum. The rats with unilateral striatal lesion with excitotoxins respond with the ipsilateral turning behavior after application of mixed dopamine
D2/D1 receptor agonists,
such as apomorphine (Schwarcz et al., 1979; Herrera-Marschitz and
Ungerstedt, 1984; Barone et al., 1986; Fricker et al., 1996). Only a
low-intensity ipsilateral turning develops after the application of
more selective dopamine D2 receptor agonists such
as bromocriptine (Schwarcz et al., 1979) or LY-171555
[(+)-3-(3-hydroxyphenyl)-N-n-propylpiperidine] (Barone et al., 1986). The induction of ipsilateral turning by D2 receptor agonist depends on the costimulation
of dopamine D1 receptors by endogenous dopamine
or by exogenously applied D1 receptor agonists
(Dziewczapolski et al., 1997). The concept of requisite synergism
of dopamine innervated dopamine receptors (LaHoste and Marshall, 1996)
could be used to explain some aspects of dopamine receptor interactions
involved in the mediation of ipsilateral turning in rats with
excitotoxic striatal lesion.
The aim of the present study was to investigate the effects of LEK-8829
on normosensitive dopamine receptors. We hypothesized the blockade of
dopamine D2 receptors and concomitant stimulation of dopamine D1 receptors by LEK-8829. The rats
with unilateral striatal lesions induced with ibotenic acid (IA) were
used to resolve this issue. The properties of LEK-8829 were analyzed
using SCH-23390, a selective dopamine D1 receptor
antagonist; haloperidol, a D2 receptor
antagonist; SKF-82958, a selective dopamine D1
receptor agonist; and bromocriptine, a clinically used
D2 receptor agonist.
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Materials and Methods |
Animals.
We used male Wistar rats (250-300 g). The animals
were maintained on a 12-h light/dark cycle (light on, 7:00 am to 7:00
pm) in a temperature-controlled colony room at 22-24°C with free
access to rodent pellets and tap water. Groups of four animals were
housed in standard plastic cages with sawdust cover on the floor
throughout the experiment.
Drugs.
Apomorphine hydrochloride (Sigma, St. Louis, MO) was
dissolved in 0.9% saline containing 0.02% ascorbic acid. LEK-8829
(LEK, Ljubljana, Slovenia) and
(±)-6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF-82958; RBI, Natick, MA) were dissolved in 0.9% saline. Haloperidol (Haldol ampules, 5 mg/ml; Krka-Jenssen, Novo Mesto,
Slovenia) was diluted with 0.9% saline. SCH-23390 (RBI) and
2-bromo-
-ergocryptine mesylate (bromocriptine; LEK) were dissolved
in dimethyl sulfoxide, with the final solution being made up with 0.9%
saline and dimethyl sulfoxide (2:1). The doses given refer to the form
indicated above except for LEK-8829, which was calculated as the base.
The drugs were administered in a volume of 2 ml/kg s.c., except for
bromocriptine, which was injected i.p..
IA-Induced Striatal Lesions.
The stereotaxic lesions were
performed on experimentally naive rats. The animals were anesthetized
with the i.p. injection of xylazine (8 mg/kg; Rompun, Bayer,
Leverkusen, Germany), ketamine (60 mg/kg; Ketanest, Parke-Davis, Wien,
Austria), and atropine (0.6 mg/kg; Belupo, Koprivnica, Croatia) and
placed in a stereotaxic frame (TrentWells, South Gate, CA). IA (RBI)
was infused at a rate of 0.15 µl/min at three different injection
sites (each site, 0.5 µl of 0.06 M ibotenic acid dissolved in 0.1 M
phosphate buffer, pH 7.4) into the right neostriatum at the following
coordinates: A = 0.2, L = 3.0, V = 4.0, and V = 5.5 and A = 1.5, L = 2.5, and V = 4.6 (all coordinates are
given in millimeters, anterior coordinate from bregma, lateral
coordinate from the midline, and ventral coordinate from the surface of
the skull at bregma; stereotaxic coordinates according Paxinos and
Watson, 1982). The incisor bar was set 2.3 mm below the interaural
line. The infusion was delivered via a 30-gauge stainless steel cannula
connected by polyethylene tubing to a 10-µl Hamilton syringe mounted
on a microdrive pump (Harvard Apparatus, South Natick, MA). At each
injection site, the cannula was left in place for 2 min before
retraction. After surgery, the lesioned animals were left for 14 days
to recover and to allow for neuronal degeneration.
Recording of Turning Behavior.
Each rat was placed in a
plastic cylindrical chamber (40-cm diameter) of the Lablinc automated
rotometer system (Colbourn Instruments, Allentown, PA) designed for the
simultaneous electromechanical recording (Ungerstedt and Arbuthnott,
1970) of the turning behavior of eight animals. The data files of the
turning profiles of each animal (i.e., the full left/right turns per
minute) recorded by the L2T2S data acquisition software (Colbourn
Instruments) were graphically represented and analyzed using standard
Lotus 1-2-3 spreadsheet, running on a PC.
Apomorphine Test.
To determine the development of striatal
degeneration and to stabilize the turning response, the IA-lesioned
animals were primed to the stimulation of dopamine
D1 and D2 receptors by the treatment with apomorphine hydrochloride (5 mg/kg) in the third and
fourth postoperative weeks. Only the apomorphine-primed IA-lesioned rats responding with at least 300 contralateral turns during the second
apomorphine session were used in subsequent experiments. The animals
were then randomly divided into experimental groups for experiments
with drugs. The experiments with drugs started 1 week after the second
priming session with apomorphine.
Dose-Response of LEK-8829-Induced Contralateral Turning.
The
dose-response of LEK-8829-induced turning (i.e., the cumulative number
of contralateral turns, duration of turning behavior, peak turning
frequency, mean turning frequency, and onset of turning) was assessed
using five groups of IA-lesioned animals. Each group received one of
the following doses of LEK-8829: 0.5, 1, 3, or 15 mg/kg or saline.
Effect of SCH-23390 on Contralateral Turning Behavior Induced by
LEK-8829.
A group of four animals was treated in three
experimental sessions as follows: 1) saline + LEK-8829 (3 mg/kg), 2)
SCH-23390 (0.5 mg/kg) + LEK-8829 (3 mg/kg), and 3) SCH-23390 (1 mg/kg) + LEK-8829 (3 mg/kg). The saline + SCH-23390 pretreatment was given 20 min before the administration of LEK-8829. Experimental sessions were
performed in weekly intervals.
Effect of Haloperidol on Ipsilateral Turning Behavior Induced by
SKF-82958.
A group of five animals was treated in four
experimental sessions as follows: 1) saline + SKF-82958 (3 mg/kg), 2)
haloperidol (0.25 mg/kg) + SKF-82958 (3 mg/kg), 3) haloperidol (0.25 mg/kg) + saline, and 4) saline + saline. The saline + haloperidol
pretreatment was given 20 min before the administration of SKF-82958 or
saline. There was a drug-free interval of 1 week between each
experimental session. The cumulative number of contralateral and
ipsilateral turns and the maximal turning frequency (peak turning
frequency) were registered during a 240-min period. The average number
of cumulative ipsilateral turns was compared with the average number of
cumulative contralateral turns using a two-tailed paired Student's t test (p < .05) to verify the statistical
significance of the direction of turning.
Dose Dependence of Bromocriptine/LEK-8829 Interaction.
In
the first experiment, a group of four animals received injections of
bromocriptine at 0 min and saline + LEK-8829 at 120 min in seven
experimental sessions as follows: 1) bromocriptine (30 mg/kg) + saline,
2) bromocriptine (30 mg/kg) + LEK-8829 (0.5 mg/kg), 3) bromocriptine
(30 mg/kg) + LEK-8829 (1 mg/kg), 4) bromocriptine (30 mg/kg) + LEK-8829
(3 mg/kg), 5) bromocriptine (30 mg/kg) + LEK-8829 (10 mg/kg), 6)
bromocriptine (30 mg/kg) + LEK-8829 (30 mg/kg), and 7) bromocriptine
(30 mg/kg) + saline. There was a drug-free interval of 1 week between
each experimental session. The cumulative number of ipsilateral turns
recorded in 2 h after the injection of LEK-8829 was calculated and
used for statistical evaluation.
In the second experiment, four groups of four animals received
injections of either bromocriptine or saline at 0 min and LEK-8829 at
120 min as follows: 1) saline + LEK-8829 (3 mg/kg), 2) bromocriptine (3 mg/kg) + LEK-8829 (3 mg/kg), 3) bromocriptine (10 mg/kg) + LEK-8829 (3 mg/kg), and 4) bromocriptine (30 mg/kg) + LEK-8829 (3 mg/kg). The
cumulative number of turns recorded in 2 h after the injection of
LEK-8829 was calculated to perform statistical evaluation of the
effects of bromocriptine on contralateral turning induced by LEK-8829.
Effect of SCH-23390 or Haloperidol on Bromocriptine/LEK-8829
Interaction.
Five groups of four animals received injections of
bromocriptine at 0 min, followed by drug/saline injections at 100 and
120 min as follows: 1) bromocriptine (30 mg/kg) + saline + saline, 2)
bromocriptine (30 mg/kg) + saline + LEK-8829 (3 mg/kg), 3) bromocriptine (30 mg/kg) + SCH-23390 (1 mg/kg) + LEK-8829 (3 mg/kg), 4)
bromocriptine (30 mg/kg) + haloperidol (5 mg/kg) + LEK-8829 (3 mg/kg),
and 5) bromocriptine (30 mg/kg) + haloperidol (5 mg/kg) + saline. The
cumulative number of ipsilateral turns recorded in 2 h after the
third injection was calculated for each experimental group and used for
statistical evaluation.
Histochemical Visualization of IA Lesions.
At the completion
of behavioral experiment, eight representative animals
(apomorphine-induced ipsilateral rotations, 472 ± 94) were
decapitated while under CO2 anesthesia, and the
brains were rapidly frozen on dry ice, wrapped in a Parafilm, and
stored at 
70°C until further processing. Six evenly spaced striatal cryostat sections (10 µm) were cut between anteroposterior
coordinates + 2.2 mm and 1.3 mm relative to bregma and thaw-mounted
onto gelatinized slides. The sections were fixed in 4%
paraformaldehide for 5 min and processed for acetylcholinesterase
(AChE) staining using 0.02 mM iso-OMPA (tetraisopropyl
pyrophosphoramide; Koch-Light, Colnbrook, Buch, England) as inhibitor
of nonspecific esterase activity (Koelle and Friedenwald, 1949).
AChE-stained sections were coverslipped with Canada balsam.
Morphometrical analysis of the extent of striatal lesions induced with
IA was performed by using the MCID, M4 image analyzer (Imaging Research
Inc., Canada). Six different levels between + 2.2 mm and 1.3 mm
relative to bregma were evaluated from eight representative animals.
The total area of the spared portion on the lesioned side was measured
(mm2) and compared with the total striatal area
of intact side.
Statistical Analysis.
All values are expressed as mean ± S.E.M. number of turns during the observation period, where
n represents the number of animals. Statistical evaluation
of the effect of treatments between experimental sessions performed on
the same group of animals and for the statistical evaluation of the
effect of treatments between different groups of animals was evaluated
using one-way ANOVA followed with Scheffe's multiple-comparison test.
A two-tailed paired Student's t test was used to verify the
statistical significance of the direction of turning in the
haloperidol/SKF experiment because the animals did not turn exclusively
in one direction.
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Results |
Dose-Response of LEK-8829-Induced Contralateral Turning in IA
Model.
The result of the dose-response experiment showing the
total number of turns and the duration of turning behavior induced by
LEK-8829 is shown in Table 1. With lower
doses of LEK-8829 (i.e., 0.5 and 1 mg/kg), the animals adopted
contralateral posture, with a few contralateral turns that were
observed for 2 h after the injection of the drug. With higher
doses of LEK-8829 (3 and 15 mg/kg), the contralateral turning become
more vigorous, with significantly increased number of cumulative number
of turns, duration of turning behavior, and turning frequency
(p < .05; ANOVA followed with Scheffe's
multiple-comparison test).
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TABLE 1
Dose-response of turning behavior after injection of LEK-8829
Five groups of IA-lesioned rats (A-D) were injected with 0.5, 1, 3, or
15 mg/kg of LEK-8829. Group E was injected with saline. Cumulative
contralateral turns, duration of turning, peak turning frequency
(maximal number of turns per minute), mean turning frequency (mean
number of turns per minute), and onset time of turning were
statistically evaluated using one-way ANOVA with Scheffe's
multiple-comparison test, p < .05. Data are expressed
as mean ± S.E.M.; n represents number of animals in
group.
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Effect of SCH-23390 on Contralateral Turning Behavior Induced by
LEK-8829.
The pretreatment with SCH-23390 resulted in a
dose-dependent inhibition of LEK-8829-induced contralateral turning.
After the pretreatment with SCH-23390 at the dose of 0.5 mg/kg, the
total number of contralateral turns induced by LEK-8829 (3 mg/kg) was nonsignificantly reduced, whereas the pretreatment with SCH-23390 with
the dose of 1 mg/kg resulted in almost complete inhibition of
contralateral turning induced by LEK-8829 (3 mg/kg) (by 70%, n = 4, p > .05, and by 90%,
n = 4, p < .05, respectively, compared with the cumulative number of contralateral turns recorded in a
control, saline-pretreatment session; one-way ANOVA with Scheffe's multiple-comparison test) (Fig. 1).
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Fig. 1.
Effect of pretreatment with saline or SCH-23390 on
LEK-8829-induced turning in IA-lesioned rats. Experimental sessions
were conducted at weekly intervals as follows: SAL + LEK-8829 (3)
(pretreatment with saline, treatment with LEK-8829 3 mg/kg); SCH (0.5) + LEK-8829 (3) (pretreatment with SCH-23390 0.5 mg/kg, treatment with
LEK-8829 3 mg/kg); and SCH (1) + LEK-8829 (3) (pretreatment with
SCH-23390 1 mg/kg, treatment with LEK-8829 3 mg/kg). Saline + SCH-23390
pretreatment was given 20 min before the administration of LEK-8829.
Columns represent mean cumulative of contralateral turns induced by
LEK-8829 (3 mg/kg), error bars indicate S.E.M. (n = 4). Statistical evaluation: (*) significantly decreased total number of
turns in group SCH (1) + LEK-8829 (3) compared with group SAL + LEK-8829 (3) (n = 4, p < .05, one-way ANOVA with Scheffe's multiple-comparison test).
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Effect of Haloperidol on SKF-82958-Induced Turning Behavior.
The treatment of saline-pretreated animals (n = 5) with
SKF-82958 has induced a weak, although significant, turning toward the
side of the lesion. During the saline treatment, the animals did not
exhibit turning behavior or recognizable postural bias. During the
haloperidol treatment, all the animals adopted contralateral posture.
The administration of SKF-82958 to haloperidol-pretreated animals
induced a significant contralateral turning of low intensity (Table
2).
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TABLE 2
Effect of haloperidol on SKF-82958-induced turning behavior
A group of five IA-lesioned rats were treated in weekly intervals as
follows: apomorphine (5 mg/kg), saline + SKF-82958 (3 mg/kg),
haloperidol (0.25 mg/kg) + SKF-82958 (3 mg/kg), haloperidol (0.25 mg/kg) + saline, and saline + saline. Cumulative number of turns
and peak turning frequency were calculated for contralateral and
ipsilateral turning after each treatment for 240 min. Data are
expressed as mean ± S.E.M. (n = 5).
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Dose Dependence of Bromocriptine/LEK-8829 Interaction.
In the
first experiment, a long-lasting ipsilateral turning behavior (greater
than 12 h) of low rotational speed with a long latency to the
onset of turning was recorded in the first bromocriptine/saline session. Saline injection did not significantly affect the
bromocriptine-induced turning (see Fig. 5A). In five experimental
sessions that followed, the interaction of bromocriptine (30 mg/kg,
injected at 0 min) with LEK-8829 (0.5, 1, 3, 10, or 30 mg/kg, injected
at 120 min) was investigated. Statistical analysis has shown a
significant, dose-dependent potentiation of bromocriptine-induced
ipsilateral turning by LEK-8829 (1, 3, and 10 mg/kg) (n = 4, p < .05, one-way ANOVA with Scheffe's
multiple-comparison test). The potentiation was most effective with the
dose of 3 mg/kg of LEK-8829 (Fig. 2). On
the other hand, the interaction of bromocriptine (30 mg/kg)/LEK-8829 (30 mg/kg) resulted in a very distinctive, polyphasic turning profile.
The cumulative number of ipsilateral turns recorded in a 2-h period
after the injection of LEK-8829 was not significantly different from
the control value (n = 4, p > .05, one-way ANOVA with Scheffe's multiple-comparison test) (Fig. 2).
Shortly after the injection of LEK-8829, there was a potentiation of
ipsilateral turning (see Fig. 5C). The initial potentiation was then
replaced by the inhibition of turning, which lasted for about 1 h.
The inhibition of turning was followed by a gradual reappearance of ipsilateral turning (Fig. 3B and see Fig.
5C). At about 4 h after the injection of LEK-8829, the animals
changed the direction of turning to the contralateral side (Fig. 3B).
The cumulative number of turns and the turning profiles recorded in the
last bromocriptine/saline session were not significantly different from
the control value obtained in the first bromocriptine/saline session
(n = 4, p > .05, one-way ANOVA with
Scheffe's multiple-comparison test) (Fig. 2).
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Fig. 2.
Bromocriptine/LEK-8829 interaction in IA-lesioned
rats. Columns represent mean cumulative ipsilateral turns recorded
during 2 h after administration of saline/LEK-8829; error bars
indicate S.E.M. (n = 4). Saline + LEK-8829
treatment was given 120 min after administration of bromocriptine.
Experimental sessions were performed at weekly intervals as follows: 1)
BRO (30) + SAL (w1) (30 mg/kg bromocriptine, saline, first week), 2)
BRO (30) + LEK (0.5) (30 mg/kg bromocriptine, 0.5 mg/kg LEK-8829), 3)
BRO (30) + LEK (1) (30 mg/kg bromocriptine, 1 mg/kg LEK-8829), 4) BRO
(30) + LEK (3) (30 mg/kg bromocriptine, 3 mg/kg LEK-8829), 5) BRO (30) + LEK (10) (30 mg/kg bromocriptine, 10 mg/kg LEK-8829), 6) BRO (30) + LEK (30) (30 mg/kg bromocriptine, 30 mg/kg LEK-8829), and 7) BRO (30) + SAL (w7) (30 mg/kg bromocriptine, saline, last week). Statistical
evaluation: significantly increased total number of ipsilateral turns
*compared with groups BRO (30) + SAL (w1), BRO (30) + LEK (30), BRO
(30) + SAL (w7), (+) compared with BRO (30) + LEK (0.5), and  compared
with BRO (30) + SAL (w1), BRO (30) + LEK (30) (n = 4, p < .05, one-way ANOVA with Scheffe's
multiple-comparison test).
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Fig. 3.
Individual recordings of turning behavior of
representative IA-lesioned rat showing effect of high dose of LEK-8829
on bromocriptine-induced ipsilateral turning. In the first experimental
session, the rat received injection of 30 mg/kg bromocriptine [BRO
(30)] at 0 min, followed by injection of saline (SAL) at 120 min (A).
In the second experimental session, the same rat was treated with 30 mg/kg bromocriptine [BRO (30)] at 0 min, followed by the injection of
30 mg/kg LEK-8829 [LEK (30)] at 120 min (B). Profiles show rotational
speed (turns/min) recorded for 10 h. Above zero line,
contralateral rotations; below zero line, ipsilateral rotations. Arrows
indicate the timing of drug/saline injections. Note initial inhibition
of ipsilateral turning induced by LEK-8829 and a change of direction of
turning (from ipsilateral to contralateral) 4 h after the
treatment with LEK-8829.
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The second experiment revealed the effect of different doses of
bromocriptine pretreatment on contralateral turning induced by
LEK-8829. Pretreatment with saline or bromocriptine at the dose of 3 mg/kg did not significantly affect the contralateral turning induced by
3 mg/kg of LEK-8829 (Fig. 4).
Bromocriptine pretreatment at the dose of 3 mg/kg per se induced only a
few ipsilateral turns (observed in 2-h period before the injection of
LEK-8829; Fig. 5E). On the other hand,
the pretreatment with bromocriptine at the dose of 10 or 30 mg/kg
induced a significant ipsilateral turning response that was potentiated
by the injection of LEK-8829 (3 mg/kg) (Fig. 5, B and F). The
cumulative number of ipsilateral turns recorded in 2 h after the
injection of LEK-8829 was dependent on the dose of bromocriptine used
in the pretreatment (Fig. 4).
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Fig. 4.
Bromocriptine/LEK-8829 interaction in IA-lesioned
rats. Columns represent mean cumulative turns recorded during 2 h
after administration of LEK-8829. Error bars indicate S.E.M.
(n = 4). IPSI, ipsilateral turning; CONTRA,
contralateral turning. Four groups of rats received LEK-8829 injections
120 min after administration of saline/bromocriptine as follows: 1) SAL + LEK (3) (saline, 3 mg/kg LEK-8829), 2) BRO (3) + LEK (3) (3 mg/kg
bromocriptine, 3 mg/kg LEK-8829), 3) BRO (10) + LEK (3) (10 mg/kg
bromocriptine, 3 mg/kg LEK-8829), and 4) BRO (30) + LEK (3) (30 mg/kg
bromocriptine, 3 mg/kg LEK-8829). *Significantly
increased total number of ipsilateral turns in group BRO (30) + LEK (3)
compared with group BRO (10) + LEK (3) (n = 4, p < .05, one-way ANOVA with Scheffe's
multiple-comparison test).
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Fig. 5.
Turning profiles of bromocriptine/LEK-8829
interaction in IA-lesioned rats. (A-F) Rotational speed (turns/min) of
individual rats recorded for 6 h. Rats were injected with
bromocriptine/saline at 0 min, followed by injection of LEK-8829/saline
at 120 min (arrows) as follows: A, 30 mg/kg bromocriptine [BRO (30)],
saline (SAL); B, 30 mg/kg bromocriptine [BRO (30)], 3 mg/kg LEK-8829
[LEK (3)]; C, 30 mg/kg bromocriptine [BRO (30)], 30 mg/kg LEK-8829
[LEK (30)]; D, saline (SAL), 3 mg/kg LEK-8829 [LEK (3)]; E, 3 mg/kg
bromocriptine [BRO (3)], 3 mg/kg LEK-8829 [LEK (3)]; and F, 10 mg/kg bromocriptine [BRO (10)], 3 mg/kg LEK-8829 [LEK (3)]. Above
zero line, contralateral rotations; below zero line, ipsilateral
rotations.
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Effect of SCH-23390 and Haloperidol on Bromocriptine/LEK-8829
Interaction.
The experiment with specific antagonists of dopamine
receptors D1 and D2 (1 mg/kg SCH-23390 and 5 mg/kg haloperidol, respectively) has shown that
both antagonists almost completely inhibited the ipsilateral turning
response mediated by the bromocriptine (30 mg/kg)/LEK-8829 (3 mg/kg)
combination (by 99% and 93%, respectively, compared with the effect
of saline in the control bromocriptine/LEK-8829 group,
n = 4, p < .05, one-way ANOVA with
Scheffe's multiple-comparison test). The experiment also revealed a
significant inhibition of bromocriptine-induced turning in response to
haloperidol (by 90%, n = 4, p < .05, one-way ANOVA with Scheffe's multiple-comparison test) (Fig.
6).
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Fig. 6.
Effect of SCH-23390 and haloperidol on turning
profile of bromocriptine/LEK-8829 interaction in IA-lesioned rats.
Columns represent mean cumulative ipsilateral turns recorded during
2 h after the administration of LEK-8829/saline; error bars
indicate S.E.M. (n = 4). Each group was treated
with bromocriptine at 0 min, followed by drug/saline injections at 100 and 120 min as follows: BRO + SAL + SAL (30 mg/kg bromocriptine,
saline, saline); BRO + SAL + LEK (30 mg/kg bromocriptine, saline, 3 mg/kg LEK-8829); BRO + SCH + LEK (30 mg/kg bromocriptine, 1 mg/kg
SCH-23390, 3 mg/kg LEK-8829); BRO + HAL + LEK (30 mg/kg bromocriptine,
5 mg/kg haloperidol, 3 mg/kg LEK-8829); BRO + HAL + SAL (30 mg/kg
bromocriptine, 5 mg/kg haloperidol, saline). Statistical evaluation:
*significantly decreased total number of turns in groups BRO + SCH + LEK, BRO + HAL + LEK, and BRO + HAL + SAL compared with BRO + SAL + LEK
group; significantly decreased total number of turns in groups BRO + HAL + SAL and BRO + SCH + LEK compared with BRO + SAL + SAL group (for
all comparisons: n = 4, p < .05, one-way ANOVA with Scheffe's multiple-comparison test).
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Visualization of Position and Extent of Striatal Lesions.
The
AChE histochemistry performed on eight representative animals has shown
extensive excitotoxic lesions positioned in the central striatum as
shown in Fig. 7. Histometrical analysis
revealed that the IA-injection destroyed 50 to 70% of the striatal
tissue (Table 3). This resulted in the
shrinkage of the striatum and enlargement of lateral ventricle on the
lesioned side. Less intense AChE staining was found in the
frontoparietal cortex overlying the lesioned striatum. Other
extrastriatal regions were not visibly affected.
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Fig. 7.
AChE-stained coronal sections from one representative
rat with IA lesion (449 ipsilateral turns induced by 5 mg/kg
apomorphine). Sections were taken from six different anteroposterior
coordinates relative to bregma: A, from + 2.2 to +1.7; B, from +1.7 to
+0.7; C, from +0.7 to 0.2; D, from 0.2 to 0.3; E, from 0.3 to
0.8; and F, from 0.8 to 1.3. Lesions of striatal tissue are
placed in right striatum shown on the left side of figures.
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TABLE 3
Morphometrical analysis of unilateral striatal lesions induced with
ibotenic acid
Six acetylcholinesterase-stained coronal sections from eight randomly
chosen rats (apomorphine-induced total ipsilateral rotations: 472 ± 94) were used for evaluation of extension of exitotoxic lesions with
MCID, M4 image analyzer. Morphometrical analyses were performed on six
different anteroposterior (A-P) coordinates relative to bregma: from
+2.2 to +1.7, from +1.7 to +0.7, from +0.7 to 0.2, from 0.2 to 0.3,
from 0.3 to 0.8, and from 0.8 to 1.3. Total area of intact and
lesioned striatum (mm2) was taken from above coordinates. Data
represent proportions (% ± S.E.M., n = 8) of spared
portion of lesioned striatum relative to striatal area of intact side.
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Discussion |
Induction of Contralateral Turning in Response to LEK-8829.
In
rats with excitotoxic striatal lesions, the dopamine receptor agonists
induce ipsilateral turning (Schwarcz et al., 1979; Herrera-Marschitz
and Ungerstedt, 1984; Dunnett et al., 1988; Norman et al., 1990). The
ipsilateral turning is generally less intense than the contralateral
turning induced in rats with unilateral dopaminergic striatal
deafferentation with 6-OHDA, which are supersensitive to dopamine
receptor agonists (Schwarcz et al., 1979, Herrera-Marschitz and
Ungerstedt, 1984). Few reports of turning induced with apomorphine or
amphetamine have shown the dependence of the direction of turning on
the localization of the excitotoxic lesion in the striatum (Kafetzopoulos et al., 1988; Norman et al., 1992; Fricker et al., 1996).
In our experiment, the lesions were positioned in the central striatum
as revealed by the AChE staining. The morphometric analysis of the
remaining striatal tissue revealed that 50 to 70% of the striatum was
destroyed by IA. Diminished AChE staining was found in the
frontoparietal cortex overlying the lesioned striatum. The decreased
AChE staining could be due to the nonspecific lesion of the cholinergic
projection from the basal forebrain to the ipsilateral cerebral cortex.
Apomorphine was used in the two priming sessions to verify the
development of striatal degeneration and to prevent further
sensitization in subsequent experiments (cf. Norman et al., 1990). When
challenged with apomorphine, all the rats used in our experiments
turned ipsilaterally.
The induction of dose-dependent contralateral turning in IA-lesioned
rats with LEK-8829 was therefore not expected. The LEK-8829-induced turning was blocked by SCH-23390, indicating a mechanism linked to
dopamine D1 receptors. To further analyze the
receptors that were potentially involved in the induction of
contralateral turning, we injected a specific dopamine
D1 receptor agonist, SKF-82958, into
haloperidol-pretreated rats. In vehicle-pretreated rats, SKF-82958
induced a weak ipsilateral turning, whereas in haloperidol-pretreated rats, SKF-82958 induced a weak contralateral turning. Haloperidol per
se induced only a contralateral posture. It has been described previously that in rats and mice with different types of unilateral striatal lesions that neuroleptics induced a postural asymmetry of the
head, neck, and body, away from the side of lesion (for review, see
Pycock, 1980).
Striatal dopamine D1 and D2
receptors seem to be located at functionally distinct sites
(Herrera-Marschitz and Ungerstedt, 1984). They may be largely separated
on the neurons of direct and indirect striatofugal pathways,
respectively (Gerfen, 1992). It is known that stimulation of the
dopamine receptors modulates the activity of the motor circuit of the
basal ganglia represented by two major pathways: transmission over the
direct pathway is facilitated via dopamine D1
receptors, and transmission over the indirect pathway is inhibited via
dopamine D2 receptors. Phasic activation of the
direct pathway results in the reduction of tonic inhibitory basal
ganglia output, resulting in the disinhibition of thalamocortical
neurons and facilitation of movement. By contrast, phasic activation of
the indirect pathway leads to increased basal ganglia output and to
suppression of movement (Wichmann and DeLong, 1996). The overall effect
of dopamine release is to reduce basal ganglia output, leading to
increased activity of thalamocortical projection neurons (Gerfen,
1995). It may be implied that D2 agonists inhibit, whereas D2 antagonists stimulate, the
striatal neurons of the indirect pathway, facilitating and suppressing
the movement, respectively. On the other hand, haloperidol does not
seem to have such effects on the IA-lesioned side. A partial loss of
striatal dopamine D2 receptors in the IA-lesioned
striatum (Mayer et al., 1990) may provide an explanation for the
diminished effects of haloperidol on the lesioned side. A preferential
degeneration of the D2 receptor-bearing neurons
of the indirect striatonigral pathway that would lead to the
disinhibition of thalamocortical neurons has been proposed to explain
the mechanism for the development of dyskinesia in Huntington's chorea
(Albin et al., 1992). The excitotoxic lesion induced with IA, however,
is not selective in this respect and also affects the
D1 receptor-bearing neurons of the direct
striatonigral efferents (Arai et al., 1987; Mansour et al., 1992). On
the other hand, a transneuronal degeneration of substantia nigra
reticulata may develop after the striatal lesion with IA (Saji et al.,
1997; DeGiorgio et al., 1998). The transneuronal degeneration of the
GABAergic nigrothalamic neurons of the pars reticulata therefore seems
to be a more likely cause for the disinhibition of thalamocortical
neurons on the IA-lesioned side. We speculate that as the result of
unilateral striatal lesion, a latent thalamocortical imbalance
develops. In untreated animals, the intact side may provide
compensation. If the thalamocortical tonus on the intact side is
decreased due to the blockade of the dopamine D2
receptors, the increased thalamocortical tonus on the lesioned side is
disclosed, leading to contralateral posture of the animals.
In kainic acid-lesioned animals, neuroleptic agents inhibited the
ipsilateral turning induced by apomorphine, whereas the induction of
contralateral turning was not described (Schwarcz et al., 1979;
Herrera-Marschitz and Ungerstedt, 1984). On the other hand, our
experiments in IA-lesioned animals pretreated with haloperidol have
shown that the stimulation of dopamine D1 receptors by SKF-82958 induces a weak contralateral turning. It appears
as if the dopamine D2 receptor blockade
determines the side of rotation, whereas the dopamine
D1 receptor stimulation determines the locomotor
component (frequency of rotation), implying some similarities with the
concept of a two-component model of rotating rodent (Kelly, 1977;
Pycock and Marsden, 1978). The same explanation could be used to
explain the effects of LEK-8829 as an D1
agonist/D2 antagonist drug. The intensity of the
LEK-8829-induced contralateral turning may, however, also depend on
additional receptor affinities of the drug (blockade of receptors
5-HT1A and/or 5-HT2, cf.
Krisch et al., 1994, 1996).
Interaction of LEK-8829 with Bromocriptine.
The treatment with
a high dose of bromocriptine induced a prolonged ipsilateral turning of
low frequency that was blocked by haloperidol. Low doses of LEK-8829
significantly potentiated the ipsilateral turning induced by a high
dose of bromocriptine. The potentiation of bromocriptine-induced
turning by LEK-8829 suggests the synergistic effects of bromocriptine
and LEK-8829 on dopamine D2 and
D1 receptors, respectively. The synergism of LEK-8829 with bromocriptine was prevented either by SCH-23390 or by
haloperidol. It was described previously that dopamine
D1 receptor agonists act synergistically with
bromocriptine (Robertson and Robertson, 1986; Weick and Walters, 1987;
Robertson et al., 1992). The ipsilateral turning mediated by dopamine
D2 receptor agonists, such as bromocriptine, may
therefore depend on the coactivation of dopamine
D1 receptors by endogenous dopamine
(Dziewczapolski et al., 1997). By analogy, the blocking effect of
haloperidol on the potentiation of bromocriptine-induced ipsilateral
turning in response to LEK-8829 may also be explained by the necessity of the costimulation of dopamine D2 receptors for
the induction of significant ipsilateral turning by the drugs acting on
dopamine D1 receptors in this model.
The potentiation of bromocriptine-induced ipsilateral turning in
response to LEK-8829, however, contradicts the previous findings (Krisch et al., 1994) regarding the antagonistic effect of LEK-8829 on
dopamine-innervated D2 receptors; namely, if
LEK-8829 is an antagonist of dopamine D2
receptors, the drug should block the agonist effects of bromocriptine
at dopamine D2 receptors and therefore inhibit
the ipsilateral turning. This apparent contradiction was resolved in
the dose-response experiments that have demonstrated the importance of
the ratio of doses of both drugs with the proposed opposing activity at
D2 receptors for the synergistic potentiation of
ipsilateral turning. The ipsilateral turning mediated by a high dose of
bromocriptine was potentiated only by the lower doses of LEK-8829,
indicating that the low dose of LEK-8829 may not prevent the
stimulation of dopamine D2 receptors by a high
dose of bromocriptine. On the contrary, our experiments have
demonstrated that it may in fact induce synergistic effects by
costimulating dopamine D1 receptors. When the
highest dose of LEK-8829 was used, the potentiation of ipsilateral
turning occurred only for a brief period of time and was followed by a
longer period of inhibition of ipsilateral turning. This was followed
by the gradual disinhibition of ipsilateral turning and switch to
contralateral turning toward the end of experiment. The complex turning
profile observed in animals pretreated with a high dose of
bromocriptine in response to a high dose of LEK-8829 may be dependent
on the pharmacokinetic factors determining the rate of displacement of
bromocriptine from D2 receptors by LEK-8829. We
speculate that during the initial displacement period, the dopamine
D1 and D2 receptors are
costimulated by LEK-8829 and bromocriptine, respectively,
synergistically potentiating the ipsilateral turning behavior for a
brief period of time. Due to the gradual displacement of bromocriptine
from the dopamine D2 receptors by LEK-8829, the
stimulation of dopamine D2 receptors eventually
becomes insufficient for the synergistic costimulation of dopamine
D1 receptors, resulting in the gradual weakening
of ipsilateral turning. When the displacement of bromocriptine is completed, the resulting blockade of D2 receptors
and stimulation of D1 receptors by LEK-8829
result in contralateral turning. However, this explanation does not
explain the apparent disinhibition of ipsilateral turning in the period
preceding the onset of contralateral turning.
Our results have shown that LEK-8829 may be an agonist of dopamine
D1 receptors and an antagonist at dopamine
D2 receptors in dopamine-innervated striatum.
This finding is in agreement with our results in the 6-OHDA model, in
which LEK-8829 was found to be an agonist on dopamine
D1 and an antagonist on dopamine D2 receptors in dopamine-depleted striatum
(ivin et al., 1996, 1998).
The recent proposals that neuroleptic agents with additional
D1 agonistic properties may be beneficial in the
treatment of the negative symptoms of schizophrenia (Kahn and Davis,
1995; Okubo et al., 1997; Lidow et al., 1998) put LEK-8829, with its D1 agonistic and
D2/5-HT2 antagonistic
properties, in a promising perspective.
IA, ibotenic acid;
AChE, acetylcholinesterase;
5-HT, serotonin;
iso-OMPA, tetraisopropyl pyrophosphoramide;
6-OHDA, 6-hydroxydopamine;
LEK-8829, 9,10-didehydro-N-methyl-(2-propynyl)-6-methyl-8-aminomethylergoline
bimaleinate;
LY-171555, (+)-3-(3-hydroxyphenyl)-N-n-propylpiperidine;
SCH-23390, (R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine
hydrochloride, SKF-82958,
(±)-6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide.
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Abstract
Introduction
