Sulpiride

The effect of DI and D2 dopamine receptor antagonists on the DNA-binding activity of the AP-1 transcription factor was studied in the rat caudate-putamen and globus pallidus following a unilateral 6-OHDA lesion of the medial forebrain bundle. In the caudate-putamen, vehicle-treated rats showed increased AP- 1 DNA-binding activity, which appeared to be reversed completely by treatment with the DI receptor antagonist SCH23390 (1 mg kg-' i. p.) and partially by the D2 antagonist sulpiride (50 mg kg-' i.p.). In the globus pallidus, vehicle and sulpiride did not induce AP-1, while SCH23390 increased it significantly. This induction was much more prominent in the 6-OHDA-lesioned hemisphere than in the intact hemisphere. The present study suggests that in the caudate-putamen, dopamine-depletion induces long-lasting enhancement of AP-1 DNA-binding activity via activation of D1 receptors and the simultaneous activation of D2 receptors facilitate it. However, in the globus pallidus of the 6-OHDAlesioned hemisphere, DI but not D2 antagonism induces AP-1 in certain cell populations which may be distinct from those expressing AP-1 upon stimulation of D2 receptors. [Neurol Res 1999; 21: 175-179]

Keywords: Dopamine antagonist; SCH23390; sulpiride; AP-1; caudate-putamen; globus pallidus

INTRODUCTION

Treatment with dopamine agonists or antagonists often results in the development of movement disorders such as clinical fluctuations with long-term levodopa therapy in Parkinson's disease patients1 and extrapyramidal symptoms with chronic neuroleptic use2. Abnormal dopaminergic transmission in the basal ganglia may underlie these disorders; however, the mechanisms of cellular responsiveness to dopamine in these areas have not been elucidated fully. For example, dopaminergic neurons can modulate the induction of activator protein1 (AP-1 ) transcription factor in target neurons in the central nervous system (CNS)3,4. We previously reported that AP-1 DNA-binding activity is increased by DI and D2 receptor agonists in the caudate-putamen and globus pallidus, respectively, of rats with an ipsilateral 6-hydroxydopamine (OHDA)-lesion of nigrostriatal neurons. Each effect was enhanced by combined administration of D2 and D1 agonists, respectively. In order to further clarify the regulatory mechanisms of cellular responsiveness by dopamine, we determined the AP-1 DNA-binding activity in the caudate-putamen and globus pallidus of rats challenged with either a D1 or D2 antagnist.

MATERIALS AND METHODS

Animal preparation

Male Sprague-Dawley rats weighing 260-280g were purchased from Charles River (Yokohama, Japan). Animals were housed two or three per cage and given free access to food and water under controlled conditions of temperature (25 deg C) and humidity, with a 12-h light-dark cycle. They were treated in accordance with Guidelines for Animal Experiment at Okayama University Medical School.

In order to destroy the nigrostriatal dopaminergic neurons, 8 (mu)g of 6-hydroxydopamine (6-OHDA) in 4 (mu)l of sterile saline containing 0.2% ascorbic acid (Sigma, St. Louis, MO, USA) was injected into the left medial forebrain bundle of each animal according to the atlas of Pellegrino et al.5 using the following coordinates: 2 mm posterior to the bregma, 1.4 mm left of midline, and 8.6 mm ventral to the dural surface. Prior to injection, animals were anesthetized with 50 mg kg-' i.p. sodium pentobarbital, and fitted to a stereotaxic frame. They were pretreated with 25 mg kg^ sub -1^ i.p. desipramine (Sigma), 30 min before surgery to prevent the uptake of 6-hydroxydopamine (6-OHDA, Sigma) by noradrenergic terminals. 6-OHDA was injected over a 5-min period using a microinfusion pump. Following a 2-week recovery period, rats were challenged with 0.5 mg kg-' s.c. apomorphine (Sigma). Animals exhibiting a rotational response of more than five rotations per min contralateral to the lesioned side 30 min after this challenge were selected for further study. Following the apomorphine challenge, animals were housed one or two in a cage, handled gently for one week, and assigned randomly into one of three treatment groups.

Drug treatment

Animals were habituated in the cage for 1 h prior to drug injection. On drug treatment, each rat received an i.p. injection of either 1 mg kg-' SCH23390 [(R)-(+)-8chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1 H-3-benzazepin-7-ol (RBI, Natic, MA, USA)], 50 mg kg sulpiride (Sigma), or vehicle. The animals were decapitated 2 h following the drug challenge; their brains were removed, frozen in dry ice, and stored at -80 deg C.

Tissue preparation

On examination, the caudate-putamen and globus pallidus were removed at -15 deg C as described previously4. The tissues were homogenized, and nuclear protein extracts were prepared according to the procedure of Schreiber et al. 6 In brief, each brain tissue sample was homogenized in 400 (mu)I of ice-cold 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesu If on ic acid (HEPES) buffer (pH 7.9) containing 10 mM KCI, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 0.1 mM ethyleneglycol-bis (Beta-amino-ethylether)-N,N,N',N'-tetraacetic acid (EGTA), 1 mM dithiothreitol (DTT) and 0.5 mM phenylmethylsulfonyl fluoride (PMSF). Following the addition of 25 (mu)l of 10% Nonidet P-40 (Sigma), the suspension was vortex mixed vigorously. The nuclear fraction was precipitated by centrifugation at 15,00xg for 5 min at 4 deg C. The pellets were suspended in 40 (mu)I of ice-cold 20 mM HEPES buffer (pH 7.9) containing 0.4 M NaCI, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF. The mixtures were left on ice for 15 min with frequent agitation. The supernatant of the nuclear extracts was collected by centrifugation at 15,000xg for 5 min at 4oC. An aliquot of the nuclear extracts was analyzed for protein concentration using a Protein assay kit II (Bio-Rad Laboratories Inc., Hercules, CA, USA); the rest of the nuclear extract was frozen at -80 deg C until assay. Gel mobility-shift assay The oligomer used in this study was AP-1, purchased from Promega (Madison, WI, USA), and contained the consensus sequences 5'-TGACTCA-3' in 5'-CGCTTGATGACTCAGCCGGAA-3'. The DNA probe was endlabeled with [ gamma32P]ATP (Du Pont, Wilmington, DE, USA) using T4 polynucleotide kinase (Pharmacia LKB, Uppsala, Sweden), and purified by Sephadex G-50 column chromatography (Pharmacia LKB).

The binding reaction was performed for 15 min at 25 deg C in a reaction medium (20 muI) containing 20 mM HEPES-NaOH (pH 7.9), 1 mM DTT, 0.3 mM EDTA, 0.2 mM EGTA, 80 mM NaCI, 2 Hg of poly[dl-dC], 10% glycerol, 0.2 mM PMSF, 0.2-0.4 ng of 32P-labeled oligonucleotide probe and nuclear extract containing 3,ug of protein. The reaction medium was then subjected to electrophoresis.

The DNA-protein complexes formed were resolved on a 4% polyacrylamide gel. Electrophoresis was carried out at 11 V/cm for 2.5 h at 4C before the gel was dried and exposed to X-ray film. The optical densities for specific AP-1 bands and for background were quantified using an image analysis system with NIH IMAGE software, version 1.41.

Data analysis The effect of each drug treatment on the induction of AP-1 complexes was evaluated in both intact and dopamine-depleted caudate-putamen or globus pallidus by use of one-way analysis of variance (ANOVA) test. When the significant differences between treatment groups were demonstrated (p

DISCUSSION

The present study revealed that vehicle-treated animals presented the greatest induction of AP-1 DNA-binding activity in the caudate-putamen suggesting persistent enhancement of AP-1 induction in this portion of the brain. Previous studies support our results and demonstrated a long-lasting increment of AP-1 DNA-binding activity and expression of Fos-like immunoreactivity in the striatum of the 6-OHDA-lesioned hemisphere37. The increase of AP-1 DNA-binding activity in the vehicletreated rats appears much more prominent in the lesioned hemisphere than in the intact side. Dopamine-depletion induced by a 6-OHDA-lesion of the medial forebrain bundle may cause sensitization of D1 and D2 receptors in the ipsilateral caudate-putamen. The residual dopamine may keep stimulating these receptors in the vehicle-treated animals to enhance AP-1 induction. Such induction of AP-1 in the lesioned hemisphere might affect AP-1 induction in the caudateputamen of the intact hemisphere.

The induction appeared to be reversed completely by treatment of rats with the D1 antagonist SCH23390 and partially by the D2 antagonist sulpiride. In the previous study, we demonstrated that the D1 agonist SKF38393 induced AP-1 binding activity in the caudate-putamen of the 6-OHDA-lesioned hemisphere and that combined treatment with the D2 agonist bromocriptine or quinpirole enhanced it, although each D2 agonist alone failed to induce AP-1(4). We hypothesized that stimulation of D1 receptors is essential for striatal AP-1 induction and that additional stimulation of D2 receptors enhances the D1 receptor-mediated effect on AP-1 induction in the dopamine-depleted caudate-putamen4. This hypothesis is consistent with the present results because the blockade of D1 receptors by SCH23390 inhibited the striatal induction of AP-1 completely. Robertson and Fibiger8 reported that i.p. injection of 1 mg kg^ sup -1^ SCH23390 in normal rats resulted in absent expression of Fos in the striatum. It is suggested that blockade of D1 receptors antagonizes the induction of Fos and AP-1 in the dopamine-depleted caudateputamen. The partial reversal effect of sulpiride on the enhanced AP-1 induction in the caudate-putamen may also be compatible with our previous report4. Because blockade of D2 receptors appears to reverse their enhancing effect of D1 receptor-mediated AP-1 induction. Several authors have reported that the D2 receptor antagonist haloperidol increased Fos expression in the striatal cells of striatopallidal neurons9. In the present study, however, we did not observe induction of AP-1 above vehicle-treated control levels by a challenge dose of sulpiride. One possible reason to explain this difference is that the dose of sulpiride was too low. In the striatum, D1 and D2 receptors are expressed predominantly on striatonigral and striatopallidal neurons, respectivelylo. D1 and D2 receptors regulate these neurons excitatory and inhibitory, respectively. Therefore, a DI antagonist may inhibit striatal AP-1 induction on striatonigral neurons, and a D2 antagonist may induce it on striatopallidal neurons. The dose of sulpiride may have been sufficient to block the enhancing effect of the D1 receptor-mediated induction of striatal AP-1 binding activity, but not enough to induce AP-1 in D2 receptorsensitive striatopallidal neurons. An alternative reason is the different pharmacologic characteristics among D2 antagonists. Sulpiride has a different relative affinity to other dopamine receptor subtypes such as D3 receptors as compared to heloperidol". The third possibility may be related to the sample location in the caudateputamen. We prepared tissue from the dorsolateral region of the caudal portion of the caudate-putamen, the same region where haloperidol-induced Fos expression reportedly was diminished'2. Another possibility is based on the different pharmacologic characteristics between Fos and AP-1. Fos is one composite protein of AP-1 and may not necessarily parallel the induction of AP-1 DNA-binding activity. Supporting this consideration, Huang and Walters reported that haloperidolinjection did not increase AP-1 DNA-binding activity in the striatum of reserpine-treated rats. The D2 antagonist may not induce, but partially reverse the enhanced AP-1 DNA-binding activity in the dopamine-depleted caudate-putamen.

In the globus pallidus, D1 antagonist SCH23390 treated-, but not sulpiride- or vehicle-treated animals showed enhanced AP-1 binding activity. Results of present study suggest that the caudate-putamen and globus pallidus appear to induce AP-1 through distinct and opposing pathways. Involvement of striatopallidal inhibitory neurons may explain this inverse AP-1 expression; that is, the activation of striatopallidal neurons inhibits pallidal cells, while their suppression activates pallidal cells. Because D1 receptors can regulate striatopallidal neurons10, D1 antagonists may be able to regulate pallidal cells via these neurons; but no evidence of activation of striatopallidal neurons was observed in the present study, because the antagonist failed to induce AP-1 in the caudate-putamen. Furthermore, this idea is challenged by our former observation that D1 agonist failed to affect pallidal AP-1 induction when given singly but enhanced the D2-mediated induction4. With regard to Fos expression in the globus pallidus, Marshall et al.13 reported that SCH23390 increased it in the intact hemisphere, but not in the 6-OHDA-lesioned side. Vargo and Marshall'4 reported that the D2 antagonist eticlopride induced Fos expression in the globus pallidus. Such inconsistent responsiveness of AP-1 DNA-binding activity or Fos expression upon treatment with D1 and D2 agonists and antagonists in the globus pallidus cannot be explained by a single common mechanism. Several authors have reported that the neurons in the globus pallidus are heterogenous in their physiologic responses1516. Kelland et al 16. divided these neurons into Type I and Type II cells based upon their extracellular wave forms in vivo. The systemic administration of the D1/D2 agonist apomorphine decreases the firing rates of Type I cells while it increases those of Type II cells16. It may be that there is a subpopulation of pallidal cells which alter AP1 DNA-binding activity or Fos expression in response to the blockade of D1 receptors. Because D1 and D2 receptors are also present on pallidal cells, such regulation can occur not only by neuronal networks, but also by locally expressed receptors in the globus pallidus' 7.

CONCLUSION

According to the results of the present study, we hypothesize that dopamine-depletion produces a longlasting induction of AP-1 DNA-binding activity in the caudate-putamen through sensitized D1 and D2 receptors. The induction can be reversed completely and partially by blockade of D1 and D2 receptors, respectively. In the globus pallidus of the dopamine-depleted hemisphere, D1 but not D2 antagonists enhance AP-1 binding activity in certain populations of cells which may be different from those expressing AP-1 upon stimulation of D2 receptors. Elucidation of single-cell responses to a challenge dose of dopamine agonists and antagonists may clarify the dopaminergic regulation of neuronal networks and consequently lead to understanding better the pathogenesis of clinical fluctuations with prolonged levodopa therapy and of tardive dyskinesia with chronic neuroleptic use.

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Kenichi Kashihara*, Kazuhumi Akiyamat^, Takeshi Ishiharat^, Yasuhiro Manabe* and Koji Abe*

*Department of Neurology, tDepartment of Neurology, i Department of Neuropsychiatry, Okayama University Medical School, Okayama, Japan

Correspondence and reprint requests to: Kenichi Kashihara, Department of Neurology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-0914, Japan. Accepted for publication September 1998.

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