Address correspondence and reprint requests to Kazuya Kannari, Third Department of Medicine, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki, 036–8216, Japan. E-mail: firstname.lastname@example.org
In order to determine whether l-DOPA-derived extracellular dopamine (DA) in the striatum with dopaminergic denervation is affected by activation of serotonin autoreceptors (5-HT1A and 5-HT1B receptors), we applied in vivo brain microdialysis technique to 6-hydroxydopamine-lesioned rats and examined the effects of the selective 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and the selective 5-HT1B receptor agonist CGS-12066 A on l-DOPA-derived extracellular DA levels. Single l-DOPA injection (50 mg/kg i.p.) caused a rapid increase and a following decrease of extracellular DA, with a peak value at 100 min after l-DOPA injection. Pretreatment with both 0.3 mg/kg and 1 mg/kg 8-OH-DPAT (i.p.) significantly attenuated an increase in l-DOPA-derived extracellular DA and the times of peak DA levels were prolonged to 150 min and 225 min after l-DOPA injection, respectively. These 8-OH-DPAT-induced changes in l-DOPA-derived extracellular DA were antagonized by further pretreatment with WAY-100635, a selective 5-HT1A antagonist. In contrast, intrastriatal perfusion with the 5-HT1B agonist CGS-12066 A (10 nm and 100 nm) did not induce any changes in l-DOPA-derived extracellular DA. Thus, stimulation of 5-HT1A but not 5-HT1B receptors attenuated an increase in extracellular DA derived from exogenous l-DOPA. These results support the hypothesis that serotonergic neurons are primarily responsible for the storage and release of DA derived from exogenous l-DOPA in the absence of dopaminergic neurons.
Oral administration of l-3,4-dihydroxyphenylalanine (l-DOPA) remains the most effective therapy for Parkinson's disease for over 30 years (Cotzias et al. 1969; Marsden and Parkes 1977; Di Rocco et al. 1996). It is generally accepted that the therapeutic effect of l-DOPA is exerted through its conversion into dopamine (DA) in the brain. A number of discussions have been made on the site of l-DOPA conversion in the brain of patients with Parkinson's disease. Although nigrostriatal dopaminergic neurons that contain a large amount of l-DOPA converting enzyme, l-aromatic amino acid decarboxylase, are remarkably denervated in Parkinson's disease (Hornykiewicz and Kish 1986), it was suggested that l-DOPA is converted to DA in remaining dopaminergic neurons (Stachowiak et al. 1987; Snyder et al. 1990; Zigmond et al. 1990). Instead, other researchers postulated that nondopaminergic elements play a major role in l-DOPA conversion (Ng et al. 1972; Hollister et al. 1979; Melamed et al. 1980; Hefti et al. 1981; Miller and Abercrombie 1999). We have recently demonstrated that DA converted from exogenously administered l-DOPA is stored in synaptic vesicles and is released by an exocytosis mechanism (Kannari et al. 2000) and that serotonergic neurons are primarily responsible for the conversion and the release of DA derived from exogenously administered l-DOPA when dopaminergic neurons are denervated (Tanaka et al. 1999). Consistent with our findings, immunohistochemical studies have revealed the existence of l-DOPA-derived DA in serotonergic neurons (Arai et al. 1994, 1995). Given that l-DOPA-derived DA stored in serotonergic nerve terminals is subject to the same regulations as serotonin (5-hydroxytryptamine, 5-HT), it is tempting to speculate that treatment with drugs that can modulate serotonergic nerve activity or 5-HT release may result in changes in l-DOPA-derived extracellular DA levels.
Autoreceptor, a receptor present on a neuron for a neurotransmitter released from that neuron, is known to play an important role in the regulation of neurotransmitter release (Brock 1995). On serotonergic neurons there exist two types of 5-HT autoreceptor: 5-HT1A receptor on the soma-dendrite (somatodendritic autoreceptor) and 5-HT1B receptor on the nerve terminal (terminal autoreceptor). Activation of 5-HT1A receptors results in a reduction in firing of serotonergic neurons (Sprouse and Aghajanian 1986; Blier and de Montigny 1987), which subsequently leads to a reduction in 5-HT release from the corresponding nerve terminals (Sharp et al. 1989; Casanovas et al. 1997), whereas activation of 5-HT1B receptors results in a direct reduction in 5-HT release from the nerve terminals (Middlemiss and Huston 1990). Taken together, from these findings it would be expected that l-DOPA-derived DA release would be reduced by 5-HT1A agonists and 5-HT1B agonists in the similar manner to 5-HT release.
In order to test this hypothesis, we applied in vivo brain microdialysis technique and monitored extracellular DA levels in the striatum of freely moving rats treated with 6-hydroxydopamine (6-OHDA), a rat model of Parkinson's disease, and determined whether pretreatment with 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), a selective 5-HT1A agonist, or CGS-12066 A, a selective 5-HT1B agonist, induces changes in l-DOPA-derived extracellular DA levels.
Materials and methods
Male Wistar rats (Japan Clea Co. Ltd, Tokyo, Japan) were housed 4–5 per cage in a temperature-controlled room (around 25°C) with a 12-h day/night cycle with free access to food and water. The rats weighed 300–350 g at the time of dialysis probe implantation. All animal experiments in this paper followed the Guidelines for Animal Experimentation of our University.
Surgical procedure for dopaminergic denervation
The animals were anesthetized with pentobarbital (50 mg/kg i.p.) and were mounted in a stereotaxic apparatus (David Kopf, Tujunga, CA, USA) with incisor bar set at 3.3 mm below horizontal. They were pretreated with desipramine (25 mg/kg i.p.) 30 min before 6-OHDA injection to prevent denervation of noradrenergic neurons. A stainless steel needle (0.4 mm o.d.) was inserted through a small burr hole on the skull and the needle tip was placed in the right medial forebrain bundle (4.5 mm posterior from the bregma, 1.2 mm lateral from the sagittal suture, and 7.8 mm ventral from the dura surface) according to the atlas of Paxinos and Watson (1986). 6-OHDA (8 µg/4 µL in saline with 0.01% ascorbic acid) was injected over 8 min and the needle was kept at the same position for more than 4 min to prevent leakage.
Two weeks later, the rats were challenged with apomorphine to verify the dopaminergic denervation. They were placed in a stainless bowl with a 36-cm diameter and apomorphine (dissolved in saline with 0.1% ascorbic acid, 0.05 mg/kg s.c.) was injected. The number of complete rotations to the left (contralateral to the lesioned side) was counted and the rats turning more than 20 times per 5 min (between 15 min and 20 min after injection) were regarded as those with extensive dopaminergic denervation and were used for the present experiments. Previously we have demonstrated that rats that met the above criteria for apomorphine-induced rotation showed more than 99% of DA depletion in the striatal tissue (Tanaka et al. 1999).
In vivo brain microdialysis
Microdialysis study was performed between 3 and 4 weeks after stereotaxic operation. A stainless steel guide cannula (Eicom Co., Kyoto, Japan) was stereotaxically implanted in the right striatum 2 or 3 days before the perfusion study. Stereotaxic coordination was 0.5 mm anterior from the bregma, 3.0 mm lateral from the sagittal suture and 3.0 mm ventral from the dura surface (Paxinos and Watson 1986). One day before the perfusion study, a microdialysis probe with 3 mm active membrane (Eicom) was implanted in the right striatum through the guide cannula. We started all the perfusion studies at around 8 : 00 in the morning. Each probe was continuously perfused with an artificial Ringer's solution (Na+ 147 mm, K+ 4 mm, Ca2+ 2.3 mm, Cl− 155.6 mm) at a constant flow rate of 2 µL/min. Dialysate was collected for every 25 min (50 µL) and automatically injected onto the HPLC column. The mobile phase consisted of 0.1 m acetate-citrate buffer (pH 3.9) containing 160 mg/L l-octanesulfonic acid, 10 mg/L Na2EDTA, and 17% methanol. Flow rate was set at 220 µL/min. DA was separated on a reverse phase analytical column (EICOMPAK MA-5 ODS; Eicom; particle size 5 µm, 2.1 × 150 mm) and detected by electrochemical detection system (Eicom), consisting of an electrochemical cell with a graphite working electrode set at + 650 mV vs. an Ag/AgCl reference electrode. Chromatogram peaks were analyzed by means of PowerChrome data recording system (ADInstruments, Pty. Ltd, Castle Hill, NSW, Australia) with a computer (Power Macintosh 8500). Detection limit of the HPLC assay for DA was about 0.1 pg (0.5 fmol) per sample.
Drugs and pharmacological manipulations
After DA levels became constant (at about 3 h from the beginning of perfusion), rats were injected with l-DOPA (50 mg/kg). Benserazide (50 mg/kg) was administered 30 min prior to l-DOPA injection to prevent decarboxylation of l-DOPA at the periphery. 8-OH-DPAT was administered 25 min prior to l-DOPA injection. CGS-12066 A (CGS-12066 1 : 1 maleate) was dissolved in the artificial Ringer's solution (10 nm or 100 nm) and was perfused through the dialysis probe in the striatum, starting 50 min prior to l-DOPA administration, until the end of the dialysis study. WAY-100635 (2 mg/kg) was administered 35 min prior to l-DOPA administration.
6-OHDA hydrobromide, benserazide hydrochloride, apomorphine, 8-OH-DPAT, CGS-12066 A, and WAY-100635 were purchased from Research Biochemicals International (Natick, MA, USA). Desipramine hydrochloride and l-DOPA methyl ester hydrochloride were purchased from Sigma (St Louis, MO, USA). l-DOPA, benserazide, 8-OH-DPAT, and WAY-100635 were dissolved in saline and were immediately administered intraperitoneally.
Dialysate DA levels in each 25-min sample (50 µL) were expressed as mean ± SEM fmol/sample. The overall effect of treatments on l-DOPA-derived DA levels was determined by two-way analysis of variance (anova) with repeated measures over time. Changes in extracellular DA levels by drug treatments at each time point were assessed by one-way anova followed by Tukey's post hoc test. Changes in the cumulative amounts of l-DOPA-derived DA induced by drug treatments were analyzed by one-way anova followed by Tukey's post hoc test.
Effect of 8-OH-DPAT on l-DOPA-derived extracellular DA levels
The mean basal extracellular levels of DA in the denervated striatum across all treatment groups were 8.1 ± 1.5 fmol/sample (n = 36). As the mean basal extracellular DA in the intact striatum was 105.3 ± 14.5 fmol/sample (n = 17) in our previous study (Kannari et al. 2000), dopaminergic denervation reduced the basal extracellular DA levels to 7.7% of controls. Single administration of 50 mg/kg l-DOPA increased extracellular DA levels to a peak value of 238.1 ± 23.5 fmol/sample at 100 min, which was followed by a rapid decrease. Pretreatment with both 0.3 mg/kg and 1 mg/kg 8-OH-DPAT attenuated l-DOPA-induced increase in extracellular DA levels (Fig. 1a). Two-way anova for repeated measures revealed a significant effect of time (p < 0.001) and dose (p < 0.001) and a significant interaction with both (p < 0.001). The peak value of extracellular DA was 126.6 ± 19.8 fmol/sample at 150 min in rats with 0.3 mg/kg 8-OH-DPAT pretreatment and 100.0 ± 9.3 fmol/sample at 225 min in those with 1 mg/kg 8-OH-DPAT pretreatment. Significantly higher levels of extracellular DA were observed in rats with 1 mg/kg 8-OH-DPAT pretreatment from 225 min to the end of the experiment than in those with 0.3 mg/kg 8-OH-DPAT pretreatment (p < 0.05, one-way anova at each time point followed by Tukey's post hoc test).
The cumulative amounts of extracellular DA in rats with both 0.3 and 1 mg/kg 8-OH-DPAT pretreatment were significantly smaller than that of the l-DOPA single injection group (53.8 ± 7.8% and 54.9 ± 6.8%, respectively; p < 0.01, one-way anova followed by Tukey's post hoc test) (Fig. 1b).
Antagonism by WAY-100635 of the effect of 8-OH-DPAT
The effects of WAY-100635, a selective 5-HT1A receptor antagonist, on 8-OH-DPAT-induced changes in l-DOPA-derived extracellular DA levels are shown in Fig. 2(a). Two-way anova for repeated measures revealed a significant effect of time (p < 0.001) and treatment (p < 0.001) and a significant interaction with both (p < 0.001). WAY-100635, when injected alone, did not alter the cumulative amount of l-DOPA-derived extracellular DA (115.0 ± 9.9% compared with the l-DOPA single injection group; not significant, one-way anova followed by Tukey's post hoc test) (Fig. 2b). By pretreatment with WAY-100635, however, the cumulative amount of l-DOPA-derived extracellular DA in rats treated with 1 mg/kg 8-OH-DPAT (54.9 ± 6.8%; p < 0.01) returned to about the same level as that of the single l-DOPA injection group (86.6 ± 8.6%; not significant).
Effect of CGS-12066 A on l-DOPA-derived extracellular DA levels
Intrastriatal perfusion of artificial Ringer's solution containing either 10 nm or 100 nm CGS-12066 A did not cause notable changes in l-DOPA-induced increase in extracellular DA levels (Fig. 3a). Two-way anova for repeated measures revealed no significant effect of time, dose, nor interaction of both. The cumulative amounts of extracellular DA were 96.7 ± 17.1% in rats with 10 nm CGS-12066 A perfusion and 110.6 ± 5.6% in those with 100 nm CGS-12066 A perfusion as compared with that of the single l-DOPA injection group (not significant, one-way anova) (Fig. 3b).
The present study demonstrated that the 5-HT1A agonist 8-OH-DPAT but not the 5-HT1B agonist CGS-12066 A attenuates an increase in extracellular DA derived from exogenous l-DOPA in the striatum of 6-OHDA-lesioned rats.
Using rats with nigrostriatal dopaminergic denervation, we have previously shown that DA in the striatum derived from exogenously administered l-DOPA is mainly stored in and released from serotonergic nerve terminals in the striatum (Tanaka et al. 1999). Accordingly, we speculated that l-DOPA-derived extracellular DA may be affected by treatments that can influence 5-HT release. In the present study, the selective 5-HT1A receptor agonist 8-OH-DPAT attenuated an increase in l-DOPA-derived extracellular DA levels. 8-OH-DPAT probably stimulated 5-HT1A receptors on the soma-dendrites of serotonergic neurons in the dorsal raphe nucleus which send efferent fibers to the striatum (Soghomonian et al. 1987; Jacobs and Azmitia 1992), and the subsequent reduction of serotonergic nerve discharge may have resulted in reduced release of l-DOPA-derived DA in the striatum. These results further support the hypothesis that DA derived from exogenously administered l-DOPA is released from serotonergic nerve terminals in the striatum with dopaminergic denervation.
Although 0.3 mg/kg has been regarded as the maximal dose for 8-OH-DPAT to reduce 5-HT release in the normal rat striatum and there is an argument that the use of 1 mg/kg 8-OH-DPAT seems inappropriate to test the functional state of 5-HT1A receptors (Casanovas et al. 1997, 1999), some differences were observed between the effect of 0.3 mg/kg and 1 mg/kg 8-OH-DPAT on l-DOPA-derived extracellular DA. Pretreatment with both 0.3 mg/kg and 1 mg/kg 8-OH-DPAT caused identical reductions in the cumulative amounts of l-DOPA-derived extracellular DA. However, the peak value of 25-min interval DA was observed much later in rats with 1 mg/kg 8-OH-DPAT. Furthermore, 1 mg/kg 8-OH-DPAT showed significantly higher levels of extracellular DA during 225 and 300 min following l-DOPA injection. It seems that a higher dose of 8-OH-DPAT tempered both the rapid increase and decrease of extracellular DA, resulting in preservation of relatively modest extracellular DA levels much longer. Under unusual conditions that the release of substitute transmitter (DA) is modulated by original autoreceptors (5-HT1A receptors) in the dopaminergic denervated striatum, dose–response effect of 8-OH-DPAT on l-DOPA-derived extracellular DA might be different from that observed on 5-HT release in the normal striatum.
In addition to 5-HT1A receptors, 8-OH-DPAT is known to interact with 5-HT7 receptors (Ruat et al. 1993) and 5-HT transporters (Shoemaker and Langer 1986). However, it is unlikely that these diverse properties of 8-OH-DPAT contribute to the changes in l-DOPA-derived DA, because the effects of 8-OH-DPAT on l-DOPA-derived extracellular DA were antagonized by the selective 5-HT1A antagonist WAY-100635. Most of the changes in l-DOPA-derived extracellular DA levels induced by 1 mg/kg 8-OH-DPAT are thought to be attributable to the stimulation of 5-HT1A receptors. While WAY-100635 blocked the effect of 8-OH-DPAT on l-DOPA-derived extracellular DA, WAY-100635 itself exerted no influence on the increase in l-DOPA-derived extracellular DA. These results are consistent with the previous study that WAY-100635 alone had no effect on extracellular 5-HT levels (Gartside et al. 1995) and indicate that, in the dopaminergic denervated state, tonic inhibitory activity of endogenous 5-HT at 5-HT1A autoreceptors that would reduce l-DOPA-derived extracellular DA is very weak or absent.
One possibility to account for the observed changes in l-DOPA-derived extracellular DA levels by 8-OH-DPAT is that 8-OH-DPAT may act on residual dopaminergic neurons. Indeed, 8-OH-DPAT has been reported to decrease DA release in the normal striatum (Rasmusson et al. 1994; Ichikawa and Meltzer 2000), although an increase in striatal DA release by 8-OH-DPAT was also reported (Benloucif and Galloway 1991; Benloucif et al. 1993). Reduction in DA release produced by a 5-HT1A agonist may be partly explained by the fact that activation of 5-HT1A receptors reduces activity of tyrosine hydroxylase, a rate-limiting enzyme of DA synthesis (Johnson et al. 1993, 1996). In the present study, however, where extracellular DA derived from exogenous l-DOPA was monitored, contribution of 8-OH-DPAT to DA synthesis from exogenous l-DOPA can be neglected since tyrosine hydroxylase is not essential for the conversion of exogenous l-DOPA into DA. Furthermore, 8-OH-DPAT is not likely to act on residual dopaminergic neurons since we have reported that the present procedure for 6-OHDA local injection induces extensive dopaminergic denervation (more than 99%) and that a large part of l-DOPA-derived DA is considered to be released from serotonergic neurons (Tanaka et al. 1999).
We assumed that activation of 5-HT1B receptors located on serotonergic nerve terminals would result in a reduction in l-DOPA-derived extracellular DA levels. However, l-DOPA-derived extracellular DA was not affected by CGS-12066 A, despite substantial amounts of CGS-12066 A (10 nm and 100 nm) being perfused through the probe in the striatum, the doses comparable to the previous reports showing significant activity on presynaptic 5-HT1B receptors located on dopaminergic nerve terminals (Yadid et al. 1994) and on cholinergic nerve terminals (Izumi et al. 1994). Therefore, 5-HT1B receptors are not likely to play an important role in the regulation of l-DOPA-derived extracellular DA.
With respect to the localization of 5-HT1B receptors, there is a discrepancy between the distribution of receptor binding sites and the messenger RNA, suggesting that 5-HT1B receptors are located predominantly on axon terminals (Boschert et al. 1994; Mengod et al. 1997). However, 5,7-DHT-induced chemical denervation of serotonergic neurons produced either no change or even an increase in 5-HT1B receptor binding sites (Vergéet al. 1986; Compan et al. 1998), indicating that presynaptic 5-HT1B autoreceptors do not account for a major proportion of the total number of these receptors. 5-HT1B receptors are presumably more important as presynaptic heteroreceptors that regulate other neurotransmitters and the physiological role of 5-HT1B receptors as presynaptic 5-HT autoreceptors may not be significant.
We used CGS-12066 A which was produced as a selective 5-HT1B receptor agonist (Neale et al. 1987). However, controversy has been raised as to its selectivity and potency for 5-HT1B receptors. For instance, CGS-12066 B (1 : 2 maleate salt of CGS-12066) has weaker activity in reducing synaptosomal 5-HT release than the other putative 5-HT1B agonist trifluoromethylphenylpiperazine (TFMPP) (Neale et al. 1987). Moreover, this compound has been shown to inhibit dorsal raphe firing (Neale et al. 1987; Sinton and Fallon 1988), which is usually observed in 5-HT1A agonists. Evidence is lacking that CGS-12066B reduces extracellular 5-HT levels in the target areas of serotonergic neurons, despite that another selective 5-HT1B agonist CP-94,253 elicited a decrease of extracellular 5-HT in the striatum of mice (Knobelman et al. 2000). Further experiments are needed to determine whether other selective 5-HT1B agonists actually have the ability to reduce l-DOPA-derived DA release.
It has been reported that intrastriatal perfusion with CGS-12066 B (Yadid et al. 1994) and systemic injection of other 5-HT1B agonists (Benloucif and Galloway 1991; Benloucif et al. 1993) reduced DA release. However, because of the same reasons argued as to 5-HT1A receptors, under the present experimental conditions showing almost total dopaminergic denervation, CGS-12066 A is unlikely to act on residual dopaminergic neurons.
Patients with advanced stages of Parkinson's disease chronically treated with l-DOPA often suffer from motor fluctuations such as dyskinesias and wearing-off phenomenon (Marsden and Parkes 1977). It is postulated that dysregulation of DA release is one of the reasons for motor fluctuations (Carlsson 1983). Indeed, in vivo microdialysis studies have revealed rapid changes in l-DOPA-induced extracellular DA levels in the striatum with dopaminergic denervation (Abercrombie et al. 1990; Sarre et al. 1992; Maeda et al. 1999). In the present study, the 5-HT1A agonist 8-OH-DPAT reduced the maximal level of l-DOPA-derived DA. Moreover, higher dose (1 mg/kg) of 8-OH-DPAT even preserved certain amount of extracellular DA in the later time course. Therefore, we propose that 5-HT1A agonists could be useful in alleviating motor fluctuations by tempering aggressive concentration changes in extracellular DA following l-DOPA administration. Although preliminary, there are clinical studies reporting that buspirone, a 5-HT1A agonist with a complex DA agonist/antagonist property, lessened the severity of l-DOPA-induced dyskinesias (Kleedorfer et al. 1991; Bonifati et al. 1994). These authors assume that the effect of buspirone may be attributable to its weak DA antagonist property and to its activity on postsynaptic 5-HT1A receptors. Alternatively, the antidyskinetic effect of the 5-HT1A agonist would be ascribed to the effect on the serotonergic somatodendritic autoreceptors.
In conclusion, we found that activation of 5-HT1A but not 5-HT1B receptors attenuates an increase in l-DOPA-derived extracellular DA levels in the striatum with nigrostriatal denervation. These results are consistent with the notion that DA derived from exogenously administered l-DOPA is released from serotonergic nerve terminals. We believe that these findings provide useful implications for the therapy of patients with Parkinson's disease suffering from motor fluctuations.