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Keywords:

  • Extracellular dopamine;
  • In vivo microdialysis;
  • L-DOPA;
  • Parkinson’s disease;
  • Reserpine;
  • Striatum

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. Effects of reserpine and L-DOPA in intact rats
  6. Effects of reserpine and L-DOPA in 6-OHDA-lesioned rats
  7. DISCUSSION
  8. Effect of L-DOPA in intact rats
  9. Effect of reserpine on basal and L-DOPA-derived DA release in the dopaminergic denervated striatum
  10. Acknowledgements
  11. REFERENCES

Abstract: The influence of L-DOPA and reserpine on extracellular dopamine (DA) levels in the striatum of intact and dopaminergic denervated rats was studied using the brain microdialysis technique. In intact rats, reserpine (5 mg/kg s.c.) reduced extracellular DA levels to 4% of basal values. L-DOPA (50 mg/kg i.p.) had no effect on extracellular DA levels in reserpine-pretreated rats. In rats with 6-hydroxydopamine-induced lesion of the nigrostriatal dopaminergic system, basal levels of extracellular DA were low but markedly increased by L-DOPA (50 mg/kg i.p.). In 6-hydroxydopamine-lesioned rats, pretreatment with reserpine (5 mg/kg s.c.) diminished L-DOPA (50 mg/kg i.p.)-induced increases in extracellular DA levels to 16% of those obtained in denervated animals not pretreated with reserpine (p < 0.01). These results suggest that in the intact striatum, extracellular DA stems mainly from vesicular storage sites and that in the striatum with dopaminergic denervation, a large part of the L-DOPA-derived extracellular DA is also derived from a vesicular pool that is released by an exocytosis mechanism.

Parkinson’s disease is characterized by a progressive loss of dopaminergic neurons in the substantia nigra pars compacta (Hornykiewicz and Kish, 1986). The mechanism of death of these neurons is still unknown, and currently there exists no possibility of preventing or modifying the neurodegenerative events. L-3,4-Dihydroxyphenylalanine (L-DOPA), the endogenous precursor of dopamine (DA), is presently the most widely used and effective drug for symptomatic treatment of Parkinson’s disease (Cotzias et al., 1969; Marsden and Parkes, 1977; Di Rocco et al., 1996). Exogenously administered L-DOPA is converted by aromatic L-amino acid decarboxylase to DA, which remains in the striatum after dopaminergic denervation (Lloyd and Hornykiewicz, 1970; Duvoisin and Mytilineou, 1978; Melamed et al., 1980a), and this converted DA is believed to exert the therapeutic effect. Indeed, recent experimental studies using the microdialysis technique revealed that even in the striatum with extensive dopaminergic denervation, exogenously administered L-DOPA can be transformed to DA and subsequently released into the extracellular space (Zetterström et al., 1986; Abercrombie et al., 1990; Orosz and Bennett, 1992; Sarre et al., 1992; Maeda et al., 1999). However, it is still unclear where exogenous L-DOPA is converted to DA and how the converted DA is stored and released into the extracellular space.

Several reports from Zigmond’s group (Stachowiak et al., 1987; Snyder et al., 1990; Zigmond et al., 1990) have shown that extensive loss of dopaminergic neurons induces compensatory changes in the residual dopaminergic neurons such as increased synthesis and release of DA and a reduction of DA inactivation. Accordingly, it is generally assumed that exogenously administered L-DOPA may be converted to DA in the remaining dopaminergic neurons that are rendered hyperactive. Using rats with extensive dopaminergic denervation, we found that the potent D2 receptor agonist quinpirole reduced endogenous DA release from the denervated striatum but did not influence L-DOPA-induced increases in DA release (Maeda et al., 1999). Thus, although autoregulation mediated by presynaptic D2 receptors of endogenous DA is preserved even in the denervated striatum in a manner comparable to that described for intact dopaminergic terminals (Stoof et al., 1982; Starke et al., 1983), this autoregulatory mechanism seems no longer able to modify DA release derived from exogenous L-DOPA. Results from positron emission tomography studies with exogenously administered L-[11C]DOPA in advanced Parkinson’s disease also suggest loss of autoreceptor control (Ekesbo et al., 1999). If L-DOPA-derived DA is stored in and released from residual dopaminergic terminals where D2 autoregulation is preserved, it should be influenced by a D2 agonist. Because this seems not the case, it is unlikely that exogenously administered L-DOPA is converted to DA and stored in remaining dopaminergic terminals in the conventional manner.

Alternatively, it is possible that L-DOPA is converted to DA in nondopaminergic neurons or sites in which presynaptic D2 receptors have no influence on DA release. Recently, we have demonstrated that serotonergic neurons contribute to the conversion of exogenous L-DOPA to DA (Tanaka et al., 1999). According to our study, >80% of L-DOPA-derived extracellular DA is considered to be released from serotonergic neurons. Although some presynaptic D2 receptors are located on nondopaminergic terminals as well as on dopaminergic ones (Sesack et al., 1994), it has been proposed that serotonin release in the striatum is not modulated through presynaptic DA receptors (Ferré et al., 1994). Likewise, it is unlikely that release of DA converted from exogenous L-DOPA in the serotonergic nerve terminals is regulated by presynaptic D2 receptors.

Another possible reason for the avoidance of D2 autoregulation may be attributed to an alteration in release mechanisms of L-DOPA-derived DA under the condition of dopaminergic denervation. Today it is recognized that there are two different patterns of neurotransmitter release. One is exocytosis, a classical, well-investigated release mechanism involving fusion of neurotransmitter-containing synaptic vesicles with the terminal membrane following depolarization-induced calcium entry into the presynaptic terminals (Kandel, 1991). The other mechanism is nonvesicular, carrier-mediated release from the cytoplasm (Attwell et al., 1993; Levi and Raiteri, 1993). Carrier-mediated release is believed to be linked to uptake carriers or transporters, is calcium-independent, and is not under presynaptic autoreceptor control but is sensitive to transport inhibitors (Levi and Raiteri, 1993). Indeed, several reports suggest that striatal DA may be released by these two mechanisms (Raiteri et al., 1979; Fairbrother et al., 1990; Olivier et al., 1995). Although the physiological significance of both release mechanisms is not yet clear, Fairbrother et al. (1990) proposed that a carrier-dependent release mechanism might operate in the absence of vesicular DA based on the results from studies where potassium-evoked striatal DA release was measured. Furthermore, Raiteri et al. (1979) speculated that carrier-mediated release of DA becomes functionally important during treatment of Parkinson’s disease with L-DOPA. Irrespective of whether L-DOPA is converted to DA in serotonergic neurons, it is possible that extracellular DA derived from exogenous L-DOPA is characteristic of carrier-mediated release and thereby not regulated by D2 receptors.

The present study was carried out to clarify whether L-DOPA-derived DA is released by exocytosis or a nonvesicular, carrier-mediated mechanism in the dopaminergic denervation state. For this purpose, we used reserpine as a pharmacological tool, because it depletes vesicular storage of catecholamines by binding to vesicular monoamine transporters (Peter et al., 1994). In the present study, rats with dopaminergic denervation were pretreated with reserpine, and the in vivo brain microdialysis technique was used to monitor changes in striatal DA release after L-DOPA administration.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. Effects of reserpine and L-DOPA in intact rats
  6. Effects of reserpine and L-DOPA in 6-OHDA-lesioned rats
  7. DISCUSSION
  8. Effect of L-DOPA in intact rats
  9. Effect of reserpine on basal and L-DOPA-derived DA release in the dopaminergic denervated striatum
  10. Acknowledgements
  11. REFERENCES

Animals

Male Wistar rats (Clea, Japan) were housed four or five per cage in a temperature-controlled room (∼25°C) on 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 article 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 mounted in a stereotaxic apparatus (David Kopf, U.S.A.) with the incisor bar set at 3.3 mm below the horizontal. They were pretreated with desipramine (25 mg/kg i.p.) 30 min before 6-hydroxydopamine (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 >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 (0.05 mg/kg) was injected subcutaneously. The number of complete rotations to the left (contralateral to the lesioned side) was counted, and the rats turning >20 times per 5 min (between 15 min and 20 min) were regarded as those with extensive dopaminergic denervation and were used for the present experiments. Our previous study demonstrated that rats that met the above criteria for apomorphine-induced rotations showed >99% depletion of DA in the striatal tissue (Tanaka et al., 1999).

In vivo brain microdialysis

The microdialysis study was performed between 3 and 4 weeks after stereotaxic operation. A stainless steel guide cannula (Eicom, Japan) was stereotaxically implanted in the right striatum ∼48 h before perfusion study. The stereotaxic coordinates were 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 a 3-mm active membrane (Eicom, Japan) was implanted in the right striatum through the guide cannula. We started all the perfusion studies between 7:00 and 8:00 a.m. Each probe was continuously perfused with an artificial Ringer’s solution (147 mM Na+, 4 mM K+, 2.3 mM Ca2+, and 155.6 mM Cl-) at a constant flow rate of 2 μl/min. Dialysate was collected every 20 min (40 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. The flow rate was set at 220 μl/min. DA and its metabolites were separated on a reverse-phase analytical column (EICOMPAK MA-5 ODS; Eicom, Japan; particle size, 5 μm; 2.1 × 150 mm) and detected by the electrochemical detection system (Eicom, Japan), consisting of an electrochemical cell with a graphite working electrode set at 650 mV versus an Ag/AgCl reference electrode. Chromatogram peaks were analyzed by means of the PowerChrome data recording system (ADInstruments, Australia) with a computer (Power Macintosh 7500; Apple Computer, U.S.A.). The detection limit of the HPLC assay for each substance was ∼100 fg (0.6-0.7 fmol) per sample.

Drugs and pharmacological manipulations

After DA levels became constant (at ∼3 h from the beginning of perfusion), reserpine (5 mg/kg) or vehicle was administered. L-DOPA (50 mg/kg) was administered 120 min after reserpine or vehicle injection. In cases of L-DOPA injection, benserazide (50 mg/kg) was administered 30 min before L-DOPA to prevent decarboxylation of L-DOPA at the periphery.

6-OHDA hydrobromide, benserazide hydrochloride, apomorphine, and reserpine were purchased from Research Biochemicals International (U.S.A.). Desipramine hydrochloride and L-DOPA methyl ester hydrochloride were purchased from Sigma (U.S.A.). Apomorphine was dissolved in saline with 0.1% ascorbic acid, and reserpine was dissolved in a minimal amount of acetic acid and diluted with 10% sucrose. All other drugs were dissolved in saline. All drugs except apomorphine were administered intraperitoneally.

Data analysis

DA, 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) levels in the dialysates were measured during 6 h. Mean basal release of each substance was determined by averaging values of three consecutive dialysates. The absolute amount of extracellular DA was expressed as the mean ± SEM value, in femtomoles per sample (40 μl), and those of DOPAC and HVA were expressed in picomoles per sample. In the experiments using intact rats, the values were relatively expressed as percentages of each mean basal release.

Changes in extracellular DA, DOPAC, and HVA levels over time within one group were analyzed by one-way ANOVA followed by Fisher’s protected least significant difference test. Effects of reserpine on cumulative amounts of extracellular DA, DOPAC, and HVA in 6-OHDA-lesioned rats were analyzed by Student’s t test.

Histological examination

At the end of the experiments, all rats were killed by decapitation. The brains were rapidly removed from the skull and fixed with 4% formaldehyde. They were cut into 20-μm-thick sections by a cryostat (Micron, Germany) and stained with 0.1% cresyl violet. Loss of nigral DA neurons by 6-OHDA and the trace of microdialysis probe in the striatum were verified histologically.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. Effects of reserpine and L-DOPA in intact rats
  6. Effects of reserpine and L-DOPA in 6-OHDA-lesioned rats
  7. DISCUSSION
  8. Effect of L-DOPA in intact rats
  9. Effect of reserpine on basal and L-DOPA-derived DA release in the dopaminergic denervated striatum
  10. Acknowledgements
  11. REFERENCES

Effect of reserpine in intact rats

In intact rats (n = 17), basal extracellular levels of DA, DOPAC, and HVA per sample were 105.3 ± 14.5 fmol, 26.3 ± 2.1 pmol, and 18.1 ± 2.3 pmol, respectively.

Reserpine produced a gradual decrease of extracellular DA levels up to 4% of basal values (from 83.2 ± 10.9 fmol per sample as basal levels to 3.1 ± 1.0 fmol per sample at 180 min after reserpine; p < 0.01, n = 5). This prominent reduction persisted for >6 h (data not shown). Extracellular DOPAC and HVA levels increased to 253 ± 34% of basal values after 40 min (p < 0.01, n = 5) and to 180 ± 13% of basal values after 80 min (p < 0.01, n = 5), respectively. Both DOPAC and HVA levels gradually decreased afterward (data not shown).

Effects of reserpine and L-DOPA in intact rats

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. Effects of reserpine and L-DOPA in intact rats
  6. Effects of reserpine and L-DOPA in 6-OHDA-lesioned rats
  7. DISCUSSION
  8. Effect of L-DOPA in intact rats
  9. Effect of reserpine on basal and L-DOPA-derived DA release in the dopaminergic denervated striatum
  10. Acknowledgements
  11. REFERENCES

In intact rats treated with saline (instead of reserpine) and L-DOPA, extracellular DA levels were transiently reduced to 65 ± 5% (p < 0.01) of basal values and then increased to 120 ± 6% (p < 0.01) of basal values (Fig. 1). Extracellular DOPAC and HVA contents were significantly increased following administration of L-DOPA, with a peak value reaching 368 ± 139% (120 min after L-DOPA, p < 0.01) and 276 ± 54% (180 min after L-DOPA, p < 0.01) of basal release, respectively.

image

Figure 1. Effect of reserpine and L-DOPA on extracellular levels of DA, DOPAC, and HVA in the striatum of intact rats. Reserpine (5 mg/kg) or vehicle (▾), benserazide (50 mg/kg), and L-DOPA (50 mg/kg) (▿) were injected intraperitoneally at 0, 90, and 120 min, respectively. Data are mean ± SEM (bars) values (% of basal values, n = 6). In rats receiving vehicle and L-DOPA (○), DA levels were transiently reduced and then showed a significant increase. In rats receiving reserpine ([UNK]), DA levels were markedly reduced to 4% of basal values in 120 min, and the low levels persisted after L-DOPA injection. DOPAC and HVA showed two peak values following injection of reserpine and L-DOPA. *p < 0.05, **p < 0.01, compared with corresponding basal values.

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When reserpine was administered before L-DOPA, reduced DA release persisted even after L-DOPA administration. DOPAC release showed two peak values: one after reserpine administration (253 ± 14% at 40 min) and the other one after L-DOPA administration (356 ± 20% at 220 min). HVA release also showed two peak values following reserpine and L-DOPA administration. However, peak values were smaller and occurred after a somewhat longer latency period than those of DOPAC (180 ± 13% at 80 min and 298 ± 35% at 280 min).

Effects of reserpine and L-DOPA in 6-OHDA-lesioned rats

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. Effects of reserpine and L-DOPA in intact rats
  6. Effects of reserpine and L-DOPA in 6-OHDA-lesioned rats
  7. DISCUSSION
  8. Effect of L-DOPA in intact rats
  9. Effect of reserpine on basal and L-DOPA-derived DA release in the dopaminergic denervated striatum
  10. Acknowledgements
  11. REFERENCES

In 6-OHDA-lesioned rats (n = 10), basal levels of DA, DOPAC, and HVA per sample were 1.8 ± 0.3 fmol, 0.2 ± 0.04 pmol, and 0.04 ± 0.005 pmol, respectively (Fig. 2).

image

Figure 2. Time course of extracellular DA, DOPAC, and HVA levels in the striatum of 6-OHDA-lesioned rats receiving reserpine and L-DOPA (left panels) and the cumulative amounts of DA, DOPAC, and HVA during 4 h after L-DOPA administration (right panels). Reserpine (5 mg/kg) or vehicle (▾), benserazide (50 mg/kg), and L-DOPA (50 mg/kg) (▿) were injected intraperitoneally at 0, 90, and 120 min, respectively. Data are mean ± SEM (bars) values (in fmol per sample for DA and pmol per sample for DOPAC and HVA, n = 5). Basal levels of DA, DOPAC, and HVA in the lesioned striatum were very low. With reserpine pretreatment, the cumulative amount of L-DOPA-derived DA was reduced to 16 ± 3% of that obtained from animals not receiving reserpine (§§p < 0.01). Cumulative amounts of DOPAC and HVA were increased to 141 ± 31 and 147 ± 35%, respectively, which were not statistically different. *p < 0.05, **p < 0.01, compared with corresponding basal values.

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In rats treated with vehicle instead of reserpine, L-DOPA administration significantly increased extracellular levels of DA, DOPAC, and HVA per sample to 147.1 ± 11.7 fmol, 8.0 ± 1.1 pmol, and 11.2 ± 1.9 pmol, respectively. These peak values were obtained at 100, 140, and 180 min after L-DOPA injection, respectively. With regard to DA, there was no initial inhibition following L-DOPA administration.

Reserpine pretreatment reduced basal DA release per sample from 1.8 ± 0.4 to 0.9 ± 0.4 fmol (difference not statistically significant), and peak values of extracellular DA per sample following L-DOPA administration amounted to only 24.4 ± 3.0 fmol at 100 min following L-DOPA administration. The cumulative amount of extracellular DA during 4 h after L-DOPA injection was 1,040.8 ± 109.8 fmol in 6-OHDA-lesioned rats with a single L-DOPA injection and 163.9 ± 26.8 fmol in those with reserpine and L-DOPA injection. Thus, pretreatment with reserpine caused an 84% reduction in L-DOPA-derived DA release (p < 0.01). DOPAC and HVA levels in rats with reserpine pretreatment were somewhat increased after L-DOPA administration compared with those in rats not treated with reserpine (DOPAC, from 60.3 ± 8.9 to 85.1 ± 18.9 pmol/4 h; HVA, from 82.6 ± 15.3 to 121.3 ± 28.5 pmol/4 h). However, these differences were not statistically significant.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. Effects of reserpine and L-DOPA in intact rats
  6. Effects of reserpine and L-DOPA in 6-OHDA-lesioned rats
  7. DISCUSSION
  8. Effect of L-DOPA in intact rats
  9. Effect of reserpine on basal and L-DOPA-derived DA release in the dopaminergic denervated striatum
  10. Acknowledgements
  11. REFERENCES

Effect of reserpine in intact rats

Reserpine produced a marked decrease of striatal DA release within the first 2 h after administration, and DA release remained at this low level for at least 6 h. The time course of this effect of reserpine on DA release is in agreement with that of other studies (Callaway et al., 1989; Fairbrother et al., 1990; Cadoni et al., 1995; Heeringa and Abercrombie, 1995). Because reserpine depletes vesicular storage of DA, basal release of DA in the intact striatum appears to originate mainly from synaptic vesicles. Therefore, the present result is compatible with the notion that under physiological conditions DA release occurs via depolarization-induced exocytosis (Kuhr et al., 1987; Westerink and De Vries, 1988; Arbuthnott et al., 1990).

Rapid increases in content of two major DA metabolites, DOPAC and HVA, following reserpine administration may reflect reserpine-induced release of DA from vesicular storage sites into the cytoplasmic extravesicular compartment, where DA is immediately metabolized by abundantly available DA-metabolizing enzymes (Agid et al., 1976). In the intact striatum, therefore, it is conceivable that there is little room for cytosolic DA to exist as a releasable pool for nonvesicular, carrier-mediated release.

Effect of L-DOPA in intact rats

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. Effects of reserpine and L-DOPA in intact rats
  6. Effects of reserpine and L-DOPA in 6-OHDA-lesioned rats
  7. DISCUSSION
  8. Effect of L-DOPA in intact rats
  9. Effect of reserpine on basal and L-DOPA-derived DA release in the dopaminergic denervated striatum
  10. Acknowledgements
  11. REFERENCES

We observed a transient decrease of DA release following administration of L-DOPA (50 mg/kg) to intact rats. This confirms previous results from Abercrombie et al. (1990), who also observed that L-DOPA induced a transient decrease in DA release depending on the amount of L-DOPA administered. They speculated that this decrease is due to stimulation of presynaptic D2 receptors by newly synthesized DA released into the extracellular space. Increases in extracellular DOPAC and HVA along with a transient reduction in extracellular DA following L-DOPA administration indicate that DA is newly synthesized but that a large part of it is metabolized to DOPAC in the cytoplasm. Although inhibition of DA release is predominant at the beginning, it is overcompensated for by the large amount of DA newly formed from exogenous L-DOPA, resulting in a subsequent increase. However, this increase amounted to only 120% of basal values. Excessive DA overflow by exogenous L-DOPA appears to be restricted because DA reuptake (Abercrombie et al., 1990; Miller and Abercrombie, 1999) and release modulation by inhibitory D2 autoreceptors (Maeda et al., 1999) are operative in the intact striatum.

Basal release of DA and its metabolites in the denervated striatum

In the present study, basal DA release in the dopaminergic denervated striatum was quite low. Although several studies have demonstrated a significant reduction of basal DA release in the striatum as a consequence of dopaminergic denervation, the extent of reduction in DA release differs between various studies. For instance, Abercrombie et al. (1990) and Andres et al. (1998) reported that basal DA levels were significantly reduced but that a small portion of DA release remained. In fact, in our previous study (Maeda et al., 1999), basal DA release in the denervated striatum amounted to ∼ 16% of control values. If some dopaminergic neurons escape from denervation, compensatory hyperactivity in the remaining dopaminergic neurons can occur and keep extracellular DA levels within a certain value (Stachowiak et al., 1987; Snyder et al., 1990; Zigmond et al., 1990). In contrast, if dopaminergic denervation is almost complete, basal extracellular DA levels in the denervated striatum should be reduced to almost undetectable levels (Zetterström et al., 1986; Robinson and Whishaw, 1988; Orosz and Bennett, 1992). Our denervation procedure seems to produce a loss of >99% of dopaminergic neurons (Tanaka et al., 1999). This virtually complete dopaminergic denervation may explain the quite low levels of DA release because compensatory events are no longer possible. In addition, the reason why an initial decrease in extracellular DA content following L-DOPA administration was not observed in denervated rats could be that most of the extracellular DA in the denervated striatum is not derived from surviving dopaminergic neurons but from other sites where D2 autoregulation is not operative. Thus, differences in basal DA release in the denervated striatum may be caused by differences in the number of dopaminergic neurons surviving experimentally induced denervation process.

Effect of reserpine on basal and L-DOPA-derived DA release in the dopaminergic denervated striatum

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. Effects of reserpine and L-DOPA in intact rats
  6. Effects of reserpine and L-DOPA in 6-OHDA-lesioned rats
  7. DISCUSSION
  8. Effect of L-DOPA in intact rats
  9. Effect of reserpine on basal and L-DOPA-derived DA release in the dopaminergic denervated striatum
  10. Acknowledgements
  11. REFERENCES

The most important finding of the present study was that L-DOPA-derived DA release in the dopaminergic denervated striatum is greatly reduced when vesicular DA storage is prevented by reserpine pretreatment. This finding suggests that >80% of L-DOPA-derived DA is stored and released from synaptic vesicles by an exocytosis mechanism. Although several hypotheses exist regarding the possible site of conversion of exogenous L-DOPA to DA (Melamed et al., 1980a,b; Hefti et al., 1981; Miller and Abercrombie, 1999), our results suggest that L-DOPA-derived extracellular DA stems from synaptic vesicles in neurons, whereas nonneuronal components such as glia cells and capillary vessels can be excluded as candidates for the origin of L-DOPA-derived extracellular DA.

It has been speculated that exogenous L-DOPA is converted to DA in dopaminergic neurons surviving a degenerative process and that the converted DA is stored in their synaptic vesicles. However, in the light of our observations this possibility seems unlikely. First, as discussed above, our experimental conditions produced an almost complete dopaminergic denervation of the striatum. Second, we found previously that L-DOPA-derived DA release is not regulated by presynaptic D2 dopaminergic receptors (Maeda et al., 1999).

Alternatively, it has been proposed that serotonergic neurons are a possible site of DA formation. Anatomical studies revealed a dense innervation of serotonergic fibers in the basal ganglia (Soghomonian et al., 1987; Jacobs and Azmitia, 1992). Serotonergic neurons possess L-aromatic amino acid decarboxylase, which is able to convert not only 5-hydroxytryptophan to serotonin but also L-DOPA to DA (Hökfelt et al., 1973; Arai et al., 1996). Furthermore, serotonergic nerve terminals are able to store DA as well as serotonin in synaptic vesicles because monoamines can be taken up nonselectively through vesicular monoamine transporters (Peter et al., 1994). Indeed, immunohistochemical studies demonstrated the presence of DA in serotonergic neurons after L-DOPA administration (Arai et al., 1995). Finally, using in vivo microdialysis, we (Tanaka et al., 1999) recently demonstrated that extracellular DA content after L-DOPA administration was strongly decreased in rats with a combined dopaminergic and serotonergic denervation, which is consistent with earlier in vitro studies using striatal tissues (Ng et al., 1972; Hollister et al., 1979). All these findings support the hypothesis that serotonergic nerve terminals play a major role in L-DOPA conversion in the absence of dopaminergic neurons. Because reserpine binds essentially to vesicular monoamine transporters, which exist in any monoaminergic synaptic vesicles (Rudnick et al., 1990), pretreatment with reserpine should deplete vesicular DA in the serotonergic nerve terminals in the dopaminergic denervated striatum, leading to a significant reduction in L-DOPA-derived DA release. Thus, based on the results of our study, we propose that when dopaminergic neurons are lost in the striatum, the conversion of exogenous L-DOPA to DA and its storage and release occur in serotonergic nerve terminals. This situation seems to be the same in L-DOPA therapy for advanced stage of Parkinson’s disease.

It is interesting that, under reserpine pretreatment, L-DOPA-derived DA release was hardly detectable in intact rats, whereas it could be detected to a certain extent in 6-OHDA-lesioned rats. Thus, nonvesicular DA release was observed only in the denervated striatum. Given that serotonergic nerve terminals are the main site of L-DOPA metabolism, possible reasons for this phenomenon may be that synthesis of DA from L-DOPA in the serotonergic terminals is so active that it exceeds the capacity for DA packaging into synaptic vesicles, and/or monoamine oxidase in the serotonergic nerve terminals is not sufficient for catabolizing the large amount of L-DOPA-derived DA in the cytosol and hence permits overflow of cytosolic DA into the extracellular space. In this study the contribution of nonvesicular carrier-mediated DA release was estimated to amount to 16% of total DA derived from L-DOPA. Nevertheless, nonvesicular DA release may play an important role in L-DOPA therapy in patients with Parkinson’s disease. As this nonvesicular DA release is not regulated by the normal physiological feedback systems, it may produce uncontrollable DA overflow responsible for motor fluctuations observed with L-DOPA therapy (Marsden and Parkes, 1977).

In summary, administration of reserpine to intact rats resulted in very low levels of extracellular DA in the striatum, and these low levels persisted also after L-DOPA administration. In the striatum of 6-OHDA-lesioned rats, L-DOPA administration increased extracellular DA levels, whereas pretreatment with reserpine produced a marked reduction in L-DOPA-derived extracellular DA levels. These results suggest that DA release in the intact striatum is derived mainly from vesicular storage sites and that in the case of dopaminergic denervation the majority of the L-DOPA-derived DA release is of vesicular origin and released by exocytosis.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. Effects of reserpine and L-DOPA in intact rats
  6. Effects of reserpine and L-DOPA in 6-OHDA-lesioned rats
  7. DISCUSSION
  8. Effect of L-DOPA in intact rats
  9. Effect of reserpine on basal and L-DOPA-derived DA release in the dopaminergic denervated striatum
  10. Acknowledgements
  11. REFERENCES
  • 1
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