Address correspondence and reprint requests to Dr. E. D. Abercrombie at Center for Molecular and Behavioral Neuroscience, Rutgers University, 197 University Ave., Newark, NJ 07102, U.S.A.
Abstract: The differential behavioral and neurochemical effects of exogenous L-DOPA in animals with intact versus dopamine (DA)-denervated striata raise questions regarding the role of DA terminals in the regulation of dopaminergic neurotransmission after administration of exogenous L-DOPA. In vivo microdialysis was used to monitor the effect of exogenous L-DOPA on extracellular DA in intact and DA-denervated striata of awake rats. In intact striatum, a small increase in extracellular DA was observed after administration of L-DOPA (50 mg/kg i.p.) but in DA-denervated striatum a much larger increase in extracellular DA was elicited. Additional experiments assessed the role of high-affinity DA uptake and impulse-dependent neurotransmitter release in the effect of exogenous L-DOPA on extracellular DA in striatum. Pretreatment with GBR-12909 (20 mg/kg i.p.), a selective DA uptake inhibitor, enhanced the ability of L-DOPA to increase extracellular DA in intact striatum. However, in DA-denervated striatum, inhibition of DA uptake did not alter the extracellular DA response to L-DOPA. Impulse-dependent neurotransmitter release was blocked by the infusion of tetrodotoxin (TTX; 1 μM), an inhibitor of fast sodium channels, through the dialysis probe. Application of TTX significantly attenuated the L-DOPA-induced increase in extracellular DA observed in striatum of intact rats pretreated with GBR-12909. In a similar manner, TTX infusion significantly attenuated the increase in extracellular DA typically observed in striatum of 6-OHDA-lesioned rats after the administration of L-DOPA. The present results indicate that DA terminals, via high-affinity uptake, play a crucial role in the clearance of extracellular DA formed from exogenous L-DOPA in intact striatum. This regulatory mechanism is absent in the DA-denervated striatum. In addition, this study has shown that DA synthesized from exogenous L-DOPA primarily is released by an impulse-dependent mechanism in both intact and DA-denervated striatum. The latter result suggests an important role for a nondopaminergic neuronal element in striatum that serves as the primary source of extracellular DA formed from exogenous L-DOPA.
Parkinson’s disease is a neurodegenerative disorder associated primarily with the loss of dopamine (DA) neurons in the nigrostriatal system. Administration of L-DOPA is the primary means for treating the symptoms of Parkinson’s disease. L-DOPA, the precursor to DA, readily is transported across the blood-brain barrier and is converted to DA by aromatic amino acid decarboxylase (AADC).
In patients with Parkinson’s disease and in rats with 6-hydroxydopamine (6-OHDA) lesions of nigrostriatal DA neurons, increases in tissue DA levels in striatum after L-DOPA administration are substantially lower compared with intact subjects (Yahr et al., 1972; Schoenfeld and Uretsky, 1973; Lloyd et al., 1975; Hefti et al., 1981). This implies that DA terminals are a major site for conversion of exogenous L-DOPA to DA in striatal tissue. However, the effect of L-DOPA on extracellular DA is significantly greater in DA-denervated striatum than in intact striatum (Abercrombie and Zigmond, 1990; Abercrombie et al., 1990; Wachtel and Abercrombie, 1994). Together, these data indicate that changes in tissue DA levels are not a valid index of the therapeutic action of exogenous L-DOPA and that DA terminals are not the primary source for L-DOPA-induced increases in extracellular DA. It is interesting that L-DOPA’s antiparkinsonian behavioral effects appear to be highly correlated with the effects of L-DOPA on extracellular DA. In animals with 6-OHDA lesions, L-DOPA readily elicits pronounced behavioral activation whereas intact animals express behavioral effects only after administration of high doses of L-DOPA (Schoenfeld and Uretsky, 1973; Hollister et al., 1979; Zetterström et al., 1986). Thus, exogenous L-DOPA has a reduced ability to increase extracellular DA in striatum and to elicit behavioral responses in intact animals compared with DA-lesioned animals.
It has been proposed that DA terminals may be more important as a site for catabolizing DA synthesized from exogenous L-DOPA in striatum rather than as a source for increases in extracellular DA levels (Abercrombie and Zigmond, 1990; Abercrombie et al., 1990; De Boer et al., 1994). Previous work has shown that monoamine oxidase type A efficiently catabolizes DA synthesized from exogenous L-DOPA within DA nerve terminals (Wachtel and Abercrombie, 1994). As discussed above, the extracellular DA concentration in intact striatum is not greatly augmented by L-DOPA administration. Rather, very large increases in extracellular 3,4-dihydroxyphenylacetic acid are observed in this condition. In addition, in intact striatum, high-affinity DA uptake can efficiently remove DA from the extracellular space for subsequent catabolism by monoamine oxidase type A (Ng et al., 1972; Trugman and Wooten, 1986; Melamed, 1988; Abercrombie et al., 1990; Wachtel and Abercrombie, 1994). Therefore, it is important to assess the role of DA terminals not only as a site for synthesis and catabolism of DA in response to exogenous L-DOPA, but in addition as a site for inactivation by high-affinity uptake of DA derived from exogenous L-DOPA.
Implicit in the working model described above is the assumption that exogenous L-DOPA is converted to DA by AADC activity of a nondopaminergic cellular component of striatum. Indeed, ∼20% of AADC activity remains in striatum after the elimination of DA terminals (Hefti et al., 1981), but the identity of the cellular source of the AADC activity responsible for increased extracellular DA levels in response to exogenous L-DOPA has yet to be determined (Melamed et al., 1980, 1981; Hefti et al., 1981; Kang et al., 1992; Nakazato and Akiyama, 1992). Theoretically, DA derived from exogenous L-DOPA could arrive in the extracellular space either by passive diffusion from a nonneuronal source or by impulse-dependent release from a nondopaminergic neuronal source.
The first goal of this study was to determine the extent to which high-affinity DA uptake influences the extracellular level of DA achieved in striatum in response to administration of L-DOPA. The effect of DA uptake inhibition on the response of extracellular DA to exogenous L-DOPA was examined in striatum of intact and 6-OHDA-lesioned rats. The second goal of this study was to assess the release mechanism underlying the appearance of extracellular DA observed in striatum after exogenous L-DOPA administration. To this end, the role of impulse-dependent neurotransmitter release in the effect of L-DOPA on extracellular DA was examined in two experimental paradigms. The first condition assessed the effect of impulse blockade on L-DOPA-induced DA release in intact striatum under conditions of inhibition of DA uptake. The second condition assessed the effect of impulse blockade in DA-denervated striatum, where DA uptake is hypothesized not to exert a strong influence on L-DOPA-induced changes in extracellular DA.
Adult male Sprague-Dawley rats (Zivic-Miller Laboratories, Pittsburgh, PA, U.S.A.) were housed individually in plastic shoebox cages. The rats were maintained under conditions of constant temperature (21°C) and humidity (40%) on a 12-h light/dark cycle (0700 h on/1900 h off). Rats were supplied with food and water ad libitum. Rats weighed 300-400 g at the time of the experiments. Animal procedures were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals (revised 1996) and all protocols were approved by the Rutgers University Institutional Animal Care and Use Committee.
6-OHDA lesions of medial forebrain bundle
Male Sprague-Dawley rats (250-300 g) were anesthetized with equithesin (2.4 ml/kg i.p.) and mounted in a stereotaxic device (David Kopf Instruments, Tujunga, CA, U.S.A.) with the skull flat between lambda and bregma. To prevent destruction of noradrenergic neurons, the animals were treated with desipramine (25 mg/kg i.p.) 30 min before the administration of the catecholaminergic neurotoxin 6-OHDA. A 30-gauge cannula (Plastics One, Roanoke, VA, U.S.A.), connected to a 10-μl syringe (Hamilton, Reno, NV, U.S.A.) via PE-50 polyethylene tubing was lowered through a small burr hole in the skull to the medial forebrain bundle at the following coordinates: AP -3.2 mm, ML ± 1.5 mm relative to bregma, and DV -7.2 mm from the dura (Paxinos and Watson, 1986). 6-OHDA (6 μg, freebase weight) in 2 μl of vehicle (0.2 mg/ml ascorbic acid in 0.9% saline) was injected at a rate of 0.5 μl/min with a syringe pump (Harvard Apparatus, South Natick, MA, U.S.A.). The injection cannula was left in place for 5 min before and 5 min after the injection. Animals recovered 2-3 weeks before implantation of a microdialysis probe into striatum.
Microdialysis probe construction and implantation
Vertical microdialysis probes were of concentric design, slightly modified from that previously described (Abercrombie and Finlay, 1991). Fused silica capillary tubing (Polymicro Technologies, Phoenix, AZ, U.S.A.), serving as an outlet line, was threaded through PE-20 tubing, which served as the inlet line. A portion of the microdialysis membrane (molecular weight cutoff, 6,000; o.d., 200 μm; Spectra/Por; Spectrum, Houston, TX, U.S.A.) was coated with a thin layer of epoxy such that a 2-mm-long active region of membrane remained at the tip of the microdialysis probe.
Artificial CSF (147 mM NaCl, 2.5 mM KCl, 1.3 mM CaCl2, and 0.9 mM MgCl2, pH 7.4) was perfused continuously through the microdialysis probe at a rate of 1.5 μl/min with a syringe pump. Before implantation, the relative recovery of each microdialysis probe was determined in vitro as previously described (Abercrombie and Finlay, 1991). In vitro recovery provided an index of consistency between microdialysis probes. The average relative recovery for DA determined in this way was 15.4 ± 0.3% (n = 47).
The animals were anesthetized with chloral hydrate (400 mg/kg i.p.) and mounted in a stereotaxic device (David Kopf Instruments) with the skull flat between lambda and bregma. The microdialysis probe was implanted into striatum at the following coordinates: AP +0.5 mm, ML ±2.5 mm relative to bregma, and DV -6.0 mm from the dura (Paxinos and Watson, 1986). The probe assembly was secured to the skull with fast-curing dental cement and three skull screws. The inlet and outlet lines of the probe were fed through a metal tether that was connected to a single-channel fluid swivel (Instech Laboratories, Plymouth Meeting, PA, U.S.A.). Experiments were conducted in awake animals and began ∼16-18 h after implantation of the microdialysis probe into striatum.
Analysis of dialysate
Dialysis samples were collected every 15 min and 20 μl was analyzed by HPLC-electrochemical detection for the quantification of DA. A Velosep RP-18 column (100 × 3.2 mm, Brownlee; Applied Biosystems, Foster City, CA, U.S.A.) was used with a mobile phase composed of 0.1 M sodium acetate buffer (pH 4.1), 0.1 mM EDTA, 1.2 mM sodium octyl sulfate, and 9% (vol/vol) methanol. The flow rate through the system was 0.7 ml/min. The system used either an Antec Model CU-04AZ (Leiden, The Netherlands) or a Waters Model 460 detector (Milford, MA, U.S.A.). The potential of the working electrode of these amperometric detectors was set at +0.6 V. Before each experiment, the system was calibrated with 20 μl of 10 nM standard solution in 0.1 M perchloric acid. DA was identified by retention time and quantified based on the peak height. The limit of detection for DA in the analyses was ∼0.4 pg. Pharmacological manipulations were not initiated until the DA level in at least three consecutive dialysate samples was stable (≤10% variability).
Drugs were dissolved in 0.9% NaCl and administered intraperitoneally (i.p.). Each rat that received L-DOPA (50 mg/kg) also received an injection of 25 mg/kg benserazide, an inhibitor of peripheral AADC, 30 min before L-DOPA, to decrease the metabolism of L-DOPA outside the CNS. A higher dose of benserazide (50 mg/kg i.p.) has been shown to have no measurable central effects by itself and to potentiate the central effect of L-DOPA (Bunney et al., 1973).
In experiments that examined the contribution of high-affinity DA uptake to the neurochemical effects of L-DOPA, GBR-12909 (20 mg/kg), a selective inhibitor of DA uptake, was administered 90 min before L-DOPA. This agent specifically blocks high-affinity DA uptake without affecting DA release (Nissbrandt et al., 1991).
Another set of experiments examined the contribution of impulse-dependent neurotransmitter release to the effects of exogenous L-DOPA on extracellular DA in striatum. This was accomplished by dissolving tetrodotoxin (TTX), a fast sodium channel blocker, in the artificial CSF perfusate at a concentration of 1 μM and then infusing it into striatum through the microdialysis probe.
Data are expressed as mean ± SEM values. Values are not corrected for the in vitro probe recovery. In cases where DA was nondetectable, a value of 0 pg/sample was assigned. Within-group effects were analyzed by using one-way ANOVA with repeated measures over time coupled to Dunn’s post hoc test for multiple comparisons (p < 0.05 criterion). Between-group effects were analyzed by using two-way ANOVA with repeated measures over time coupled to Fisher’s protected least significant difference post hoc test (p < 0.05 criterion).
Severity of 6-OHDA lesions was determined by measuring the tissue DA content in striata of all animals. To allow washout of any neurochemical changes due to drug treatment, the rats were killed by decapitation and the probe assembly was removed at least 1 week after the dialysis experiments. The striata from the intact and lesioned side were rapidly dissected on ice, wrapped in aluminum foil, labeled, and frozen at -80°C until analysis. The wet weight of each striatum was measured and striata were then homogenized in 0.1 M perchloric acid containing 100 μM EDTA (20 μl/mg of wet tissue). Homogenates were centrifuged at 29,200 g for 25 min at 2-8°C. The amount of DA in 10-μl samples of the resulting supernatant was quantified by HPLC-electrochemical detection in a manner similar to that described above for the analysis of DA in dialysate. The extent of striatal DA depletion produced by the 6-OHDA lesion was determined as the percent decrease in the concentration of DA in striatal tissue on the lesioned versus intact side. Only animals that had extensive striatal DA depletion (98.4 ± 1.0%, n = 18) were included in the present experiment.
GBR-12909 was purchased from Research Biochemicals (Natick, MA, U.S.A.). Chloral hydrate, L-DOPA methyl ester, and TTX were purchased from Sigma Chemical (St. Louis, MO, U.S.A.). Benserazide was generously donated by Hoffmann-La Roche (Nutley, NJ, U.S.A.). All other reagents and chemicals were of the highest purity commercially available (Fisher Scientific, Springfield, NJ, U.S.A.).
Effect of L-DOPA on extracellular DA in intact and DA-denervated striatum
In intact rats, systemic administration of L-DOPA significantly increased extracellular DA from an average baseline level of 6.1 ± 1.0 pg/sample to a peak level of 7.9 ± 1.2 pg/sample (F8,40 = 11.4, p < 0.001, n = 6) (Fig. 1). In 6-OHDA-lesioned rats, treatment with L-DOPA produced a significant increase in striatal extracellular DA from an average baseline level of 0.7 ± 0.3 pg/sample to a peak level of 35.5 ± 3.5 pg/sample (F8,40 = 45.9, p < 0.001, n = 6) (Fig. 1). The L-DOPA-induced increase in extracellular DA was significantly greater in DA-denervated than in intact striatum (F7,70 = 27.6, p < 0.001).
Role of high-affinity DA uptake in the appearance of extracellular DA after exogenous L-DOPA
The administration of GBR-12909, a selective inhibitor of DA uptake, significantly increased extracellular DA in intact striatum from an average baseline level of 6.6 ± 0.7 pg/sample to a peak level of 46.8 ± 2.5 pg/sample (F4,16 = 92.8, p < 0.001, n = 5) (Fig. 2). In a separate group of rats, this effect of DA uptake blockade was significantly augmented from 46.6 ± 5.4 pg/sample to a peak level of 69.6 ± 14.7 pg/sample by the administration of L-DOPA 90 min after GBR-12909 treatment (F8,48 = 6.5, p < 0.001, n = 7) (Fig. 2). The absolute magnitude of this L-DOPA-induced change was not significantly different from the absolute change in extracellular DA observed in DA-denervated striatum after L-DOPA alone (F7,77 = 0.17, p = 0.98). The effect of L-DOPA administration in DA-lesioned rats was not significantly altered by pretreatment with GBR-12909 (F7,70 = 0.15, p = 0.99) such that L-DOPA significantly increased extracellular DA in these latter animals from 1.1 ± 0.2 to 41.8 ± 9.9 pg/sample (F8,40 = 12.0, p < 0.001, n = 6) (Fig. 2).
Impulse dependence of L-DOPA-induced DA release in striatum
In intact striatum, TTX infusion significantly lowered extracellular DA from an average baseline level of 4.4 ± 0.5 pg/sample to below the detection limit of the assay (F4,16 = 36.4, p < 0.001, n = 5) (data not shown). As shown in Fig. 2, exogenous L-DOPA potentiates extracellular DA levels in intact striatum when high-affinity DA uptake is inhibited. In intact rats treated with GBR-12909, TTX infusion significantly decreased this response of extracellular DA to L-DOPA treatment (F7,77 = 4.2, p < 0.001) (Fig. 3). In this condition, exogenous L-DOPA increased extracellular DA from 4.2 ± 0.4 pg/sample to a peak level of 11.6 ± 1.5 pg/sample (F8,40 = 19.3, p < 0.001, n = 6) compared with the change from 46.6 ± 5.4 pg/sample to a peak level of 69.6 ± 14.7 pg/sample in the absence of TTX (Fig. 3).
TTX infusion significantly attenuated the ability of exogenous L-DOPA to increase DA efflux in DA-denervated striatum (F7,70 = 22.5, p < 0.001). When TTX was infused into DA-denervated striatum, exogenous L-DOPA significantly increased extracellular DA from 0.3 ± 0.3 pg/sample to a peak level of 6.2 ± 0.9 pg/sample (F8,40 = 13.9, p < 0.001, n = 6) compared with the change from 0.7 ± 0.3 pg/sample to a peak level of 35.5 ± 3.5 pg/sample in the absence of TTX (Fig. 3).
The present study was designed to determine the roles of high-affinity DA uptake and impulse-dependent neurotransmitter release in the appearance of DA in striatal extracellular fluid after the administration of exogenous L-DOPA to intact and DA-lesioned rats. Systemic administration of L-DOPA elicited a robust increase in extracellular DA in DA-denervated striatum whereas in intact striatum only a very small increase in extracellular DA was observed. These results are in agreement with previous findings that extracellular DA is increased to a greater extent in DA-denervated striatum than in intact striatum in response to exogenous L-DOPA (Abercrombie and Zigmond, 1990; Abercrombie et al., 1990; Wachtel and Abercrombie, 1994). The intense lesion severity in the 6-OHDA-treated rats used in these experiments (∼98% depletion of tissue DA) indicates that any substantial contribution of residual DA terminals to L-DOPA’s effect on extracellular DA in striatum is unlikely. As suggested previously, this effect of exogenous L-DOPA on extracellular DA therefore is likely to be mediated by a nondopaminergic cellular element of striatum (Ng et al., 1972; Trugman and Wooten, 1986; Melamed, 1988; Abercrombie et al., 1990; Wachtel and Abercrombie, 1994).
We hypothesized that the lesser effect of exogenous L-DOPA on extracellular DA level in intact striatum relative to lesioned striatum is due, in part, to the fact that DA terminals efficiently clear extracellular DA derived from exogenous L-DOPA. To examine this hypothesis, the effect of exogenous L-DOPA on extracellular DA was assessed in intact striatum in the presence of GBR-12909, a selective DA uptake inhibitor. In intact striatum, GBR-12909 elicited an increase in extracellular DA and a further increase was produced by administration of L-DOPA. In a similar manner, previous studies have revealed that L-DOPA’s effect on extracellular DA in intact striatum is potentiated by pretreatment with nomifensine, a relatively nonselective inhibitor of catecholamine uptake (Abercrombie and Zigmond, 1990; Abercrombie et al., 1990). The absolute magnitude of the change in extracellular DA observed in intact striatum after the combination of GBR-12909 and L-DOPA was not different from the absolute change in DA observed in DA-denervated striatum after L-DOPA alone. In addition, DA uptake blockade did not alter L-DOPA’s effect on extracellular DA in DA-denervated striatum. Together, these results indicate the importance of DA terminals for removing from the extracellular space DA derived from exogenous L-DOPA.
As discussed above, extracellular DA level does not substantially increase in striatum of intact rats after administration of exogenous L-DOPA. This phenomenon is coincident with a negligible behavioral response (Schoenfeld and Uretsky, 1973; Hollister et al., 1979; Zetterström et al., 1986). In contrast, significant increases in extracellular DA and behavioral effects of exogenous L-DOPA are highly correlated in DA-lesioned rats. In intact striatum, it is likely that increases in extracellular DA in response to exogenous L-DOPA are modest because of the small diffusion coefficient for DA in extracellular space of striatum that results from the effects of high-affinity DA uptake (Nicholson and Rice, 1991; Garris and Wightman, 1994). Likewise, in intact striatum, the DA derived from exogenous L-DOPA apparently does not reach the DA receptors responsible for DA-mediated behavioral changes. It appears that DA uptake effectively clears DA synthesized from exogenous L-DOPA from the extracellular fluid of intact striatum such that diffusion of DA either to the microdialysis probe or to the appropriate postsynaptic receptors is quite restricted. Unrestricted diffusion of DA in the extracellular fluid of DA-denervated striatum allows both the detection of extracellular DA at the microdialysis probe and the activation of DA receptors, which in turn elicits behavioral changes.
These neurochemical and behavioral data discussed above are consistent with the hypothesis that increased DA release at DA synapses may not be a major site for DA release in response to exogenous L-DOPA. When DA uptake was inhibited in intact striatum, administration of L-DOPA elicited a large increase in extracellular DA that was comprised of a significant TTX-sensitive portion. Likewise, in striatum of 6-OHDA-lesioned rats, the ability of exogenous L-DOPA to increase extracellular DA was significantly attenuated by TTX infusion. The release of DA formed from exogenous L-DOPA in both intact and DA-denervated striatum therefore has two components, (1) a large TTX-sensitive component (∼80%) and (2) a smaller TTX-insensitive component (∼20%). The impulse dependence and impulse independence of extracellular DA observed after exogenous L-DOPA have previously been revealed to occur in intact striatum with various ratios between these two components (Abercrombie and Zigmond, 1990; Acquas et al., 1992; Koshimura et al., 1992). However, there is no prior evidence for an impulse-dependent component in DA formed from L-DOPA in DA-lesioned striatum. It is proposed that most released DA formed from exogenous L-DOPA apparently is derived from a nondopaminergic neuronal component in rat striatum.
The cellular constituents underlying the two components of DA release in response to exogenous L-DOPA are not known (Hefti et al., 1981; Melamed et al., 1980, 1981; Kang et al., 1992; Nakazato and Akiyama, 1992). The most likely neuronal mediators for the impulse-dependent component of DA release in striatum after exogenous L-DOPA are the neurons that contain AADC activity such as the GABAergic medium-spiny projection neurons (Hefti et al., 1981; Melamed et al., 1981) and the serotonin afferent terminals (Arai et al., 1996). Studies that have examined the effects of lesions of these different types of neurons on the tissue DA response to exogenous L-DOPA have been inconclusive (Hefti et al., 1981; Melamed et al., 1980, 1981). Microdialysis studies in our laboratory, however, suggest the possibility that serotonin terminals in DA-denervated striatum are the source for the increase in extracellular DA after exogenous L-DOPA (Miller and Abercrombie, 1998). The impulse-independent component of extracellular DA formed from exogenous L-DOPA could potentially result from passive neurotransmitter efflux from glia, as these latter cells express the AADC gene and contain AADC (Li et al., 1992). In addition, some neuronal DA derived from exogenous L-DOPA may diffuse to the microdialysis probe from striatal areas beyond the influence of TTX applied locally via the microdialysis probe.
In conclusion, administration of L-DOPA increases extracellular DA in DA-denervated striatum. This phenomenon is under the regulation of high-affinity DA uptake in intact striatum. In addition, striatal DA release in response to exogenous L-DOPA occurs largely by an impulse-dependent mechanism. This suggests that a non-dopaminergic neuronal constituent of striatum likely is responsible for the therapeutic effects of L-DOPA in Parkinson’s disease.
We thank William S. Cobb and Gareth R. I. Barker for their technical assistance in the tissue analyses. We also thank Dr. Christina R. McKittrick and William S. Cobb for critical reading of the manuscript. The gift of benserazide from Hoffmann-La Roche (Nutley, NJ, U.S.A.) is greatly appreciated. This research was supported by USPHS grants NS19608 (E.D.A.) and MH11826 (D.W.M.).