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Journal ofNeurochemistryLippincott—Raven Publishers, Philadelphia© 1997 International Society for Neurochemistry

Neurotrophic Effects of L-DOPA in Postnatal MidbrainDopamine Neuron/Cortical Astrocyte Cocultures

*~M~j.jaAngeles Mena, *Viviana Davila, and *David Suizer

* Departments of Neurology and Psychiatry, Columbia University and Department of Neuroscience, New York State

Psychiatric Institute, New York, New York, U.S.A.; and ~Departamento de Investigación,Hospital Ramón y Cajal, Madrid, Spain

Abstract: L-DOPA IS toxic to catecholamine neurons inculture, but the toxicity is reduced by exposure to astro-cytes. We tested the effect of L-DOPA on dopamine neu-rons using postnatal ventral midbrain neuron/corticalastrocyte cocultures in serum-free, glia-conditioned me-dium. L-DOPA (50 tiM) protected against dopamine neu-ronal cell death and increased the number and branchingof dopamine processes. In contrast to embryonically de-rived glia-free cultures, where L-DOPA is toxic, postnatalmidbrain cultures did not show toxicity at 200 1iM L-

DOPA. The stereoisomer D-DOPA (50—400 btM) was notneurotrophic. The aromatic amino acid decarboxylase in-hibitor carbidopa (25 ~zM)did not block the neurotrophiceffect. These data suggest that the neurotrophic effect ofL-DOPA is stereospecific but independent of the produc-tion of dopamine. However, L-DOPA increased the levelof glutathione. Inhibition of glutathione peroxidase by L-buthionine sulfoximine (3 1iM for 24 h) blocked the neuro-trophic action of L-DOPA. N-Acetyl-L-cysteine (250 1iMfor 48 h), which promotes glutathione synthesis, had aneurotrophic effect similar to that of L-DOPA. These datasuggest that the neurotrophic effect of L-DOPA may bemediated, at least in part, by elevation of glutathione con-tent. Key Words: L-DOPA—Glia—Glutathione—Parkin-son‘s disease—Dopamine neurons.J. Neurochem. 69, 1398—1408 (1997).

The effects of L-DOPA on dopamine (DA) neuronsare quite different in vivo and in vitro. Whereas rela-tively low levels (25 jiM) of L-DOPA are toxic inculture (Melamed, 1986; Mena et al., 1992, 1993; My-tilineou et al., 1993; Pardo et al., 1995), the drug hasnot been seen to damage DA neurons in healthy ani-mals (Hefti et al., 1981; Perry et al., 1984; Yurek etal., 1991; Blunt et al., 1993) or humans (Quinn et al.,1986). As L-DOPA is the most effective compoundfor symptomatic treatment of Parkinson‘ s disease(PD), its influence on the progression of the diseaseis particularly important. Although some reports haveindicated increased fluctuations and dyskinesia relatedto the duration of the L-DOPA treatment (Lesser etal., 1979), other studies have found no evidence of

negative impact (Markham and Diamond, 1986; Blintet al., 1988; Uitti et al., 1993).

Previous studies on L-DOPA toxicity in culture haveused embryonically derived cells grown in the presenceof a very low level of glia (<5%), although in thebrain DA neurons are enveloped by glia. To investigatethe modulatory effects of glia on L-DOPA toxicity, wehave used a coculture system of postnatal midbrainDA neurons and cortical astrocytes, a target area ofmidbrain DA projections. The protective role of gliahas been demonstrated in experiments showing thatmedium preconditionedby glia protects embryonicallyderived midbrain cultures from L-DOPA toxicity(Mena et al., 1996). The midbrain neurons/corticalglia coculture system used in the present report is ad-vantageous for this study because it produces a highnumber of mature midbrain neurons generally 30—40% of the neurons are tyrosine hydroxylase (TH) -

positive, as compared with <4% typical of embryoni-cally derived cultures]. We suggest that this system,which has previously been used to provide insightsinto the mechanisms of methamphetamine neurotoxic-ity (Cubells et al., 1994) and the role of superoxidedismutase (SOD) in protection from cell death(Przedborski et al., 1996), is appropriate for studyingthe effects on cell survival and the mechanisms ofaction of L-DOPA.

MATERIALS AND METHODS

MaterialsL- and o-DOPA, L-N-acetylcysteine (L-NAC), L-buthio-

nine- S,R-sulfoximine (BSO), glutathione [as its reduced

Received April 7, 1997; revised manuscript received May 16,1997; accepted May 16, 1997.

Address correspondence and reprint requests to Dr. M. A. Menaat Departamento de Investigación, Hospital Ramón y Cajal, Can deColmenar Km 9, Madrid 28034, Spain.

Abbreviations used: BSO, L-buthionjne-S,R-sulfoximine; DA, do-pamine; GSH, glutathione (reduced form); GSSG, glutathione (oxi-dized form); L-NAC, L- N-acetylcysteine; PD, Parkinson‘s disease;SOD, superoxide dismutase; TH, tyrosine hydroxylase.

1398

L-DOPA AND POSTNATAL DOPAM1NE COCULTURE 1399

form (GSH)], 3,3 ‘-diaminobenzidine tetrahydrochloride,and 5,5 ‘-dithio-bis(2-nitrobenzoic acid) were obtained fromSigma Chemical (St. Louis, MO, U.S.A.). Biotinylated horseanti-mouse secondary antibody was from Vector Labora-tories (Burlingame, CA, U.S.A.).

Coculture of postnatal DA neurons/corticalastrocytes

Cultures of ventral midbrain neurons from postnal day 1—2 rats were prepared as in related studies (Rayport et al.,1992; Rayport and Suizer, 1995; Pothos et al., 1996; Przed-borski et al., 1996) except that serum-freemedium was usedas recently described (Mena et al., 1997). Astrocyte mono-layers were prepared from the rostral half of the cerebralcortex in ice-cold calcium- and magnesium-free phosphate-buffered salineon postnatal day 2. The chunks of tissue weredissociated with 20 U/ml papain in 1 mM cysteine, 0.001%phenol red, 500 jiM kynurenate, 116 mM NaCl, 5.4 mMKO, 26 mM NaHCO5, 2 mM NaH2PO4, 1 mM magnesiumphosphate, 500 jiM EDTA, and 25 mM glucose, pH 7.3.Cells were plated a density of 150,000 per well and fed withmedium containing 90% minimal essential medium, 10%calf serum, 1.83 mM glucose, 5 jig/ml bovine pancreaticinsulin, 500 jiM glutamine, 0.6 U/mI penicillin, and 60 jig!ml Streptomycin. 5-Flurodeoxyuridine (11 jiM with 55 jiMuridine) was added 4—5 days after plating, to prevent prolif-eration of oligodendrocytes.

After 2 weeks in culture, midbrain neurons were platedonto glial monolayers. Twenty-four hours before plating, theglial medium wasreplaced by serum-free medium containing47% minimal essential medium, 40% Dulbecco‘s modifiedEagle‘s medium, 10% Ham‘s F-12 nutrient medium, 18.9mM glucose, 0.25% albumin, 500 jiM glutamine, 100 jig!ml transferrin, 15 jiM putrescine, 30 nM triiodothyronine,25 jig/ml insulin, 200 nM progesterone, 125 nM corticoste-rone, 5 j.tg/ml SOD, 10 j.tg/ml catalase, and500 jiM kynure-nate. On the next day, the tissue was dissociated, and 80,000cells, resuspended in serum-free glia conditioned medium,were plated per well. During the following 24 h, cultureswere exposed to 5-fluorodeoxyuridine and maintained in se-rum-free medium at 37°Cin 5% CO2.

Experimental designsDose-dependent effects of L-DOPA were studied in post-

natal cocultures grown in serum-free medium and treatedwith L-DOPA (0, 25, 50, 100, 200, and 400 jiM) for 48 hat day 5 in vitro.

The effects of L-DOPA and D-DOPA were compared incultures grown as above and treated with one of the twostereoisomers at concentrations of 50 and 400 jiM for 48 h.These two concentrations were chosen because we foundthat L-DOPA shows neurotrophic activity with the lowerconcentrationandneurotoxic properties at the higherconcen-tration (see below).

The neurotrophic properties of L-DOPA on cell survivalwere further investigated in cultures treated on the third dayafter plating with 50 jiM L-DOPA for either 24 h or 4 days.We investigated whether the neurotrophic activity of L-

DOPA is due to itself or to its metabolite DA, in culturestreated with 50 jiM L-DOPA or 50 ‚aM L-DOPA with 25pM carbidopa. Previously, we found by HPLC with electro-chemical detection that 25 ‚aMcarbidopa blocked the conver-sion of L-DOPA to DA in our culture conditions (Mena etal., 1997).

The interaction betweenL-DOPA andGSH wasexamined.

Cells maintained in culture for 5 days were treated with GSH(200 and 400 ‚aM) and with the GSH precursor, L-NAC(250 and 500 ‚aM), for 2 days or with a GSH synthesisinhibitor, BSO (3 ‚aM), for 24 h. The effects of L-DOPA(50 ‚aM) on GSH intracellular levels were examined in co-cultures treated with L-DOPA for 24 h.

Measurement of levels of quinonesExtracellular quinones, which indicate extracellular L-

DOPA autooxidation, were evaluated according to the spec-trometric measurement of the optical density at 490 nm inthe culture medium (Mena et al., 1992, 1993).

Cell culture immunocytochemistryDA neurons in ventral midbrain cultures were identified

by TH immunohistochemistry as previouslydescribed (Ray-port et al., 1992; Przedborski et al., 1996). In brief, cultureswere fixed with 4% paraformaldehyde, washed in Tris-buf-fered saline (0.1 M Tris-HC1 in 9 g/L NaC1, pH7.4), perme-abilized with 0.1% Triton X-100 (Sigma), and incubated at4°C for 48 h in a 1:640 dilution of monoclonal anti-THantiserum (Boehringer Mannheim) in Tris-buffered salinecontaining0.1% Triton X-100 and 10% normal horse serum.The secondary antibody was a biotinylate-conjugated mono-clonalanti-mouse antibody (1:200) and ahorseradish-conju-gated avidin/biotin complex (Vector Laboratories); immu-nostaining was revealed by 3,3 ‘-diaminobenzidine/H2O2.To assess the specificity of TH immunostaining, the primaryor the secondary antibody was omitted.

Glutathione level measurementsGlutathione (GSH) levels were measured according to the

method of Tietze (1969). In brief, 2 X 106 cells were washedtwice with phosphate-buffered saline, lysed in 3% perchloricacid for 15 min at 4°C,and centrifuged, and supernatantswere neutralized with 9 volumes of 5 mM EDTA in 0.1 MNaH2PO4, pH 7.5. Glutathione content was measured byaddition of 5,5 ‘-dithio-bis (2-nitrobenzoïc acid), and the re-action was monitored at 412 nm. GSH content is expressedas a function of total proteins in the cell extract. Proteinswere quantified in the pellet by the method of Bradford(1976).

Cell counts and statisticsResults are expressed as mean ±SEM values for three to

six independent experiments, and each data point corre-sponds to four or more cultures derived from the same litter.In each culture, eight to 10 consecutive fields (magnificationof 100 or 200 X) were counted under phase optics; boththe total number of neurons and TH-stained neurons werecounted. The number of cells was corrected for area. Resultswere statistically evaluated for significance by ANOVA formultiple groups followed by Student‘ s t test. The differencewas considered significant when p < 0.05.

RESULTS

Dose-dependent effect of L-DOPA on postnatalcocultures

To observe the effects of L-DOPA, we incubatedthe cultures for 7 days in serum-free culture mediumand added L-DOPA (0, 25, 50, 100, 200, and 400 ‚aM)for 48 h on day 5. In contrast to previous observationswith embryonically derived cultures, there was no tox-icity or enhanced quinone formation at concentrations

J. Neurochem., Vol. 69, No. 4, 1997

1400 M. A. MENA ET AL.

FIG. 1. Effect of L-DOPA on TH-positive neurons. Micrographs show TH immunostaining of postnatal ventral midbrain cells treatedwith L-DOPA. Cells were treated for 48 h after 5 days in culture with (A) vehicle, (B) L-DOPA (50 pM), (C) L-DOPA (100 pM), or (D)L-DOPA (200 pM). Bar = 30 jim.

of L-DOPA up to 200 ‚aM. Moreover, L-DOPA at 50‚aM increased the number of surviving TH-positiveneurons by 166 ±9% (Fig. 1 and Table 1).

L-DOPA also markedly increased the proliferationof TH-positive neuntes. To compare these effects, weadapted semiquantitative methods for estimating theelaboration of processes in culture (Denis-Donini et

al., 1983). L-DOPA (50 ‚aM) increased the number ofTH-positive processes that extended >80 ‚am from apoint in the center of the cell body by 177 ± 14%and increased the number of TH-positive neurons thatdisplayed more than three primary processes by 162±12% (Fig. 2). This process outgrowth was attenu-ated at levels> 100 ‚aM, but there was no neurotoxicity

TABLE 1. Effect of L-DOPA (48 h) on neuronal survival and quinone formation

Cells/cm2

% TH-positiveQuinones

(OD X 10~)TH-positive TH-negative

Controls 645 ±34 (100%) 1,251 ±123 (100%) 34.2 ±1.54 369 ±19 (100%)L-DOPA

25 pM 949 ±516 (147%) 1,130 ±88 (90%) 45.3 ±3.6° 367 ±15 (100%)50 pM 1,071 ±58~(166%) 1,076 ±75 (86%) 49.8 ±l.3l~ 328 ±II (89%)100 pM 774 ±65 (120%) 1,451 ±10 (116%) 34.8 ±0.92 343 ±11 (93%)200 pM 710 ±71 (110%) 1,426 ±11 (114%) 33.2 ±1.3 373 ±11 (101%)400 pM 534 ±49 (82.8%) 919 ±87° (73.4%) 36.6 ±3.1 635 ±26~(172%)

Data are mean ±SEM values. Numbers of TH-positive and -negative cells/cm2 are from two to sixexperiments (n > 6 for each data point). Cultures were treated with L-DOPA for 48 h after 5 days inculture.

°p< 0.05, b~< 0.01, ‘~p< 0.001 versus controls.


L-DOPA AND POSTNATAL DOPAMINE COCULTURE 1401

FIG. 2. Effects of 50 pM L-DOPA on process outgrowth of TH-positive neurons. L-DOPA (50 pM) increased the number ofTH-positive processes that extended >80 pm from a point in thecenter of the cell body and increased the number of TH-positiveneurons that displayed more than three primary processes. Dataare expressed as mean ±SEM (bars) percentages of TH-posi-tive neurons that displayed more than three primary processesversus total 1H-positive neurons (% TH + MP) and as mean±SEM (bars) percentages of TH-positive neurons with pro-cesses >80 pm versus total TH-positive neurons (% TH + LP).*p <0.01 for L-DOPA versus controls.

up to the highest concentration tested, and quinoneformation was observed only at 400 ‚aML-DOPA (Ta-blei and Fig. 1).

Effect of L-DOPA on cell survival of all neuronsand TH-positive cells and on quinone formation

From the previous experiment, we selected 50 ‚aML-DOPA as the optimal concentration for neurotrophiceffects. Cultures were treated after 3 days in vitro with50 ‚aM L-DOPA or solvent for 24 h or 4 days and fixedat 2 h or 4 and 7 days after plating. In both cases,L-DOPA significantly increased the survival of TH-positive labeled neurons versus controls (Fig. 3). Nochanges in number of TH-unlabeled neurons or in qui-none formation were observed (data not shown).

FIG. 3. Effect of 50 pM L-DOPA on TH-positive neuronal sur-vival. Cultures were exposed at 3 days postplating to vehicle or50 pM L-DOPA for 24 h or 4 days. The cells were fixed after 2h or 4 or 7 days postplating. Data are expressed as mean ±SEM(bars) values (n = 6 cultures) of TH-positive neurons. °p<0.05,**p < 0.01 for L-DOPA versus controls.

Comparison of D- and L-DOPA effectsTwo experiments were performed to study the dose-

and time-dependent effects of L- and D-DOPA on post-natal midbrain neuron/cortical astrocyte cocultures. Toexamine shorter-term effects, L- or D-DOPA (0, 50,and 400 ‚aM) was added at day 5 for 48 h. L-DOPA(50 ‚aM) increased the number and percentage of TH-positive neurons without altering quinone formation ornumber of TH-unlabeled neurons. In contrast, D-DOPA(50 ‚aM) decreased levels ofTH-positive and -negativeneurons to 83 and 64% of controls, respectively.Higher levels of L-DOPA (400 ‚aM) decreased levelsof TH-positive and -negative neurons to 83 and 74%,whereas 400 ‚aM D-DOPA decreased levels of TH-positive and -negative cells to 60 and 49% of controls.Quinone formation was elevated with L-DOPA (400‚aM) to 173% of controls and to 498% of controls withD-DOPA (Fig. 4 and Table 2).

TABLE 2. Effect of L-DOPA and D-DOPA (48 h)on neuronal survival and quinone formation

Cells/cm2


(OD X I 0~)1H-positive TH-negative

Controls 560 ±33 (100%) 1,086 ±51 (100%) 34.2 ±1.4 290 ±1.2 (100%)L-DOPA

50 pM 846 ±78° (151%) 1,109 ±48 (102%) 43.1 ±1.8° 300 ±19 (103%)400 pM 464 ±43 (83%) 804 ±82 (74%) 36.6 ±3 514 ±72~ (173%)

n-DOPA50 pM 465 ±18°~(83%) 697 ±6.9°-°(64%) 40.1 ±0.9 305 ±3.3 (102%)400 pM 338 ±63~~(60%) 530 ±7‘~°(49%) 38.3 ±5.4 1,444 ±99‘° (498%)

Data are mean ±SEM values. Numbers of 1H-positive and -negative cells /cm2 are from two to sixexperiments (n > 6 for each data point). Cultures were treated with L- and D-DOPA (0, 50, and 400 pM)for 48 h after 5 days in culture.

“p < 0.05, b~< 0.01, Cp < 0.001 versus controls.~p < 0.05, “p < 0.001 for cultures treated with D-DOPA versus L-DOPA.


1402 M. A. MENA ET AL

FIG. 4. Comparison ofthe effect of L- and D-DOPA on TH-positive neurons. TH immunostaining was done of postnatal ventral midbraincells treated with L- and D-DOPA. Cells were treated at 5 days postplating for 48 h with (A) vehicle, (B) L-DOPA (50 pM), (C) D-DOPA(50 pM), (D) L-DOPA (400 pM), or (E) D-DOPA (400 pM). Bar = 30 pm.



TABLE 3. Effect of L-DOPA and D-DOPA (8 days)on neuronal survival and quinone formation

Cells/cm2

% 1H-positiveQuinones

(OD X 10~)TH-positive TH-negative

Controls 302 ±17 (100%) 510 ±15 (100%) 36.9 ±1.7 352 ±11 (100%)L-DOPA (200 pM) 260 ±33 (86%) 501 ±27 (98%) 34 ±4 556 ±25‘ (160%)D-DOPA (200 pM) 89 ±26~“’(30%) 192 ±33“ (38%) 26.6 ±2“ 1,144 ±25“ (325%)

Data are mean ±SEM values. Numbers of 1H-positive and -negative cells/cm2 are from two experiments(n = 8 for each data point). Cultures were treated with L- and o-DOPA (200 pM) for 8 days after 5 daysin culture.

“p < 0.05, b~< 0.01, ‘p < 0.001 versus controls.“p < 0.001 for cultures treated with o-DOPA versus L-DOPA.

To examine longer-term effects, cocultures weretreated withL-DOPA or D-DOPA (200 ‚aM) for 8 days.L-DOPA (200 ‚aM) did not altercell survival, althoughit produced a 158% increase in levels of extracellularquinones. D-DOPA (200 ‚aM) for 8 days decreased thelevel of TH-positive neurons to 28% of controls andthat of TH-negative neurons to 38% of controls. D-DOPA increased quinone levels to 325% of controls,producing a distinct red-brown cast to the medium anddamaged cellular morphology (Fig. 5 and Table 3).We conclude that D-DOPA has no neurotrophic effectand is toxic at a dose where L-DOPA is neuroprotec-tive.Effect of carbidopa on L-DOPA response

To examine if the neurotrophic effect that we reportwas dependent on the dopamine formation from L-DOPA, we cotreated the cultures with L-DOPA withcarbidopa. Cocultures exposed to a combination of 25‚aM carbidopa [a level that blocks DA formation fromL-DOPA in culture (Basma et al., 1995; Mena et al.,1997)] and 50 ‚aM L-DOPA had a neurotrophic effectsimilar to the effect of L-DOPA alone. Therefore, con-version of L-DOPA toDA is not required for the neuro-trophic effect. However, carbidopa significantly in-creased the levels of quinones produced (Table 4).Effect of L-BSO, L-NAC, and glutathione onL-DOPA response

A different mechanism by which L-DOPA could ex-ert neurotrophic effects is by up-regulating oxygen rad-

ical scavenging systems. In particular, L-DOPA hasbeen shown to increase GSH formation in humans(Tohgi et al., 1995) and in culture (Han et al., 1996).We designed experiments to test whether GSH synthe-sis itself could provide the neurotrophic effects.

We added exogenous GSH (200 and 400 ‚aM at 5days in vitro for 48 h) and found an increased elabora-tion of TH-positive processes identical to that by L-DOPA (157 and 176%, respectively, of control lev-els). The GSH precursor, L-NAC (250 and 500 ‚aM),also induced process outgrowth identical to that with L-DOPA or GSH and increased the level of TH-positiveneurons to 146 and 182%, respectively, of control lev-els (Fig. 6 and Table 5).

IfL-DOPA exerts aneurotrophic effect by potentiat-ing the GSH pathway, process outgrowth should beinhibited by GSH synthesis inhibition. We found thata GSH synthesis inhibitor, BSO, at a level that had noeffect itself (3 ‚aM for 24 h) completely inhibited theneurotrophic effects of 50 ‚aM L-DOPA (Table 6).

Effect of L-DOPA on glutathione levelsTo determine whether L-DOPA increases intracellu-

lar GSH content, we examined GSH intracellular levels24 h after treatment with 50 ‚aM L-DOPA using aprocedure by Tietze (1969). The assay method doesnot distinguish between the reduced (GSH) and oxi-dized (GSSG) forms of glutathione. However, GSSGnormally only represents a small fraction (0.2%) of

TABLE 4. Effect of L-DOPA and carbidopa (CBD; 48 h)on neuronal survival and quinone formation

Cells/cm2

% 1H-positiveQuinones

(OD X I 0~)1H-positive TH-negative

Controls 509 ±25 (100%) 1,357 ±100 (100%) 27.3 ±2.6 544 ±16 (100%)L-DOPA (50 pM) 824 ±27‘ (162%) 1,485 ±101 (109%) 35.7 ±1.4“ 582 ±14 (107%)CBD (25 pM) 529 ±25 (104%) 1,194 ±48 (88%) 30.7 ±1.5 555 ±18 (102%)L-DOPA/CBD 794 ±38‘ (156%) 1,397 ±68 (103%) 36.2 ±1“ 674 ±14“ (124%)

Data are mean ±SEM values. Numbers of TH-positive and -negative cells/cm2 are from two experiments(n = 10 for each data point). Cultures were treated with L-DOPA (50 pM) and/or CBD (25 pM) for 48 hafter 5 days in culture.

“p < ~o5, bp < 0.01, ‘p < 0.001 versus controls.


1404 M. A. MENA ET AL

DISCUSSION

FIG. 5. Comparison of the effect of long-term L- and D-DOPAon TH-positive neurons. Micrographs show TH immunostainingof postnatal ventral midbrain cells treated with L-DOPA (200 pM)or D-DOPA (200 pM). Cells were treated at 5 days postplatingfor 8 days with (A) vehicle, (B) L-DOPA (200 pM), or (C)o-DOPA (200 pM). Bar = 30 pm.

the total glutathione (Pan and Perez-Polo, 1993; Ferrariet al., 1995), and it was previously found that L-DOPA-induced elevated glutathione levels in midbraincultures were due predominantly to an increased GSHcontent (Mytilineou et al., 1993). We found that 50‚aM L-DOPA increased GSH content to 137% of con-trol levels in midbrain neurons/cortical glia cocultures(from 20.8 ± 1.3 to 28.6 ± 2.2 nmol/mg of protein;n = 8, two experiments; p < 0.01).

This study demonstrates that L-DOPA has neuro-trophic effects on DA neurons, stimulates elaborationof neuntes, and protects DA neurons from cell death.These results contrast with previous studies using em-bryonic neurons cultured without an astrocyte layer,where L-DOPA is toxic at relatively low levels (Menaet al., 1993; Mytilineou et al., 1993; Pardo et al.,1995). In a previous study, we found that glia-condi-tioned medium protects against this toxicity (Mena etal., 1996). The present report points to a further in-sight, that in a coculture system using postnatal neu-rons and glia, L-DOPA is neurotrophic. In untreatedcells the death of TH-positive neurons was similar tothat in previous studies (Brugg et al., 1996). Thisdeath rate was reduced by >50% by L-DOPA. Thismay provide an explanation for discrepancies betweenthe results of L-DOPA in the various models. The neu-rotrophic actions of L-DOPA may underlie the long-term improved motor control after the cessation of drugadministration.

Previous studies indicate that DA added to culturemedium increases the expression of TH and aromaticamino acid decarboxylase in primary cultures of fetalneurons (De Vitry et al., 1991), and the neurotransmit-ter itself has been postulated as a neurotrophic factor(Leslie, 1993). Normally, L-DOPA inneuronal cultureis rapidly converted to DA and taken up into synapticvesicles (Pothos et al., 1996). If conversion of L-

DOPA to DA is required for its neurotrophic effects,the response should be blocked by the aromatic aciddecarboxylase inhibitor carbidopa (Basma et al.,1995). Our study reveals therefore that the mechanismresponsible for the neurotrophic effect of L-DOPA isindependent of its conversion to DA.

Astrocytes produce several neurotrophic and neu-rite-promoting agents (Engele et al., 1991; Nagata etal., 1993; Hoffen et al., 1994; Takeshima et al., 1994;Muller et al., 1995) that influence development, sur-vival, neunte extension, and neurotoxin resistance(Engele et al., 1991; Nagata et al., 1993; Takeshimaet al., 1994). Among the systems identified are a gluta-mate uptake activity that protects against excitotoxicity(Rosenberg et al., 1991) and stimulation of GSH syn-thesis and enzymes involved in GSH metabolism (Nis-ticè et al., 1992). GSH plays a major role in protectionagainst oxidative stress (Meister, 1988; Makar et al.,1994) and functions to remove the H202 generated bymonoamine oxidase (Werner and Cohen, 1993). GSHis produced by GSH peroxidase in culture (Raps etal., 1989). GSH peroxidase is mainly a mitochondrialenzyme (Victonica et al., 1984) exclusively located inthe human midbrain within glial cells (Slivka et al.,1987; Damier et al., 1993). GSH levels decrease withage in neurons but remain stable in astrocytes in culture(Sagara et al., 1996). However, neurons can maintainan intracellular glutathione poo1 by taking up cysteineprovided by glial cells (Sagara et al., 1993).



FIG. 6. Effects of L-NAC and GSH on postnatal ventral midbrain cells. Cultures were treated at 5 days postplating for 48 h with (A)vehicle, (B) L-NAC (0.25 mM), (C) L-NAC (0.5 mM), (D) L-NAC (1 mM), (E) GSH (200 pM), or (F) GSH (400 pM). Bar = 30 pm.

Protection from oxidation enhances the survival ofmesencephalic cultures (Mena et al., 1993, 1996;Pardo et al., 1995), and the number of TH-positiveneurons is increased if SOD, USH peroxidase, or L-NAC is addedto the medium (Colton et al., 1995). Wehave shown that TH-positive neurons that overexpressSOD display increased neutite outgrowth and survival(Przedborski et al., 1996) and are protected from L-DOPA toxicity (Mena et al., 1997). Recently, Han etal. (1996) have reported that L-DOPA raises GSH lev-els in cultures of fetal rat mesencephalon, a mouseneuroblastoma line (Neuro-2A), a human neuro-blastoma (SKNMC), and glia from newborn rat brainbut not in midbrain neuronal cultures grown withoutglia.

In the present study, the effects of L-DOPA were notassociated with an elevation in content of extracellularquinones, in contrast with the observations in embry-onically derived, glial-free cultures (Mena et al.,1996), suggesting that astrocytes may inhibit L-DOPAquinone formation by providing an antioxidant system.D-DOPA was not neurotrophic, although it was moreeffective in inducing formation of extracellular qui-nones. Moreover, we found that the GSH synthesispromoter L-NAC and GSH itself reproduced L-DO-PA‘s effects, whereas the GSH synthase inhibitor BSOblocked L-DOPA‘s effect. Therefore, the present studysuggests that L-DOPA is neurotrophic owing to stimu-lation of GSH peroxidase. It is possible that L-DOPAis sequestered intracellularly in glia by L-amino acid



TABLE 5. Effect of L-NAC and GSH (48 h)on neuronal survival

Cells/cm2

%TH-positiveTH-positive TH-negative

Controls 342 ±17 (100%) 919 ±46 (100%) 27.3 ±2.1L-NAC

1 mM 365 ±42 (107%) 1,030 ±97 (111%) 26.2 ±3.10.5 mM 500 ±46“ (146%) 1,119 ±203 (121%) 31.2 ±3.20.25 mM 623 ±49“ (182%) 1,061 ±97 (115%) 37.2 ±3.1“

GSH200 pM 539 ±16“ (157%) 1,026 ±106 (112%) 34.1 ±1.9“400 pM 603 ±62‘ (176%) 946 ±92 (103%) 39.1 ±1.1‘

Data are mean ±SEM values. Numbers of TH-positive and -negativecells/cm2 are from two experiments (n = 10 foreach data point). Cultureswere treated with L-NAC or GSH for 48 h after 5 days in culture,

“p < 0.05, h~< 0.01, ‘p < 0.001 versus controls.

transport systems and in turn stimulates astrocytic GSHperoxidase.

It appears unlikely that L-DOPA provides its neuro-trophic effect by acting as an antioxidant itself becausehigh doses of L-DOPA do not provide neuroprotectionand increase quinone formation; however, a cautionarynote is provided by the finding that L-NAC, previouslyassumed to act as an antioxidant due to its role as asubstrate for GSH synthesis, may protect PC12 cellsby acting directly as an oxygen radical scavenger (Yanet aI., 1995). L-NAC is a pluripotent protector thatelevates the level of intracellular glutathione, preventsagainst apoptotic death of neuronal cells, and enhancestrophic factor-mediated cell survival (Mayer and No-ble, 1994; Ferrari et al., 1995). Indeed, depletion ofbrain GSH with BSO in rats is accompanied by im-paired mitochondrial function and decreased complexIV activity (Heales et al., 1995) leading to energycrisis as a mechanism of nigral cell death (Jenner,1993).

In clinical studies, GSH content is decreased in thesubstantia nigra of patients with idiopathic PD (Ried-

erer et al., 1989; Sofic et al., 1992; Sian et al., 1994).L-DOPA treatment reverses GSH depletion (Tohgi etal., 1995). In normal subjects, the nigrostriatal DAneurons, the most important disease target associatedwith PD, are normally surrounded by a low density ofglia (Damier et al., 1993; Sagara et al., 1993; Makaret al., 1994), suggesting that these neurons are lessprotected from oxidative stress. It is striking that, inPD, the density of GSH peroxidase-positive glia sur-rounding the surviving DA neurons is increased, sug-gesting a compensatory proliferation of glia that pro-tects surviving neurons against pathological death (Da-mier et al., 1993).

Although L-DOPA is toxic for catecholamine neu-rons in other in vitro systems (Melamed, 1986; Menaet aI., 1992, 1993; Pardo et al., 1995), catecholaminesare required for the development of the nervous systemand survival. Inactivation of the TH gene produced thedeath of 90% of mutant mice fetuses, whereas adminis-tration of L-DOPA to pregnant females resulted incomplete rescue of mutant mice (Zhou et al., 1995).Transgenic animals with mutations of the TH genehave a poorer prognosis than those with mutations ofthe dopamine /3-hydroxylase gene (Thomas et al.,1995), suggesting that DA is essential for developmentof the nervous system. It is likely that L-DOPA andcatecholamines are critical factors for promoting thesurvival and differentiation of DA neurons and thatthe effects of L-DOPA on DA cells in vivo might beneurotrophic or neurotoxic depending on the local en-vironment produced by glial cells.

In summary, conclusions from this study and thatof Mena et al. (1996) indicate that a long-term potenti-ation of DA release by L-DOPA may occur owingto promotion of neunitic arbonization and protectionagainst cell death. The neurotrophic effects are inde-pendent of conversion to DA and dependent on thepresence of astrocytes and may result from antioxidantmechanisms such as up-regulation of GSH. Significantquestions that need to be addressed include whetherthe effect is due to L-DOPA itself acting as an antioxi-

TABLE 6. Effect of L-DOPA and BSO (48 h)on neuronal survival and quinone formation

Cells/cm2


(OD X l0~)TH-positive TH-negative

Controls 450 ±18 (100%) 1,303 ±60 (100%) 25.7 ±2.1 285 ±1.8 (100%)L-DOPA (50 pM) 720 ±72“ (160%) 1,408 ±143 (108%) 33.9 ±0.8~‘ 284 ±7 (99.7%)BSO (3 pM) 436 ±17 (96.8%) 1,160 ±72 (89%) 26.7 ±1.05 293 ±3 (103%)L-DOPA + BSO 441 ±40“ (98%) 1,329 ±11 (102%) 24.6 ±0.8 276 ±4 (96.7%)

Data are mean ±SEM values. Numbers of TH-positive and -negative cells/cm2 are from two experiments(n = 10 for each data point). Cultures were treated after 5 days in vitro with 50 pM L-DOPA (for 48 h)or 3 pM L-BSO alone or in combination. Pretreatment with BSO for 24 h was followed by washout withneuronal medium, before L-DOPA or solvent was added.

b~ < 0.01 versus controls.“p < 0.001 for cultures treated with L-DOPA + BSO versus L-DOPA.



dant or if it is due to up-regulation of a cellular systemsuch as the GSH peroxidase pathway, whether seques-tration of DA by synaptic vesicles plays a role in pro-tection from L-DOPA toxicity, and the precise path-ways of exchange of cysteine or other protective mech-anisms between glial cells and DA neurons.

Acknowledgment: This work was funded by the Parkin-son‘s Disease Foundation, grants 10154 and 07418 from theNational Institute on Drug Abuse (to D.S.), and grants PR95-098 and SAF 96-1099 from theSpanish MEC. Our thanksto Johanna Bogulavsky and Irma Ryjak for expert technicalassistance and to Mrs. Liselotte Gulliksen for editorial help.

REFERENCES

Abercrombie E. D., Bonatz A. E., and Zigmond M. J. (1990) Effectsof L-DOPA on extracellular dopamine in striatum of normaland 6-hydroxydopamine-treated rats. Brain Res. 52, 536—544.

Basma A. N., Morris E. J., Nicklas W. J., and Geiler H. M. (1995)L-DOPA cytotoxicity to PC 12 cells in culture is via its autoxida-tion. J. Neurochem. 64, 825—832.

Blint J., Bonnet A. M., and Agid Y. (1988) Does L-DOPA aggravateParkinson‘s disease? Neurology 38, 1410—1416.

Blunt S. B., Jenner P., and Marsden C. D. (1993) Suppressive effectofL-DOPA on dopamine cells remaining in the ventral tegmen-tal area of rats previously exposed to the neurotoxin 6-hydroxy-dopamine. Mov. Disord. 8, 129—133.

Bradford M. M. (1976) A rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein—dye binding. Anal. Biochem. 72, 248—254.

Brugg B., Michel P. P., Agid Y., and Ruberg M. (1996) Ceramideinduces apoptosis in cultured mesencephalic neurons. J. Neuro-chem. 66, 733—739.

Colton C. A., Pagan F., Snell J., Colton J. S., Cummins A., andGilbert D. L. (1995) Protection from oxidation enhances thesurvival of cultured mesencephalic neurons. Exp. Neurol. 132,54—6 1.

Cubells J. F., Rayport S., Rajendran G., and Sulzer D. (1994)Meth-amphetamine neurotoxicity involves vacuolation of endocyticorganelles and dopamine-dependent intracellular oxidativestress. J. Neurosci. 14, 2260—2271.

Damier P., Hirsch E. C., Javoy-Agid F., Zhang P., and Agid Y.(1993) Protective role of glutathione peroxidase against neu-ronal death in Parkinson‘s disease. Neuroscience 52, 1—6.

Denis-Donini S., Glowinski J., and Prochiantz A. (1983) Specificinfluence of striatal target neurons on the in vitro outgrowth ofmesencephalic dopaminergic neuntes: a morphological quanti-tative study. J. Neurosci. 3, 2292—2299.

De Vitry F., Hillion J., Catelon J., Thibault J., Benoliel J., andHamon M. (1991) Dopamine increases the expression of tyro-sine hydroxylase and aromatic amino acid decarboxylase inprimary cultures of fetal neurons. Dey. Brain Res. 59, 123—131.

Di Porzio U., Daguet M. C., Glowinski J., and Prochiantz A. (1980)Effect of striatal cells on in vitro maturation of mesencephalicdopaminergic neurons in serum-free conditions. Nature 288,370—374.

Du X. and lacovitti L. (1995a) Synergy between growth factors andcatecholamines (CA) required for novel expression of tyrosinehydroxylase in non-CA brain neurons. Soc. Neurosci. Abstr. 21,1053.

Du X. and lacovitti L. (l995b) Synergy between growth factors andtransmitter required for catecholamine differentiation in brainneurons. J. Neurosci. 15, 5420—5427.

Engele J., Schubert D., and Bohn M. C. (1991) Conditioned mediaderived from glial cell lines promote survival and differentiationof dopaminergic neurons in vitro: role of mesencephalic glia.J. Neurosci. Res. 30, 359—371.

Ferrari G., Irene C. Y., and Greene L. A. (1995) N-Acetylcysteine(D-and L-stereoisomers) prevents apoptotic death of neuronalcells. J. Neurosci. 15, 2857—2866.

Garns P. A., Ciolkowski E. L., Pastore P., and Wightman R. M.(1994) Efflux of dopamine from the synaptic cleft in the nucleusaccumbens of the rat brain. J. Neurosci. 14, 6084—6093.

Gasser T., Wszolek Z. K., Trofatter J., Ozelius B. S., Uitti R. J., LeeC. S., Gusella J., Pfeiffer R. F., Calne D. B., and BreakefieldX. 0. (1994) Genetic linkage studies in autosomal dominantparkinsonism: evaluation of seven candidate genes. Ann. Neu-roi. 36, 387—396.

Han S-K., Mytilineou C., and Cohen G. (1996) L-DOPA up-regu-lates glutathione and protects mesencephalic cultures againstoxidative stress. J. Neurochem. 66, 501—510.

Harden D. C. and Grace A. A. (1995) Activation of dopamine cellfiring by repeated L-DOPA administration to dopamine-depletedrats: its potential role in mediating the therapeutic response toL-DOPA treatment. J. Neurosci. 15, 6157—6166.

Heales S. J. R., Davies S.E.C., Bates I.E., and Clark J. B. (1995)Depletion of brain glutathione is accompanied by impaired mi-tochondnial function and decreased N-acetyl aspartate concen-tration. Neurochem. Res. 20, 3 1—38.

Hefti F., Melamed E., Bhawan J., and Wurtman R. J. (1981) Long-term administration of L-DOPA does not damage dopaminergicneurons in the mouse. Neurology 31, 1194—1195.

Hirano T. and Kasano K. (1992) Spatial distribution of excitatoryand inhibitory synapses on a Purkinje cell in a rat cerebellarculture. Soc. Neuro.sci. Abstr. 18, 1112.

Hoffen B. J., Hofman A., Bowenkamp K., Huettl P., Hudson J.,Martin D., Lin L. F. H., and Gerhardt G. A. (1994) Glial cellline-derived neurotrophic factor reverses toxin-induced injuryto midbrain dopaminergic neurons in vivo. Neurosci. Lert. 182,107—111.

lacovitti L. A. (1996) Factors regulating the induction of a dopamin-ergic phenotype in neurons grown in culture, in MesencephalicDopaminergic Geil Cultures Congress, p. 13. National Institutesof Health, Bethesda, Maryland.

Jenner P. (1993) Altered mitochondrial function, iron metabolismand glutathione levels in Parkinson‘s disease. Acta Neurol.Scand. 145, 6—13.

Leslie F. M. (1993) Neurotransmitters as neurotrophic factors, inNeurotrophic Factors (Fallon J. H. and Loughlin S. E., eds),pp. 565—598. Academic Press, San Diego.

Lesser R. P., Fahn S., Snider S. R., Cote L. J., Isgreen W. P., andBarretR. E. (1979) Analysis of the clinical problems in parkin-sonisms and the complications of long-term L-DOPA therapy.Neurology 29, 1253—1260.

Makar T. K., Nedergaard M., Preuss A., Gelbard A. S., PerumalA. S., and Cooper A. J. L. (1994) Vitamin E, ascorbate, gluta-thione, glutathione disulfide, and enzymesof glutathione metab-olism in cultures ofchick astrocytes and neurons: evidence thatastrocytes play an important role in antioxidative processes inthe brain. J. Neurochem. 62, 45—53.

Markham C. H. and Diamond S. G. (1986) Long term follow up ofearly DOPA treatment in Parkinson‘s disease. Ann. Neural. 19,365 —372.

Mayer M. and Noble M. (1994) N-Acetyl-L-cysteine is a pluripotentprotector against cell death and enhance of trophic factor-medi-ated cell survival in vitro. Proc. Natl. Acad. Sci. USA 91, 7496—7500.

Meister A. (1988) Glutathione metabolism and its selective modifi-cation. J. Biol. C/scm. 263, 17205—17208.

Melamed E. (1986) Initiation of L-DOPA therapy in parkinsonianpatients should be delayed until the advanced stages of thedisease. Arch. Neurol. 43, 402—405.

Mena M. A., Pardo B., Casarejos M. J., Fahn S., and Garcia deYebenes J. (1992) Neurotoxicity of L-DOPA on catecholamine-rich neurons. Mov. Disord. 7, 23—31.

Mena M. A., Pardo B., Paino C. L., and Garciade Yebenes J. (1993)L-DOPA toxicity in foetal rat midbrain neurones in culture:modulation by ascorbic acid. Neuroreport 4, 438—440.

Mena M. A., Casarejos M. J., Paino C. L., and Garcia de Yebenes



J. (1996) Glia conditioned medium protects fetal rat midbrainneurons in culture from L-DOPA toxicity. Neuroreport 7, 441—445.

Mena M. A., Khan U., Togasaki D. M., Sulzer D., Epstein C. J., andPrzedborski S. (1997) Effects of wild-type and mutated copper/zinc superoxide dismutase on neuronal survival and L-DOPA-induced toxicity in postnatal midbrain culture. J. Neurochem.69, 21—33.

Muller H. W., Junghans U., and Kappler J. (1995) Astroglial neuro-trophic and neunite-promoting factors. Pharmacol. Ther. 65, 1—18.

Mytilineou C., Han S-K., and Cohen G. (1993) Toxic and protectiveeffects of L-DOPA on mesencephalic cell cultures. J. Neuro-c/scm. 61, 1470—1478.

Nagata K., Takei N., Nakajima K., Saito H., and Kohsaka S. (1993)Microglial conditioned medium promotes survival anddevelop-ment of cultured mesencephalic neurons from embryonic ratbrain. J. Neurosci. Res. 34, 357—363.

Nisticè G., Ciniolo M. R., Fiskin K., lannone M., De Martino A.,and Rotillo G. (1992) NGF restores decrease in catalase activityand increase superoxide dismutase and glutathione peroxidaseactivity in the brain of the aged rats. Free Radie. Biol. Med.12, 177—181.

Nutt J. G. (1995) Long-duration response to levodopa. Neurology45, 1613—1616.

Pan Z. and Perez-Polo R. (1993) Role of nerve growth factor inoxidant homeostasis: glutathione metabolism. J. Neurochem.61, 1713—1721.

Pardo B., Mena M. A., Casarejos M. J., Paino C. L., and Garcia deYebenes J. (1995) Toxic effects of L-DOPA on mesencephaliccell cultures: protection with antioxidants. Brain Res. 682, 133—143.

Perry T. L., Wee Yong V., Ito M., Foulks J. G., Wall R. A., GodinD. V., and Clavier R. M. (1984) Nigrostriatal dopamine neu-rons remain undamaged in rats given high doses of L-DOPAand carbidopa chronically. J. Neurochem. 43, 990—993.

Pothos E., Desmond M., and Sulzer D. (1996) L-3,4-Dihydroxyphe-nylalanine increases the quantal size of exocytotic dopaminerelease in vitro. J. Neurochem. 66, 629—636.

Przedborski S., Khan U., Kostic V., Carlson E., Epstein C. J., andSulzer D. (1996) Increased superoxide dismutase activity im-proves survival of cultured postnatal midbrain neurons. J. Neu-rochem. 67, 1383—1392.

Quinn N., Parker D., Janota I., and Marsden C. D. (1986) Preserva-tion of the substantia nigra and locus coeruleus in a patientreceiving L-DOPA (2Kg) plus decarboxylase inhibitor over afour-year period. Mov. Disord. 1, 65—68.

Raps S. P., Lai J., Hertz I., and Cooper A. J. L. (1989) Glutathioneis present in cultured astrocytes but flot in cultured neurons.Brain Res. 493, 398—401.

Rayport S. and Sulzer D. (1995) Visualization of antipsychotic drugbinding to living mesolimbic neurons reveals D2 receptor, aci-dotropic, and lipophilic components. J. Neurochem. 65, 691—703.

Rayport S., Sulzer D., Shi W-X., Sawasdikosol S., Monaco J., Bat-son D., and Rajendran G. (1992) Identified postnatal mesolim-bic dopamine neurons in culture: morphology and electrophysi-ology. J. Neurosci. 12, 4264—4280.

Riederer P., Sofic E., Rausch W-D., Schmidt B., Reynolds G. P.,Jellinger K., and Youdim M. B. H. (1989) Transition metals,

fernitin, glutathione, and ascorbic acid in parkinsonian brains.J. Neurochem. 52, 5 15—520.

Rosenberg P. A., Loning R., Xie Y., Zaleskas V., and Aizenman E.(1991) 2,4,5-Trihydroxyphenylalanine in solution forms a non—N-methyl-D-aspartate glutamatergic agonist and neurotoxin.Proc. Nati. Acad. Sei. USA 88, 4865—4869.

Sagara J., Miura K., and Bannai S. (1993) Maintenance of neuronalglutathione by glial cell. J. Neurochem. 61, 1672—1676.

Sagara J., Makino N., and Bannai S. (1996) Glutathione efflux fromcultured astrocytes. J. Neurochem. 66, 1876—1881.

Sian J., Dexter D. T., Lees A. J., Daniel S. E., Agid Y., Javoy-Agid F., Jenner P., and Marsden C. D. (1994) Alterations inglutathione levels in Parkinson‘s disease and other neurodegen-erative disorders affecting basal ganglia. Ann. Neurol. 36, 348—355.

Slivka A., Mytilineou C., and Cohen G. (1987) Histochemical evalu-ation of glutathione in brain. Brain Res. 409, 275—284.

Sofic E., Lange K. W., Jellinger K., and Riederer P. (1992) Reducedand oxidized glutathione in the substantia nigra of patients withParkinson‘s disease. Neurosci. Lert. 17, 128—130.

Sundaresan M., Yu Z-X., Ferrans V. J., Irani K., and Finkel T.(1995) Requirement for generation of H

2O2 for platelet-derivedgrowth factor signal transduction. Science 270, 296—299.

Takeshima T., Shimoda K., Sauve Y., and Commissiong J. W.(1994) Astrocyte-dependent and independent phases ofthe de-velopment and survival of rat embryonic day 14 mesencephalicneurons in culture. Neuroscience 60, 809—823.

Thomas S. A., Matsumoto A. M., and Palmiter R. D. (1995) Nor-adrenaline is essential for mouse fetal development. Nature 374,643—646.

Tietze F. (1969) Enzymic method for quantitative determination ofnanogram amounts oftotal and oxidized glutathione: applicationto mammalian blood and other tissue. Anal. Biochem. 27, 502—522.

Tohgi H., Abe T., Saheki M., Hamato F., Sasaki K., and TakahashiS. (1995) Reducedandoxidized forms of glutathione and alpha-tocopherol in the cerebrospinal fluid of parkinsonian patients:comparison between before and after L-dopa treatment. Neu-rosci. Lett. 184, 21—24.

Uitti R. J., Ahlskog J. E., Maraganore D. M., Muentner M. D., At-kinson E.J., Cha RH., and O‘Brien P.C. (1993) L-DOPAtherapy and survival in idiopathic Parkinson‘s disease: OlmstedCounty project. Neurology 43, 1918—1926.

Victorica J., Machado A., and Satndsteguï J. (1984) Age-dependentvariations in peroxide-utilizing enzymes from rat brain mito-chondnia and cytoplasm. J. Neurochem. 42, 351—356.

Werner P. and Cohen G. (1993) Glutathione disulfide (GSSG) asa marker of oxidative injury to brain mitochondria. Ann. NYAcad. Sei. 679, 364—369.

Yan C. Y. I., Ferrari G., and Greene L. A. (1995) N-Acetylcysteine-promoted survival of PC12 cells is glutathione-independent buttranscription-dependent. J. Biol. Chem. 270, 26827—26832.

Yurek D. M., Steece-Collier K., Collier T. J., and Sladek J. R.(1991) Chronic L-DOPA impairs the recovery of dopamineagonist-induced rotational behavior following neural grafting.Exp. Brain Res. 86, 97—l07.

Zhou Q. Y., Quaife C. J., and Palmiter R. D. (1995) Target disrup-tion of the tyrosine hydroxylase gene reveals that catechol-amines are required for mouse fetal development. Nature 374,640—643.


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