Effects of 6-hydroxydopamine on primary cultures of substantia

nigra: specific damage to dopamine neurons and the impact of glial

cell line-derived neurotrophic factor

Yun Min Ding,1 Juliann D. Jaumotte, Armando P. Signore and Michael J. Zigmond

Department of Neurology and the Pittsburgh Institute for Neurodegenerative Disease, University of Pittsburgh School of Medicine,

Pittsburgh, Pennsylvania, USA

Abstract

6-Hydroxydopamine (6-OHDA)-induced loss of dopamine

(DA) neurons has served to produce an animal model of DA

neuron loss in Parkinson’s disease. We report here the use of

6-OHDA to produce an in vitro model of this phenomena using

dissociated cultures prepared from neonatal rat mesenceph-

alon. Cultures were exposed to 6-OHDA (40–100 lM, 15 min)

in an antioxidant medium, and DA and GABA neurons eval-

uated by immunocytochemistry. 6-OHDA induced morpholo-

gical and biochemical signs of cell death in DA neurons within

3 h, followed by loss of tyrosine hydroxylase immunoreactive

neurons within 2 days. In substantia nigra (SN) cultures, DA

neurons were much more affected by 6-OHDA than were

GABA neurons. In contrast, DA neurons from the ventral

tegmental area were only lost at higher, non-specific

concentrations of 6-OHDA. The effects of 6-OHDA on nigral

DA neurons were blocked by inhibitors of high affinity DA

transport and by z-DEVD-fmk (150 lM), a caspase inhibitor.

Glial cell line-derived neurotrophic factor (GDNF) treatment

reduced TUNEL labeling 3 h after 6-OHDA exposure, but did

not prevent loss of DA neurons at 48 h. Thus, 6-OHDA can

selectively destroy DA neurons in post-natal cultures of SN,

acting at least in part by initiating caspase-dependent apop-

tosis, and this effect can be attenuated early but not late by

GDNF.

Keywords: apoptosis, caspase, neuroprotection, Parkin-

son’s disease, trophic factor, ventral tegmental area.

J. Neurochem. (2004) 89, 776–787.

Parkinson’s disease is characterized by the progressive

degeneration of dopamine (DA) neurons in the substantia

nigra (SN). Although the cause of this loss is unknown,

evidence suggests a role for oxidative stress (see reviews by

Olanow and Tatton 1999; Stokes et al. 1999). Thus, an

understanding of the mechanisms by which oxidative stress

kills DA neurons and how specific agents can attenuate this

neurodegeneration may provide insights into the etiology and

treatment of the disease. The intracerebral administration of

6-hydroxydopamine (6-OHDA) has been widely used to

cause oxidative stress to DA neurons. Under the right

conditions, this neurotoxin can cause the selective loss of DA

neurons, thereby producing an animal model of certain key

aspects of Parkinson’s disease (reviewed in Zigmond and

Keefe 1997).

6-OHDA also has been widely used to damage DA

neurons in vitro. However, unlike the case for in vivo models,

these in vitro effects of 6-OHDA have never been shown to

be restricted to DA neurons. Indeed, in those cases where it

has been examined, the in vitro effects of 6-OHDA have been

observed to be non-specific (Table 1). As a result, it has been

difficult to draw conclusions regarding the effects of selective

oxidative stress in DA neurons from such studies. Yet, an

Received December 31, 2003; revised manuscript received January 14,

2004; accepted January 16, 2004.

Address correspondence and reprint requests to Michael J. Zigmond,

Department of Neurology, S-526 Biomedical Science Tower, University

of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.

E-mail: zigmond@pitt.edu1The present address of Yun Min Ding is SBRI J-217, MC 2030,

Department of Neurology, University of Chicago, Chicago, IL 60637,

USA.

Abbreviations used: BME, basal medium eagle; DA, dopamine; DAT,

dopamine transporter; DETAPAC, diethylenetriaminepentaacetic acid;

GABA, gamma-amino butyric acid; GDNF, glial cell line-derived

neurotrophic factor; HEPES, N-(2-Hydroxyethyl)piperazine-N¢-(2-ethanesulfonic acid); 6-OHDA, 6-hydroxydopamine; MPTP,

1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine; PBS, phosphate-buffered

saline; SN, substantia nigra; TH, tyrosine hydroxylase; TH+, TH-im-

munostained cells; VTA, ventral tegmental area; z-DEVD-fmk, Z-As-

p(OCH3)-Glu(OCH3)-Val-Asp (OCH3)-fluoromethylketone.

Journal of Neurochemistry, 2004, 89, 776–787 doi:10.1111/j.1471-4159.2004.02415.x

776 � 2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 776–787

in vitro model of selective DA neuron loss would be of great

benefit in attempts to understand the basis of cell death that

can be triggered by oxidative stress and to develop strategies

for promoting cell survival.

In this report we describe a cellular model in which post-

mitotic, primary DA neurons from the SN are selectively

damaged by 6-OHDA through a process that involves

apoptosis. We then use this model to examine the effects of

glial cell line-derived neurotrophic factor (GDNF), which has

been shown to enhance the survival of developing DA neurons

in culture and protect these neurons from 6-OHDA in vivo.

Materials and methods

Materials

All reagents were of the highest available grade and were purchased

from Sigma/Aldrich (St Louis, MO, USA), unless otherwise noted.

SN neuron culture

All procedures were carried out in accordance with the NIH

Guide to the Care and Use of Animals and approved by the

University of Pittsburgh Animal Care and Use Committee.

Cultures of primary mesencephalic cells were made using the

method of Cardozo (1993) with minor modifications, including

some suggested by Burke, Sulzer and co-workers (Burke et al.

1998). Timed pregnant Sprague-Dawley rats (Hilltop Laboratory

Inc., Scottdale, PA, USA) were housed for 2–5 days and given

free access to water and food under a 12 : 12 h light-dark cycle.

Post-natal pups (P0) were killed during the light phase by

decapitation under aseptic conditions, and brains were removed

from the skulls and placed in cold Gey’s Balanced Salt Solution

(Life Technologies Inc., Grand Island, NY, USA) containing 0.5%

D-glucose. Under a dissecting microscope, a 0.8–1.0-mm thick

coronal section of the mesencephalon was made using a scalpel,

and the regions containing SN were isolated (Fig. 1). Unless

otherwise noted, the regions containing the ventral tegmental area

(VTA) were excluded from the culture. In initial studies, similar

slices were fixed and stained for tyrosine hydroxylase (TH), the

rate-limiting enzyme for DA biosynthesis, to verify the dissected

regions of the midbrain.

Tissue was digested in a solution of papain (15 units/mL,

Worthington Biochemical Corporation, Lakewood, NJ, USA) with

0.45 mg/mL L-cysteine in a dissociation media containing 90 mM

Na2SO4, 30 mM K2SO4, 5.8 mM MgCl2, 0.25 mM CaCl2, 10 mM

Table 1 Summary of selected literature on cytotoxicity of 6-OHDA in vitro

Reference Conclusion Specificity

Age at onset

of culturing

and species

Brain

region

DIV

before

6-OHDA

Exposure

duration

Vehicle

for

6-OHDA

Barkats et al. (2002) 50 lM induced 65%

DA neuron death

– E14 rat Ventral midbrain 3 2 h –

Bronstein et al. (1995) 30 lM damaged DA neurons – E16 rat Ventral midbrain 4–6 48 h 0.1% ascorbate

Cerruti et al. (1993) 10 nM changed DA

cell morphology

– E14 rat Ventral midbrain 2 or 7 48 h Culture medium

Ibi et al. (2001) 100 lM induced 40%

DA cell death

– E16 rat Ventral midbrain 9 24 h Distilled water

Grothe et al. (2000) 100 lM caused 50%

DA cell loss

– E14 rat Ventral midbrain 6 24 h DMEM

Han et al. (2003) 20 lM caused 30–40%

apoptotic loss

of DA cells

– E 14 rat Ventral midbrain 5 6–24 h Ascorbate

Holtz and

O’Malley (2003)

100 lM induced changes

suggesting apoptosis

– E 14 mouse Ventral midbrain 6 6–18 h Ascorbate

Kramer et al. (1999) 10–50 lM caused

widespread cell death

Non-specific E15 rat Ventral midbrain 5 30 min 200 lM ascorbate

Lotharius et al. (1999) 60 lM affected non-DA

neurons

Non-specific E14 CF1

mouse

Ventral midbrain 7 24 h –

Michel and Hefti (1990) 10–100 lM killed DA

and GABA neurons

Non-specific E15 rat Ventral midbrain 6–7 24 h –

Mytilineou and

Danias (1989)

0.1 mM results in greater

toxicity to DA neurons

– E14 rat Ventral midbrain 21 1 h 0.1 mM ascorbate

Pietz et al. (1996) 3 lg/lL induced 40%

DA cell death

– E14 rat Ventral midbrain 7 30 min,

1–24 h

0.2% ascorbate

Rosenberg (1988) 25 lM was non-selective Non-specific E15 rat Cortex 2 48 h –

von Coelln et al. (2001) 40 lM induced 50%

DA cell death

– E14 rat Ventral midbrain 4 24 h –

Effects of 6-OHDA and GDNF on primary SN neurons 777

� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 776–787

HEPES (N-2-hydroxyethylpiperazine)-N¢-(2-ethanesulfonic acid),

0.5% phenol red and 20 mM glucose (pH 7.4) for 1 h at 35�C in a

rotating incubator (Incudrive-S, Schutt Labortechnik, Gottingen,

Germany). After washing twice with dissociation medium, the tissue

was mechanically dissociated with a fire-polished glass Pasteur

pipette in a trituration medium containing 0.1% trypsin inhibitor,

0.1% bovine serum albumin and 10 mM HEPES in basal medium

Eagle (BME; Life Technologies Inc, Carlsbad, CA, USA). To

remove the lighter weight cellular debris, the cell suspension was

collected, layered on top of centrifugation medium containing 1%

trypsin inhibitor and 1% bovine serum albumin in BME and

centrifuged for 4 min at 12.5 g. (GS-6R centrifuge, Beckman

Coulter Inc., Fullerton, CA, USA). The pellet was re-suspended with

trituration medium and cell counts performed with trypan blue to

determine the concentration of dye-excluding, viable cells. The cell

suspension was then adjusted with trituration medium to yield a

concentration of 400 000 cells per mL and 50 lL (20 000 cells) was

added to each well. Culturing was done on glass slides provided

with removable chamber walls that formed 16 wells, each of which

was approximately the size of those on a standard 96-well plate

(0.4 cm2 bottom surface area) (#178599, Nalge Nunc International,

Naperville, IL, USA). The slides were pre-coated with 20 ng/lLpoly D-lysine and 2 ng/lL laminin and contained 200 lL feeding

medium consisting of 0.225% D-glucose, 0.67 mM glutamine,

67 units/mL penicillin, 6.7 mg streptomycin, 0.5% N2 supplement

(Life Technologies Inc.), 2% rat or calf serum (see below) and

10 mM HEPES in BME. Each rat pup produced enough cells for 2–3

wells; thus, a litter of 10–16 pups yielded about 20–32 culture wells

or up to two slides. Cultures were maintained at 37�C in an

atmosphere containing 5% CO2 in equilibrium with H2O.

Rat serum was used in most of our studies. Initial experiments

used commercially produced rat serum. However, rat serum of tissue

culture quality became unavailable from commercial sources during

our studies, prompting us to prepare our own rat serum. For this

purpose, whole blood was collected via cardiac puncture from adult

female rats (generally those that had fostered our pups) killed by

CO2 (Banker and Goslin, 1991) and the blood was kept at 4�C for

24 h followed by centrifugation at 900 g, 4�C, for 10 min. The

supernatant was saved and centrifuged at 20 000 g, 4�C, for 10 min

and the resulting supernatant frozen. Serum prepared in this way

from 10 to 12 rats was then thawed, mixed, aliquoted and stored at

)20�C for up to 12 weeks.

Measurement of breakdown of 6-OHDA under culture

conditions

6-OHDA was dissolved in BME and the solution incubated at 37�Cin an atmosphere maintained at 5% CO2. In an initial series of

experiments, the amount of 6-OHDA remaining in the culture

medium was measured by removing aliquots of solution at several

time points and placing them in a tube containing an equal volume

of 0.1 N HClO3 in order to stop oxidation. Samples then were

analyzed by HPLC coupled with electrochemical detection with

methods for the analysis of DA that we have previously described

(Smith et al. 2002) and employing a 100 lM 6-OHDA standard

made in 0.1 N HClO3 acid to identify the proper peak. The detection

limit of the system was 5 nM. Two to three experiments were

performed for each condition.

Exposure of SN cultures to experimental conditions

Exposure to 6-OHDA was initiated after 3–4 days in vitro (DIV).

Except where noted, the following protocol was employed: Medium

was withdrawn from the wells and replaced with 180 lL BME

without serum followed by the addition of 20 lL of freshly prepared

6-OHDA in vehicle. Vehicle was freshly prepared containing the

metal chelator diethylenetriaminepentaacetic acid (DETAPAC;

10 mM) and ascorbic acid (0.15%, Fisher Scientific, Pittsburgh,

PA, USA) and flushed with N2 for 10 min while the solution was

sitting on ice. Then, a predetermined amount of 6-OHDAwas added

to the vehicle and 20 lL of the 6-OHDA solution or the vehicle

alone was added to the cultures. After 15 min, the medium was

removed, the cultures were gently washed twice with 300 lL BME

plus 10 mM HEPES, 200 lL of feeding medium (described above)

was added to each culture and the cultures were further incubated for

3–48 h at 37�C.

Recombinant human GDNF (100 ng/mL, Upstate Biotechnology,

Lake Placid, NY, USA) was added to the medium in specified

experiments. In those cases where cells were exposed to both GDNF

and 6-OHDA, cultures were pre-treated with GDNF for 1 h prior to

and during exposure to 6-OHDA. Cultures were then gently washed

twice with BME containing 10 mM HEPES, after which feeding

medium containing GDNF was added to the cultures. In studies

attempting to block uptake of 6-OHDA, the DAT inhibitors

nomifensine (10–50 lM) or GBR12909 (2 lM) (Sigma/RBI) were

added 1 h before 6-OHDA exposure and remained until the toxin

was removed and the cultures washed. In studies utilizing the

caspase inhibitor, z-Asp(OCH3)-Glu(OCH3)-Val-Asp (OCH3)-fluor-

omethylketone (z-DEVD-fmk; Enzyme Systems Products, Liver-

more, CA, USA), the inhibitor was dissolved in dimethylsulfoxide

(DMSO) and then 2.5 lL were added to the culture approximately

18 h before exposure to 6-OHDA so as to produce a final

concentration of 150 lM. This z-DEVD-fmk was present in the

cultures during the 6-OHDA exposure procedure and for the 48-h

post-toxin incubation. For the control group, only DMSO was

added.

Fig. 1 Photomicrograph of a coronal section of post-natal rat mes-

encephalon immunostained for tyrosine hydroxylase (TH) after uni-

lateral removal of the SN region. On the left, the entire substantia nigra

(SN) and ventral tegmental area (VTA) are visible and TH-stained cells

appear white against a darker background of unstained tissue. On the

right, TH stained cells of VTA are still apparent, whereas the SN tissue

on this side was dissected for the culture. Scale bar ¼ 500 lm.

778 Y. M. Ding et al.

� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 776–787

Immunocytochemical staining

The cultures were fixed using cold 4% paraformaldehyde in 0.1 M

phosphate-buffered saline (PBS, pH 7.4) for 1 h on ice. Following

several washes of PBS, the cultures were blocked for 30 min in a

blocking solution containing 5% normal donkey serum (Jackson

Immuno-Research, West Grove, PA, USA), 1% bovine serum

albumin and 0.3% Triton X-100 in 0.1 M PBS (pH 7.4), followed by

an overnight incubation at 4�C in blocking solution containing

mouse anti-TH (1 : 1000, Chemicon International, Temecula, CA,

USA) and rabbit anti-GABA (1 : 1000, Sigma/Aldrich). Following

three washes of PBS containing 0.1% Tween-20, the cultures were

incubated in the blocking solution containing Cy-3 (donkey anti-

mouse) and FITC (donkey anti-rabbit) conjugated secondary

antibodies (1 : 200, Jackson Immuno-Research) for 30 min on

ice, followed by staining with Hoechst 33258 reagent (1 : 1000,

Sigma/Aldrich). The chamber walls were then removed and the

slides were coverslipped using Fluoromount-G (Southern Biotech-

nology Associates, Inc., Birmingham, AL, USA) and visualized by

fluorescence microscopy. Staining for TH and GABA were both

negative when the relevant primary or secondary antibodies were

omitted.

Our analysis of the mechanism of cell death included the

measurement of activated caspase-3, typically an executioner

caspase. Rabbit anti-activated caspase-3 (1 : 200, Cell Signaling

Technology, Beverly, MA, USA) was examined using a procedure

similar to that for TH and GABA. Staining for caspase-3 was

negative when the relevant primary or secondary antibodies were

omitted. Double staining for TH and in situ end-labeling of DNA

breaks was achieved by first immunostaining against TH followed

with staining by the method of terminal deoxynucleotidyl transf-

erase (TdT)-mediated dUTP nick end-labeling (TUNEL). The

ApopTag Fluorescein In situ Apoptosis Detection Kit was used

following the instructions provided by the manufacturer (Intergen

Molecular & Cell Biology, Purchase, NY, USA).

Data analysis

The number of positive TH and/or GABA neurons in each well was

counted under 20 · magnification using a Zeiss Axiophot micro-

scope (Carl Zeiss Inc., Germany). Unless otherwise noted, the total

number of TH- or GABA-positive neurons from four random optical

fields was calculated for each culture well. Each of these fields was

1.25 mm in diameter and collectively the four fields represented

approximately 17% of the 0.4 cm2 well. Cells positive for both TH

and TUNEL were identified by counting under 40 · magnification.

Statistical significance was tested using two-way analysis of

variance (Intercooled Stata 7, Stata Corporation, College Station,

TX, USA), followed by multiple comparisons using a Bonferroni

correction. p-values less than 0.05 were considered significant. To

assess the effect of GDNF at 48 h, we used linear contrast to

compare the difference between the means for vehicle control and

6-OHDA-treated samples with the difference between the means for

samples treated with GDNF alone and those treated with 6-OHDA

plus GDNF. Unless otherwise noted, studies were carried out in

triplicate with the means of the three wells being considered a single

number. Such studies were then repeated 3–8 times to produce

means ± SEM. All statistical analyses were then performed on these

untransformed means. However, unless described otherwise, the

values presented in the report are expressed as a per cent of values

obtained from vehicle-treated control cultures. In two experiments,

counting was performed independently by two individuals. We

observed no significant differences (p > 0.9) between the two sets of

counts.

Results

Spontaneous cell death during culturing

DA neurons routinely comprised 5–6% of the total popula-

tion of cells in our SN cultures as defined by immunostaining

for TH at DIV 4 (i.e. 1000–1200 DA cells per 20 000 cells

plated). As will be seen in subsequent figures (e.g. Fig. 3a),

most of these cells displayed multiple, highly branched

processes. The cultures also contained GABA-positive

neurons, which were present in roughly equivalent numbers

to the DA neurons and exhibited a comparable somal size

and degree of process outgrowth. Based on analyses of

cultures from ventral mesencephalon (Michel et al. 1999;

J. D. Jaumotte & M. J. Zigmond, unpublished observations),

we assume that the majority of the remaining cells in the SN

cultures were glia.

The number of TH+ neurons declined over the first 3 days

of incubation. At DIV 2 the number of detectable DA

neurons was about 60% of those present 1 h after plating,

while at DIV 3 and 4 about 40% of DA neurons remained.

Therefore, we selected DIV 4 to begin the remaining studies

as the number of viable DA neurons appeared to plateau at

that time point.

Effect of culture conditions on the response to 6-OHDA

In our initial studies, 6-OHDA was dissolved in BME and

added to cultures, which then were evaluated 48 h later.

Under these conditions, we observed an extensive loss of

both DA and GABA neurons at concentrations of the toxin as

low as 40 lM. Many other cells in the culture also showed

signs of cell death. As 6-OHDA can rapidly oxidize in

solution, we reasoned that the non-specific damage produced

by 6-OHDA was likely due to the action of reactive oxygen

species forming within the culture medium. To test this

hypothesis, 6-OHDA was first dissolved in one of several

different vehicles and incubated in BME (but without the

addition of cells) at 37�C for 5–30 min. We observed that

6-OHDA dissolved in BME alone broke down almost

completely within 5 min. In contrast, when we dissolved

6-OHDA in a vehicle containing BME plus the antioxidant

ascorbic acid (0.15%), the breakdown of 6-OHDA was

significantly slowed such that 82 ± 6.6% of the toxin was

still intact at 20 min (Fig. 2). Further addition of the metal

chelator DETAPAC (1 mM; Berman et al. 1996) followed by

flushing with N2 for 10 min before the addition of 6-OHDA,

further reduced the loss of 6-OHDA during incubation such

that 96 ± 2.0% of the 6-OHDAwas now present after 20 min

Effects of 6-OHDA and GDNF on primary SN neurons 779

� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 776–787

(Fig. 2). Moreover, using this protocol, 6-OHDA (40 lM)

caused a selective and concentration-dependent decline in the

number of DA neurons present 48 h later compared with

cultures exposed to vehicle alone (Fig. 3). For example, after

exposure to 40 lM 6-OHDA, only 47 ± 4.7% (mean ±

SEM) of the DA neurons remained (p < 0.01), whereas

there was no significant loss of GABA neurons (95 ± 3.3%

of control; NS) (Figs 3b and d). At 100 lM 6-OHDA GABA

neurons were reduced to 78 ± 6.4 (p < 0.05); however, this

was significantly less than the loss of DA neurons, which

were reduced to 22 ± 9.5% of control (Figs 3c and d;

p < 0.01). In contrast, 500 lM 6-OHDA, which caused a

near-total loss of DA neurons (3.7 ± 9.2% of control) did

cause a significant loss of GABA cells (41 ± 1.4% of

control; p < 0.01) (Fig. 3d).

Because of these findings, 6-OHDA was dissolved in

vehicle flushed with N2 and containing DETAPAC and

ascorbic acid in all subsequent experiments. However, even

with these precautions, we observed some variability in the

neuronal responses of our cultures to 6-OHDA. For

example, although 100 lM 6-OHDA generally caused a

near total and selective loss of DA neurons, in some cases,

cultures failed to show a loss of DA neurons, whereas

occasionally this concentration of 6-OHDA caused a

significant loss of GABA neurons as well as DA neurons.

Although we are uncertain of the source of this variability,

we hypothesize that it was due primarily to variations in the

amount of actual 6-OHDA present in the culture medium

in any given experiment. Therefore, in each subsequent

study we included wells containing 100 lM 6-OHDA and

used the results of that exposure as an index of the

specificity of the toxin in that particular experiment. Only

when the 100 lM 6-OHDA produced at least a 50% loss of

DA cells and less than a 20% loss of GABA cells did

we proceed with the analysis. This resulted in the exclusion

of approximately 25% of our experiments from further

consideration.

(a) (b)

(c) (d)

Fig. 3 The effects of 15-min exposure to 6-OHDA on the survival of

DA neurons. At DIV 3–4, cultures were exposed to 6-OHDA for

15 min. After 48 h, the cells were fixed and immunostained for TH

(red) and for GABA (green). Representative photomicrographs show

cells incubated with (a) vehicle alone, (b) 40 lM 6-OHDA or (c) 100 lM

6-OHDA. Note the specific loss of DA neurons, but not GABA neurons

in (b) and (c). In (d) data were quantified for 6-OHDA effects on DA

neurons (open bars) and GABA neurons (filled bars) by cell counts of

immunostained neurons. Data represent the mean ± SEM for eight to

10 experiments, performed in duplicate or triplicate. The data were

analyzed by a 2-way analysis of variance and a post-hoc Bonferroni

test on the untransformed data. *p < 0.01 versus control (0 lM 6-

OHDA). Scale bars in a–c ¼ 100 lm.

Fig. 2 6-OHDA degradation as a function of time and different vehi-

cles. Samples were prepared with 6-OHDA (100 lM) in BME alone

(j), BME plus 0.015% ascorbic acid (*), or vehicle containing ascorbic

acid, DETAPAC, and flushed with N2 (m). Samples were incubated for

up to 30 min at 37�C and analyzed by HPLC. Data represent the

mean ± SEM of two to three replicates for each condition.

Fig. 4 The effects of an inhibitor of the high affinity DA transporter on

the toxic effects of 6-OHDA. SN cultures at DIV 3–4 were exposed to

40 lM (open bars) or 100 lM (filled bars) 6-OHDA for 15 min. After

48 h, cultures were fixed and TH+ neurons were counted. DA neurons

treated with 2 lM GBR12909 showed enhanced survival relative to DA

neurons that did not receive GBR12909. Data represent the means

± SEM for five experiments performed in triplicate. The data were

analyzed by a 2-way analysis of variance and a post-hoc Bonferroni

test on the untransformed data. *p < 0.01 for differences between 40

or 100 lM 6-OHDA and 40 or 100 lM + 10 lM GBR12909.

780 Y. M. Ding et al.

� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 776–787

The effect of DAT inhibitors on 6-OHDA-induced

damage

It is generally assumed that the selectivity of 6-OHDA is due

to the specific uptake of the toxin into DA neurons, after

which the 6-OHDA oxidizes to form DA quinone and

reactive oxygen species (see Zigmond and Keefe 1997). This

would be consistent with our observation that specific

damage by 6-OHDA only occurred when care was taken to

prevent the breakdown of 6-OHDA within the culture

medium. To test the assumption that specific damage

required the uptake of 6-OHDA via the high affinity DA

transporter (DAT), we examined the impact of two DAT

inhibitors on the response to 6-OHDA, GBR12909 and

nomifensine. A moderate concentration of GBR12909

(2 lM) completely blocked the damage of DA neurons

normally caused by 40 lM 6-OHDA (p < 0.01) and signi-

ficantly increased the survival of DA neurons from exposure

to 100 lM 6-OHDA (Fig. 4; p < 0.01). Higher concentra-

tions of GBR12909 (‡ 10 lM) were found to be toxic to all

cultured neurons. Likewise, incubation with a low concen-

tration of nomifensine (10 lM) prior to and during exposure

to 100 lM 6-OHDA significantly attenuated the 6-OHDA-

induced loss of DA neurons measured 48 h later without

affecting DA neurons in control cultures. However, this

concentration of nomifensine did not alter the neurotoxic

effects of 100 lM H2O2 or 500 lM 6-OHDA, treatment that

presumably caused non-specific toxicity (data not shown).

Thus, it appeared that the selective effects of 6-OHDA on

DA neurons resulted from an intracellular action of the toxin

subsequent to its uptake through the high affinity DA

transporter.

Comparison of SN with VTA

In vitro studies of DA neurons usually examine cultures

prepared from a large portion of the ventral mesencephalon.

Yet, this area of brain contains at least two groups of DA

neurons, the SN and the VTA. Previous reports suggest that

DA neurons from the SN are more vulnerable to 6-OHDA

than those from the VTA (Hedreen and Chalmers 1972;

Okamura et al. 1995), a finding that parallels the observa-

tion that the SN is much more affected than the VTA in

patients with Parkinson’s disease (Uhl et al. 1985; German

et al. 1989; Hirsch et al. 1997). Therefore, in all of our

studies presented in this report thus far, we had restricted

tissue to the SN region, specifically excluding the VTA. To

determine whether this restriction was necessary, we

compared 6-OHDA-induced toxicity of DA neurons in

cultures prepared from SN with those prepared from VTA.

We observed that DA neurons from the SN were signifi-

cantly more affected by 6-OHDA than were those from the

VTA (Fig. 5). This difference could not be explained in

terms of differences in the density of TH+ cells between SN

and VTA cultures as it persisted even when this density

was equalized through the dilution of VTA cultures with

cells for an adjacent region (Fig. 5b). Therefore, DA

neurons in cultures of the SN appeared to be inherently

more sensitive to 6-OHDA than were DA neurons in

cultures of the VTA.

Contribution of apoptotic cell death to 6-OHDA-induced

DA neuron damage

Having established a specific model for DA neuron death due

to oxidative stress, we next explored the nature of that cell

death. First, we examined the morphology of the DA neurons

3 h after exposure to 6-OHDA. At this time, there was not

yet any reduction in the number of TH+ cells. However,

about half of the DA neurons exposed to a low concentration

of 6-OHDA (40 lM) exhibited fragmented neurites or a

reduction in the number of neurites, as well as a marked

reduction in somal size (Fig. 6a). We also observed immu-

noreactivity for the activated form of caspase-3 in

78 ± 19.6% of the DA cell bodies exhibiting somal shrink-

age (Fig. 6b). This staining was particularly evident in the

34 ± 13.7% of the damaged DA neurons that displayed

condensed nuclei as determined using the Hoechst stain

(Fig. 6c). In addition, 38 ± 21.3% of the DA cells exhibiting

morphological signs of distress also showed positive TUNEL

staining (Fig. 6d; p < 0.001). Caspase-3 activation, con-

densed nuclei and TUNEL labeling were seen at a much

lower frequency in vehicle-treated cultures, and at a much

higher frequency in the DA neurons of cultures exposed to

100 lM 6-OHDA (data not shown).

Finally, we examined the impact of the caspase inhibitor

z-DEVD-fmk on 6-OHDA-induced damage to DA neurons.

Cultures were incubated with z-DEVD-fmk (150 lM) begin-

ning 18 h prior to 6-OHDA exposure and continuing

throughout the experiment. The number of surviving DA

Fig. 5 Effect of 6-OHDA on DA neurons in cultures prepared from SN

and VTA. (a) Equal total numbers of cells from SN and VTA were

exposed to 6-OHDA (40 or 100 lM) for 15 min at DIV 4 and then

examined by immunocytochemistry 48 h later. (b) VTA preparations

were first diluted with tissue dorsal to the VTA in order to approximate

the density of TH+ cells observed in the SN preparations. The

experiment then proceeded as in (a). *p < 0.05 versus control.

Effects of 6-OHDA and GDNF on primary SN neurons 781

� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 776–787

neurons was assessed 48 h after the 15-min exposure to

6-OHDA. The inhibitor completely blocked the DA neuronal

loss typically seen after exposure to 40 lM 6-OHDA and

significantly reduced the effects of 100 lM 6-OHDA (Fig. 7;

p < 0.01). Comparable results have recently been reported

(Han et al. 2003). Collectively, the data suggest that

6-OHDA appears to kill DA neurons in culture through a

caspase-dependant mechanism that is at least partly

associated with apoptosis. Unfortunately, however, the site

of action of z-DEVD-fmk is not entirely clear. The agent was

originally designed to target the asp-glu-val-asp (DEVD) site

on caspase-3, inhibiting activation of the protease. Yet,

though the agent does inhibit caspase-3 (e.g. Masuda et al.

1997), a key executioner protease (Hengartner 2000), it also

is associated with a reduction in the activity of caspase-6, -7,

-8 and -10, albeit at high concentrations (Ki ¼ 500 lM)

(Margolin et al. 1997; Garcia-Calvo et al. 1998).

Impact of GDNF on the survival of DA neurons in

response to 6-OHDA

GDNF has been reported to both protect and rescue DA

neurons from 6-OHDA-induced damage (reviewed by Con-

nor and Dragunow 1998; Bohn 1999) and has recently been

shown to have a ameliorative effect on DA neurons in a

preliminary clinical trial (Gill et al. 2003). We therefore

wished to examine its effects on 6-OHDA-induced toxicity in

our cultures. Fresh GDNF (100 ng/mL) was added to SN

cultures at each of three time points: 1 h prior to, during and

immediately after a 15-min exposure to 6-OHDA. We

observed that 3 h following exposure to 40 and 100 lM

Fig. 6 Effects of 6-OHDA on morphological and cytochemical indices

of apoptosis. Cultures at 3–4 DIV were exposed to 40 lM 6-OHDA for

15 min. Three hours after toxin treatment, cultures were fixed and the

cells were stained using Hoechst reagent, immunostained for TH and

evaluated morphologically for neurite fragmentation and somatal size.

In a representative single culture, triple-labeled cells (arrow heads)

that stained for (a) TH (green) also exhibited neurite fragmentation, (b)

caspase-3 activation (red) and (c) condensed nuclei as revealed by

Hoechst staining (blue). (d) An additional culture treated as in (a–c)

was double labeled for TH and TUNEL. Several DA neurons with

damaged neurites were also TUNEL positive (arrowhead) 3 h after 6-

OHDA (co-labeling for TH and TUNEL appears yellow). All experi-

ments were repeated a minimum of three times. Scale bar ¼ 20 lm

(a–c) or 40 lm (d).

Fig. 7 Effect of z-DEVD-fmk on DA neurons treated with 6-OHDA. SN

cultures exposed to 6-OHDA for 15 min were analyzed 48 h later for

DA neuronal survival by TH immunocytochemistry. SN cultures were

treated with 40 lM (open bars) or 100 lM (filled bars) 6-OHDA plus

DMSO (left bars) or 150 mM z-DEVD-fmk (right bars). Data are

expressed as a percentage of the number of control neurons, and

represent the means ± SEM for five to six experiments, each carried

out in triplicate. *p < 0.01 for differences between 40 lM 6-OHDA and

40 lM + 150 lM z-DEVD-fmk, and between 100 lM 6-OHDA and

100 lM + 150 lM z-DEVD-fmk.

Fig. 8 GDNF effects on DNA fragmentation in DA neurons treated

with 6-OHDA. SN cultures at DIV 3–4 were exposed to 6-OHDA for

15 min and analyzed 3 h later by TUNEL. Control conditions were

compared with 40 or 100 lM 6-OHDA. Cultures either received 100 lM

GDNF 1 h prior to, during and immediately after 6-OHDA or vehicle (+)

or only media changes at comparable times (–). Data represent a

percentage of the mean total number of TH+ neurons that were

TUNEL positive in each well cultured in the presence of the 6-OHDA

vehicle. All means are derived from three to five independent experi-

ments performed in triplicate and counted at 40 · magnification.

*p < 0.01 for differences between cultures receiving 6-OHDA and

those receiving 6-OHDA plus GDNF.

782 Y. M. Ding et al.

� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 776–787

6-OHDA GDNF had markedly attenuated the number of DA

neurons that were TUNEL labeled (Fig. 8; p < 0.01). GDNF

also augmented the total number of TH neurons normally

present 48 h after exposure to 6-OHDA (40 lM). However,

this increase could be attributed to the general effect of

GDNF on DA cell survival that we and others have observed.

Therefore, in the presence of GDNF, 6-OHDA (40 lM)

reduced the number of TH+ neurons to 51 ± 1.1% of control

cultures that also received GDNF alone. This effect was not

significantly different from the effect seen when 40 lM 6-

OHDA was added without GDNF present (53 ± 7.3%)

(Fig. 9). We concluded therefore that whereas GDNF

protected against initial apoptosis caused by 6-OHDA, it

did not protect DA neurons against the longer-term cell death

caused by the toxin.

Discussion

Specificity of the toxic effects of 6-OHDA

We have examined the effects of exposing cultures of

dissociated post-natal cells from the ventral mesencephalon

to 6-OHDA, focusing primarily on cultures from the SN

region. Our initial studies indicated that 6-OHDA caused the

loss of GABA neurons and glia, as well as DA neurons.

These findings are consistent with previous studies using

6-OHDA, in which non-specific damage was observed in

mesencephalic cultures (Michel and Hefti 1990; Bronstein

et al. 1995; Kramer et al. 1999; Lotharius et al. 1999;

Grothe et al. 2000; Zhou et al. 2000), as well as cerebellar

granule neurons (Dodel et al. 1999), cortical cells (Rosen-

berg 1988), thymocytes (Tsao et al. 1996) and microglia

(Takai et al. 1998) (see Table 1). However, we were able to

reduce the non-specific damage produced by 6-OHDA using

five modifications to the standard paradigm: (i) We added

ascorbic acid to diminish the oxidation of 6-OHDA. (ii) We

added the metal chelator DETAPAC, which has been shown

to promote DA uptake into DA terminals in the presence of

ascorbate (Berman et al. 1996). (iii) The medium was

flushed with N2 to reduce dissolved O2 prior to use. (iv)

We limited to 15 min the period during which 6-OHDA was

present and then carefully washed the cells so as to remove as

much of the 6-OHDA as possible from the medium. (v)

Finally, we restricted the source of the DA cells to the SN

region of mesencephalon, excluding cells from VTA, which

would otherwise represent most of the dopaminergic neu-

rons. In the great majority of experiments, this protocol

allowed us to observe a significant amount of 6-OHDA-

induced cell death among nigral DA neurons under condi-

tions in which GABA neurons were unaffected.

The specific effects of 6-OHDA presumably involved its

uptake into DA neurons via DAT and the subsequent

intracellular oxidation of 6-OHDA, as inhibitors of the

transporter blocked toxicity without affecting the non-

specific damage caused by H2O2. These findings are

consistent with the assumption that non-specific damage to

cultured mesencephalic cells caused by 6-OHDA, whether

documented or not, is the result of the non-specific oxidation

of the toxin in the culture medium and the subsequent actions

of reactive oxygen species. Still further complications are

likely to have been introduced into many previous experi-

ments by the failure to separate DA neurons of the SN from

those of the VTA, as the latter appeared to be highly

insensitive to 6-OHDA.

The nature of DA neuron cell death in our model

6-OHDA has been reported to cause the death of DA neurons

by apoptosis, necrosis, or a combination of the two (Marti

et al. 1997; Woodgate et al. 1999; He et al. 2000; Zuch

et al. 2000; Neystat et al. 2001; Han et al. 2003; Holtz and

O’Malley 2003). However, these previous studies have

examined the effects of long exposures of 6-OHDA (> 6 h)

and thus are likely to have measured both specific, intracel-

lular effects of 6-OHDA taken up into DA neurons and non-

specific, extracellular effects of reactive oxygen species

formed in the medium. Under our conditions, 6-OHDA

appeared to produce its specific effects on DA neurons at

least in part through the initiation of apoptosis. This

conclusion is based on four sets of observations. First, 3 h

after exposure to 6-OHDA (40–100 lM), DA neurons in SN

cultures displayed fragmented neurites, shrunken somata and

condensed chromatin. This was followed by a loss of DA

cells 48 h after 6-OHDA exposure. Secondly, we observed

DNA fragmentation at this time as evidenced by nuclear

Fig. 9 The effects of GDNF (100 ng/mL) on the viability of DA neu-

rons exposed to 6-OHDA (40 lM) as assessed 48 h post treatment.

Cultures were treated with 6-OHDA (filled bars) or its vehicle (open

bars). Cultures exposed to GDNF (right pair of bars) received GDNF

for 1 h prior to 6-OHDA, as well as during and after 40 lM 6-OHDA

exposure. The data represents three to five experiments performed in

triplicate. *p < 0.01 for differences between control and 6-OHDA-

treated cultures, and between cultures treated with GDNF and

6-OHDA plus GDNF. Note, however, that 6-OHDA reduced the

number of viable cells by approximately 50% in both the presence and

absence of GDNF.

Effects of 6-OHDA and GDNF on primary SN neurons 783

� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 776–787

fragmentation and positive TUNEL staining. Thirdly, by 3 h

after exposure to 6-OHDA we also detected the activation of

caspase-3, a key executioner protease in the apoptotic

pathway. Fourthly, z-DEVD-fmk, a caspase inhibitor,

blocked the effects of 6-OHDA as measured 48 h after

exposure to the toxin.

The effects of z-DEVD-fmk deserve special attention.

First, this caspase inhibitor also can protect cultured DA

neurons against MPP+ (Bilsland et al. 2002), suggesting a

final common pathway for the death induced by these two

toxins despite several differences observed in the their effects

(e.g. Choi et al. 1999). Secondly, whereas z-DEVD-fmk

prevented 6-OHDA-induced death of DA neurons, the other

indices of cell death – condensed nuclei, TUNEL labeling

and caspase-3 activation itself – were not seen 3 h after

exposure to 6-OHDA in all affected DA neurons. This

suggests either that the onset of these indices of apoptosis

requires more than 3 h to appear, or that not all 6-OHDA-

induced cell death involves conventional apoptotic process

toxicity despite being caspase-dependent.

DA neuron death in Parkinson’s disease

We cannot know whether our 6-OHDA model mimics the

nature of DA neuron death in Parkinson’s disease. The DA

neurons we examined were very young and in culture.

Moreover, the death was triggered by a very brief incubation

to a specific toxin to which patients with Parkinson’s disease

presumably are not exposed, although at least one group has

reported the detection of 6-OHDA in the brains of Parkin-

son’s disease patients (Jellinger et al. 1995). Finally, the

nature of cell death occurring in Parkinson’s disease is still a

matter of controversy. Although oxidative stress is widely

believed to be a key factor in this process, there has been

considerable debate regarding the relative contributions of

apoptosis and necrosis (e.g. see reviews by Burke and

Kholodilov 1998; Graeber et al. 1999; Jellinger 2000;

Hartmann and Hirsch 2001; Graeber and Moran 2002;

Zigmond and Burke 2002). Nonetheless, there are reports at

both the light and electron microscopic level showing

evidence of apoptotic-like morphological and biochemical

changes in the SN of post-mortem material from patients

who suffered from Parkinson’s disease (e.g. Mochizuki et al.

1996; Anglade et al. 1997; Hirsch et al. 1999; Hartmann

et al. 2000, 2001; Tatton 2000). Therefore, we believe it is

reasonable to assume that apoptosis plays a role in the DA

neuron loss that occurs in Parkinson’s disease, and thus that

our in vitro model may be relevant to at least a component of

the pathophysiology of that disease.

Effects of GDNF on 6-OHDA toxicity

The cell death caused by 6-OHDA in our system was altered

by the addition of GDNF. Attention was first drawn to GDNF

as a neuroprotective factor by studies indicating that it

promotes the survival of developing DA neurons in culture

(Engele et al. 1991; Lin et al. 1993). Subsequently, it was

shown that GDNF, as well as vital vectors that contained the

GDNF gene, blocked another kind of DA neuron death – that

which is produced in vivo by 6-OHDA (e.g. Hoffer et al.

1994; Kearns and Gash 1995; Sauer et al. 1995; Tomac et al.

1995; Choi-Lundberg et al. 1997). GDNF also has been

shown to protect against other insults, including lesioning of

motor neurons (Hottinger et al. 2000) and cerebral ischemia

(Miyazaki et al. 1999). Therefore, there is reason to believe

that GDNF can serve as a survival factor in several different

instances.

There also are several studies indicating that GDNF

protects DA neurons from neurotoxins in vitro (Hou et al.

1996; Eggert et al. 1999; Sawada et al. 2000), although

others have reported variable or little protection of cultured

DA neurons (Hou et al. 1997; Kramer et al. 1999). Unfor-

tunately, it is difficult to determine the significance of either

set of observations as, at least in the case of exposure to

6-OHDA, the toxicity may not have been limited to the

dopaminergic cells (see above). Nonetheless, we observed

that GDNF prevented 6-OHDA-induced apoptotic responses

in DA neurons examined 3 h after exposure under conditions

in which we were able to verify the specific effect of the

toxin. We anticipated that these neuroprotective effects of

GDNF would be long lasting, which appears to be the case

in vivo. However, GDNF did not appear to block DA cell

loss as measured after 48 h once the inherent pro-survival

effects of GDNF on control cultures were taken into

consideration. There are several possible explanations for

this discrepancy. For example, the neurotoxic consequences

of 6-OHDA may persist after GDNF or the signaling

mechanism that mediates the effects of GDNF is active.

Alternatively, GDNF may block the apoptosis caused by

6-OHDA, but allow other neurotoxic processes to persist,

processes that were not detected by such measures as nuclear

condensation, TUNEL labeling or caspase activation. Such

processes might normally exist side by side with apoptosis or

only emerge when apoptosis is blocked. It also is possible

that these GDNF-insensitive effects of 6-OHDA are different

from developmental cell death. This would explain the

common observation that GDNF does attenuate the sponta-

neous DA neuron loss normally seen over several days in

culture, even though it did not block the 6-OHDA-induced

cell loss. Finally, GDNF may only delay any given toxic

event rather than block it entirely.

There are some precedents for our finding that GDNF was

ultimately ineffective in blocking toxin-induced cell death.

For example, GDNF (Koeberle and Ball 1998), like BDNF

(Vidal-Sanz et al. 1987), fails to produce a long-term blockade

of retinal ganglion cell death following axotomy. Moreover,

we have recently observed that GDNF blocks some but not all

indices of 6-OHDA-induced cell death in MN9D cells (Ugarte

et al. 2003). In interpreting the significance of the failure of

GDNF to block cell death, it is noteworthy that z-DEVD-fmk

784 Y. M. Ding et al.

� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 776–787

did block the toxic effects of 6-OHDA as measured at 48 h

post exposure to 6-OHDA. Our results may therefore suggest

that GDNF acts upstream of caspase-3. Further investigation

will be necessary to determine the precise nature of the effects

of this trophic factor as well as their biological significance.

Acknowledgements

We thank David L. Cardozo for his invaluable assistance in

establishing our method for ventral mesencephalic cultures, David

Sulzer for helpful discussions regarding culturing and the use of

GDNF, Teresa G. Hastings and Adrian C. Michael for their advice

on reducing the oxidization of 6-OHDA, Sandra M. Pearl for her

participation in the early phase of these studies, Eric A. Falke for his

participation in initial studies comparing SN and VTA neurons,

Susan L. Slagel for performing the analyses of 6-OHDA, Richard J.

Clarke for images prepared in the Center for Biologic Imaging

directed by Simon C. Watkins and Howard E. Rockette, Department

of Biostatistics, for assistance with the statistical analysis, Ruth G.

Perez and Susan D. Giegel for helpful comments on the manuscript,

Emma L. Culligan for preparation of the final draft and Michael E.

Greenberg, in whose laboratory these studies were initiated while

one of the authors (MJZ) was on sabbatical leave. Preliminary

reports of this research have been presented at meetings of the

Society for Neuroscience (New Orleans, LA, November 2000; San

Diego, CA, Nov 2001; Orlando, FL, 2002), the International

Parkinson’s Disease Congress (Helsinki, Finland, July 2001), the

10th meeting of the Israeli Society for Neuroscience (Eliat, Israel,

December, 2001) and Dopamine 2002 (Portland, OR, July 2002).

The research was supported in part by grants from the Scaife Family

Foundation, the National Parkinson Foundation and the USPHS

(NS19608 and NS39267).

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