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Experimental Neurology 189 (2004) 78–93

Sparing of behavior and basal extracellular dopamine after

6-hydroxydopamine lesions of the nigrostriatal pathway

in rats exposed to a prelesion sensitizing

regimen of amphetamine

Isabella Anna Moroz,a,1 Susana Pecina,b,1 Timothy Schallert,c,d and Jane Stewarta,*

aCenter for Studies in Behavioral Neurobiology, Concordia University, Montreal, PQ, CanadabDepartment of Psychology, University of Michigan, Ann Arbor, MI 48109, USA

cDepartment of Psychology and Institute for Neuroscience, University of Texas, Austin, TX 78712, USAdDepartment of Neurosurgery, University of Michigan, Ann Arbor, MI 48109, USA

Received 6 December 2003; revised 1 May 2004; accepted 7 May 2004

Abstract

Repeated administration of amphetamine leads to enduring augmentation of its behavioral-activating effects, enhanced dopamine (DA)

release in striatal regions, and morphological changes in DA target neurons. Here we show that exposure to a 2-week escalating-dose regimen

of amphetamine prevents behavioral asymmetries of forelimb use and spontaneous (drug-independent) turning behavior following unilateral

6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal pathway made 7–14 days after termination of amphetamine treatment

(Experiments 1–3). Exposure to three nonescalating injections of amphetamine 7 days before 6-OHDA lesions had no effect (Experiment 2).

Prelesion amphetamine treatment led to normalization of basal extracellular levels of striatal DA as measured by microdialysis on days 11–

14 and 25–28 after lesioning (Experiment 3). However, there were no significant differences between treatment groups in postmortem tissue

levels of DA and its metabolites, indicating a dissociation between the DA depletion and the extracellular levels of DA as measured by

microdialysis. Finally, rats exposed to the escalating amphetamine regimen had reduced lesion-induced loss of TH-IR cells in the ipsilateral

DA cell body regions (Experiment 3). Thus, prelesion exposure to the escalating doses of amphetamine may render the cells resistant to the

consequences of damage after subsequent 6-OHDA lesions, possibly by accelerating the development of compensatory changes in the DA

neurons that typically accompany behavioral recovery. The potential role of amphetamine-induced endogenous neurotrophic factors in the

behavioral sparing and normalization of basal extracellular DA levels observed after subsequent 6-OHDA lesions is discussed.

D 2004 Elsevier Inc. All rights reserved.

Keywords: Amphetamine; Prelesion; Behavioral sparing; Striatal dopamine; 6-OHDA; Nigrostriatal pathway; Neurotrophic factors; Exercise

Introduction activating effects that are associated with heightened

Exposure to psychostimulant drugs leads to changes in

brain and behavior that outlast the drugs’ acute neurophar-

macological effects. Repeated administration of amphet-

amine results in an enduring enhancement of its behavioral-

0014-4886/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.expneurol.2004.05.012

* Corresponding author. Center for Studies in Behavioral Neurobiology,

Department of Psychology, Concordia University, 7141 Sherbrooke Street.

W., Montreal, Quebec, Canada H4B 1R6. Fax: +1-514-848-2817.

E-mail address: stewart@csbn.concordia.ca (J. Stewart).1 These authors contributed equally to these independent experiments.

Isabella Anna Moroz is currently a postdoctoral fellow at the Centre for

Stroke Recovery, Ontario Health Research Institute, University of Ottawa,

Ottawa, Ontario, Canada.

dopamine (DA) overflow in striatal regions in response

to acute drug challenges (Kalivas and Stewart, 1991;

Robinson and Becker, 1986). This phenomenon of ‘‘be-

havioral sensitization’’ is very different from ‘‘amphetamine

neurotoxicity’’ that develops after continuous infusion or

frequent injection of multiple high doses of amphetamine

and leads to depletion of striatal DA presumably due to

terminal degeneration (for a review, see Robinson and

Becker, 1986). The behavioral and neurochemical effects

of repeated administration of amphetamine develop gradu-

ally and have been observed for months after the termination

of drug treatment (Paulson et al., 1991). Enduring enhance-

ment of function within the midbrain DA system has also

Table 1

Two-week escalating-dose treatment with amphetamine

Monday Tuesday Wednesday Thursday Friday Saturday Sunday

Week

1

1 1 2 2 2 – –

Week

2

3 4 4 4 4 – –

The numbers represent mg/kg of d-amphetamine sulfate per injection on

each day. All animals received two injections per day.

I.A. Moroz et al. / Experimental Neurology 189 (2004) 78–93 79

been observed after partial unilateral 6-hydroxydopamine (6-

OHDA) lesions of the nigrostriatal neurons. In these models,

basal extracellular levels of dopamine released from the

surviving terminals in the lesioned striatum, as measured

using in vivo microdialysis, normalize gradually, corres-

ponding in time to behavioral recovery from such lesions

(Abercrombie et al., 1990; Altar et al., 1994; Castaneda et al.,

1990; Robinson and Whishaw, 1988; Robinson et al., 1994).

The gradual and long-lasting nature of behavioral sensitiza-

tion and recovery from partial lesions has been proposed to

occur as a result of neurochemical (Robinson and Becker,

1986; Robinson et al., 1994) and structural modifications

(Finkelstein et al., 2000; Robinson and Kolb, 1997) in neural

circuitry, and alterations in patterns of synaptic connectivity,

most probably brought about by the actions of neurotrophic

factors (Flores and Stewart, 2000a; Moroz et al., 2003). We

hypothesized that repeated administration of amphetamine

before 6-OHDA lesions of the nigrostriatal DA system may

mobilize neurotrophic factors and other precursors to struc-

tural adaptations at the time of lesioning that could, in turn,

enhance DA neuron functioning and thus reduce the impact

of the lesions on behavior, that is, facilitate behavioral

sparing after such lesions.

In intact rats, repeated treatment with amphetamine has

been shown to increase dendritic length, density of dendritic

spines, and the number of branched spines on the major DA

output neurons in the nucleus accumbens (NAcc), striatum,

and prefrontal cortex (Li et al., 2003; Robinson and Kolb,

1997, 1999). Furthermore, repeated administration of am-

phetamine has also been shown to induce increases in

expression of neurotrophic factors known to affect the

survival, maintenance, and morphological plasticity of adult

neurons. Treatments with amphetamine have resulted in

increases in astrocytic basic fibroblast growth factor (FGF-

2) in the DA cell body regions, ventral tegmental area (VTA)

and substantia nigra pars compacta (SNc; Flores et al., 1998),

and DA striatal terminal regions (Flores and Stewart, 2000b),

as well as increases in brain-derived neurotrophic growth

factor (BDNF) in the basolateral amygdala and its target

regions including medial NAcc and dorsal medial striatum

(Meredith et al., 2002). More importantly, the number of

FGF-2-immunoreactive astrocytes in the VTA and SNc has

been strongly and positively correlated with the magnitude

of behavioral sensitization, and infusions of an FGF-2-

neutralizing antibody into the VTA before amphetamine

administration have been shown to prevent the development

of sensitization (Flores et al., 2000). Thus, the long-lasting

behavioral and neurochemical changes induced by repeated

administration of amphetamine could be mediated through

the actions of neurotrophic factors such as FGF-2 and BDNF.

In view of findings that increases in neurotrophic factor

expression before brain injury have the potential to protect

against subsequent insults (Altar et al., 1994; Choi-Lund-

berg et al., 1998; Mandel et al., 1997; Shults et al., 1996,

2000), we hypothesized that exposure to amphetamine

before 6-OHDA lesions might serve to attenuate behavioral

and neurochemical deficits after these lesions. We report

here two independent experiments, conducted in separate

laboratories, using different strains of rats and different

placements of 6-OHDA infusions within the nigrostriatal

pathway, both showing that exposure to an escalating-dose

regimen of amphetamine prevents subsequent 6-OHDA

lesion-induced asymmetries of forelimb use and turning

behavior. In addition, we report that this behavioral sparing

coincides with normalization of basal extracellular DA

levels in the striatum as assessed using in vivo microdialysis.

Materials and methods

Experiment 1

Subjects

Adult male Long–Evans rats (Charles River, Wilming-

ton, MA; 250–300 g), housed on a reversed light–dark

cycle with free access to food and water served as subjects.

Amphetamine regimen

d-Amphetamine sulfate (Sigma, St. Louis, MO), dis-

solved in physiological saline, was given in a 2-week

escalating-dose regimen (esc/amph; n = 10), as described

previously (Flores and Stewart, 2000b). This regimen in-

volved two daily intraperitoneal (ip) injections of amphet-

amine in the colony room, 7–8 h apart, 5 days a week, for 2

weeks. The dose of amphetamine began with 1 mg/kg and

escalated to 4 mg/kg for the last 4 days of treatment (see

Table 1). The saline-treated control group (sal; n = 12)

received 0.9% saline (1 ml/kg). To control for amphetamine-

induced increases in motor activity, four rats from the sal

group were housed 24 h/day in cages with running wheels

(34 cm diameter) during the amphetamine administration

period (Robinson and Kolb, 1999).

Intrastriatal 6-OHDA lesions

Unilateral 6-OHDA (6-hydroxydopamine hydrochloride,

Sigma) infusions (4 Al of 10 Ag/4 Al in 0.05% ascorbic acid

solution; 0.5 Al/min) into the striatum (stereotaxic coordi-

nates with flat skull: 1.7 mm anterior, 2.9 mm lateral to

bregma, and 4.0 mm ventral to skull) were performed under

Equithesin (25 mg/kg pentobarbital; Sigma) and 150 mg/kg

chloral hydrate anesthesia (0.35 ml/100 g ip) followed by

atropine sulfate (0.1 mg/kg ip; Sigma).

tal Neurology 189 (2004) 78–93

Behavioral testing and limb-use observation

Use of each forelimb for upright support and for landing

when descending from a rearing position was analyzed both

pre- and postoperatively in the cylinder test, as previously

described (Tillerson et al., 2001). Occurrences of forelimb

use for wall exploration and landing were determined sep-

arately, and each was expressed in terms of (1) the percent

ipsilateral limb use [ipsi / (ipsi + contra + both) � 100] and

(2) the percent contralateral limb use [contra / (ipsi + contra +

both) � 100]. The percent contralateral limb use was then

subtracted from the percent ipsilateral limb use for both the

wall behavior and landing. These two scores (wall and

landing) were averaged to obtain a single overall limb-use

asymmetry score that corrected for variability in the absolute

number of landing movements versus wall movements

among animals or between groups. This asymmetry score

has been shown previously to be highly correlated with the

degree of 6-OHDA-induced striatal DA depletion (Schallert

and Tillerson, 1999).

Designs and procedures

The timing of the experimental manipulations in Exper-

iment 1 is outlined in Fig. 1A. Briefly, 2 weeks after

termination of the 2-week escalating-dose regimen of am-

phetamine, rats received unilateral intrastriatal 6-OHDA

I.A. Moroz et al. / Experimen80

Fig. 1. Diagram outlining the timing of the experimental manipulations in

Experiments 1, 2, and 3.

infusions. Asymmetry of forelimb use was tested in the

cylinder test before 6-OHDA lesioning (pretest) and 1, 3, 7,

and 14 days after lesioning.

Statistical analyses

Data were analyzed by mixed-factor analysis of variance

(ANOVA) with drug (sal, esc/amph) as the between factor

and day (pretest, d1, d3, d7, d14) as the repeated factor.

Experiments 2 and 3

Subjects

Adult male Wistar rats (Charles River, QC; 325–350 g),

housed on a reversed light–dark cycle with free access to

food and water served as subjects.

Amphetamine regimens

d-Amphetamine sulfate (SmithKline Beecham Pharma,

Oakville, ON), dissolved in physiological saline, was given

either in a 2-week escalating-dose regimen (esc/amph, see

Table 1; Experiment 2, n = 5; Experiment 3, n = 7), or once

a day, every second day, for a total of three injections (3

amph; 3 mg/kg ip, n = 5). Saline-treated animals for each

amphetamine regimen (sal, Experiment 2, n = 4/group;

Experiment 3, n = 5) received 0.9% saline (1.0 ml/kg).

Intra-MFB 6-OHDA lesions

Unilateral 6-OHDA (6-hydroxydopamine hydrochloride,

Sigma, Oakville, ON) infusions (2 Al of 8 Ag/4 Al of 0.9%saline containing 0.05% ascorbic acid solution; rate: 0.2 Al/min) into the medial forebrain bundle (MFB; stereotaxic

coordinates with flat skull: 2.9 mm posterior, 1.7 mm

lateral to bregma, and 7.6 mm ventral to dura) were

performed under sodium pentobarbital anesthesia (30 mg/

kg ip) supplemented with isoflurane (Biomeda MTC, Cam-

bridge, ON). Atropine sulfate (0.5 mg/ml, 0.2 ml/rat sc)

was given to reduce bronchial secretions. Desmethyli-

mipramine (15 mg/kg ip; RBI Biochemicals, Oakville,

ON), a norepinephrine reuptake inhibitor, was given 30

min before infusion of 6-OHDA to protect the noradre-

nergic cells from the neurotoxin.

In Experiment 3, after 6-OHDA infusion, guide cannulae

(22-gauge stainless steel) were implanted bilaterally into the

striatum (stereotaxic arms angled 10j from the vertical

plane, stereotaxic coordinates with flat skull: 1.2 mm

anterior, 3.0 mm lateral to bregma, and 3.4 mm ventral to

skull surface). The cannulae were anchored to the skull with

stainless steel screws and secured to the surface with dental

cement.

Behavioral testing and limb-use observation

Forelimb use for upright support and for landing when

descending from a rearing position was analyzed pre- and

postoperatively in the cylinder test, as described in Exper-

iment 1. Rats were also tested for spontaneous ipsiversive

turning (turning toward the side of the lesion) in a novel

I.A. Moroz et al. / Experimental Neurology 189 (2004) 78–93 81

environment, as previously described (Moroz et al., 2003).

The number of compact (within the diameter of approxi-

mately 20 cm) 360j turns and 180j half turns ipsilateral and

contralateral to the side of the lesion was recorded and

summed across 5-min testing sessions. The number of

ipsilateral turns was presented as a percent of the total

number of turns displayed by an animal [ipsi / (ipsi +

contra) � 100%].

Microdialysis

Microdialysis was conducted as described previously

(Emmi et al., 1996, 1997) in hexagonal testing chambers

(42 � 39 � 33.5 cm) built from Plexiglas with wooden

ceilings and stainless steel rod floors. The cages were

individually enclosed in wooden cubicles where lighting

was provided on a reversed cycle by overhead light bulbs

(15 W). The dialysis probe consisted of a 2.2-mm length of

semipermeable dialysis membrane (Fisher Scientific, 240

Am OD, 13000 M.W. cutoff), closed at one end and attached

to a 21-mm-long, 26-gauge stainless steel length of tubing.

A 40- to 50-cm length of PE-20 tubing connected the other

end of the stainless steel shaft to an infusion swivel

stationed above the testing chamber, which was connected

via PE-20 tubing to a variable speed infusion pump. A small

diameter, fused silica tube extended internally through the

probe with one end resting 0.5 mm from the tip of the probe

and the other end exiting the PE tubing 35 cm below the

infusion swivel. The probes were secured by brass collars

that screwed onto the guide cannulae. The external length of

PE-20 tubing was protected from damage by steel spring

casings. The probes were designed so that the entire length

of semipermeable membrane extended below the guide

cannula tip.

The probes were inserted on the evening preceding the

microdialysis testing. To prevent occlusion, artificial cereb-

rospinal fluid (ACSF, 145 mM Na+, 2.7 mM K+, 1.22 mM

Ca2+, 1.0 mM Mg2+, 150 mM Cl�, 0.2 mM ascorbate, 2

mM Na2HPO4, pH, 7.4) was perfused overnight at a rate

of 0.25 Al/min. Dialysate sampling and activity monitoring

began the next morning. Half of the animals from each

treatment condition were dialyzed on the lesioned side on

the first day of dialysis and on the intact side on the

second day of dialysis; for the other animals, the con-

ditions were reversed. The dialysate flow rate was in-

creased to 0.6 Al/min, and baseline dialysate samples

(approximately 12 Al/sample) were collected every 20

min. A 10-Al volume of dialysate was extracted from each

sample and immediately analyzed using one of two similar

high-performance liquid chromatography systems with

electrochemical detection (HPLC-EC). The samples were

loaded onto reverse-phase columns (15 � 4.6 mm; Hasil

C18, 5 Am; S.P.E. Limited, Concord, ON, Canada) through

manual injection ports (Rheodyne 7125; 20 Al loop);

reduction and oxidation currents for DA and its metabo-

lites, dihydroxyphenylacetic acid (DOPAC) and homova-

nillic acid (HVA), were measured with dual-channel ESA

coulometric detectors (Coulochem 5100, with a model

5011 analytical cell). The currents for DA were measured

independent of those for DOPAC and HVA using separate

channels of the Coulochem detectors. The mobile phases

[15% acetonitrile, 0.076 M SDS, 0.1 M ethylenediamine-

tetraacetic acid (EDTA), 0.058 M NaPO4, 0.27 M citric

acid, pH = 3.35] were circulated through each closed

system at a flow rate of 1.3 ml/min by Waters 515 HPLC

pumps. The peaks obtained for DA, DOPAC, and HVA

were integrated and quantified by EZChrom Chromatog-

raphy Data System (S.P.E. Limited). Dialysate samples

from individual rats always were analyzed with the same

HPLC-EC system, and the assignment of animals to each

system was counterbalanced across all treatment groups.

Food was removed from the chambers before sampling,

but a water drinking tube was available.

Postmortem tissue analysis

Animals were killed by decapitation and their brains

were rapidly removed, placed in isopentane, cooled on dry

ice, and frozen overnight at �80jC. The following day, the

brains were sliced on a cryostat into 200 Am sections.

Punches were taken from the dorsal striatum (1 � 2 mm

diameter) of the lesioned and nonlesioned hemisphere, from

two sections about 1.2 and 1.5 mm anterior to bregma, and

from the SN (2 � 1 mm diameter) of the lesioned and

nonlesioned hemisphere, from two sections about 5.2 and

5.5 posterior to bregma. The neurochemical assessment of

DA, DOPAC, and HVA was then performed as described

previously (Moroz et al., 2003). Punches were suspended in

phosphate buffer (PB) and frozen overnight. The following

day, samples were thawed and centrifuged at 4000 rpm for

15 min. Pellets were suspended in 0.1 M NaCl and analyzed

for protein content. The supernatant was removed and

assayed for DA, DOPAC, and HVA using HPLC-EC. The

supernatant was injected into a 15-cm C18 column (5 mm

particle size, Scientific Products and Equipment, Ontario).

The mobile phase consisted of 30 mM citric acid, 60 mM

sodium phosphate monobasic, 0.10 mM EDTA, 14% ace-

tonitrile, and 0.08 mM sodium dodecyl sulphate, pH 3.35.

The mobile phase was pumped through the system at 1.2 ml/

min using a Waters 515 HPLC pump. Compounds were

detected and quantified with an ESA coulochem detector

(model 5100A) equipped with an analytical cell (model

5011; E1 = +0.35 V, E2 = �0.3 V, ESA, Inc.). The

concentrations were estimated from peak heights by com-

parison with injections of known amounts of pure standards

(Sigma) and expressed as micrograms per milligram of

protein.

TH immunohistochemistry

Brains were removed and stored overnight in the

fixative solution [paraformaldehyde (w/v) and 15% picric

acid (v/v) in PB (pH 6.9)] at 4jC. Coronal 50 Am sections

were cut on a vibratome and stored overnight in PB at

4jC. Before slicing, a small mark was cut in each brain to

I.A. Moroz et al. / Experimental Neurology 189 (2004) 78–9382

allow discrimination of the hemispheres in each section.

Sections were then processed for TH immunoreactivity, as

described previously (Moroz et al., 2003). Free-floating

tissue sections were preincubated in 0.3% Triton X-100 PB

and 1% normal goat serum (NGS) for 1 h at room

temperature. They were then incubated for 24 h at 4jCwith the rabbit anti-TH polyclonal antibody diluted to

1:5000 (Chemicon) in PB and 1% NGS. After incubation

in the primary antibody, sections were rinsed three times

for 5 min in cold PB and incubated for 1 h at room

temperature (RT) in a solution of rat adsorbed biotinylated

anti-rabbit antibody (Vector) diluted to 1:200 with PB and

1% NGS. After three 5-min washes in cold PB, sections

were incubated in an avidin–horseradish peroxidase com-

plex (Vectastain Elite ABC Kit, Vector, Burlington Canada)

for 30 min at RT and rinsed again three times for 5-min in

cold PB. Next, sections were incubated for 10 min at room

temperature and under constant agitation in a solution of

0.05% 3,3V-diaminobenzidine (Sigma) in PB. Then, with-

out washing, the sections were transferred to a 2,3V-diaminobenzidine-PB solution, pH 7.8, with 0.01% H2O2

to catalyze the reaction. To obtain an orange-brown reac-

tion product, NiCl2 was withheld from the 3,3V-diamino-

benzidine-PB-H2O2 solution. This incubation was

terminated 5 min later by washing the sections three times

for 10 min in cold PB. Tissue from all groups was included

in each batch of immunocytochemical processing. Sections

were then mounted on gelatin-coated slides, dried for at

least 24 h, hydrated in distilled water (1 min), and

gradually dehydrated in 70%, 95%, and 100% ethanol.

Slides were then cleared in Hemo-De and cover slipped

with Permount (Fisher Scientific).

Image analysis

Immunostained sections were analyzed under a Leica

microscope (Leitz DMRB). The number of TH-immunore-

active (IR) cells per squared millimeter was estimated from

digitized images of sample areas within SNc and VTA using

a computerized image analysis system (NIH Image 1.6).

Structure boundaries were defined according to the Paxinos

and Watson (1997) stereotaxic atlas. TH-IR cells were

manually counted in a minimum of eight images taken from

SNc and four from VTA at two different levels from

bregma: �5.2 and�5.3 mm, in each hemisphere. Only

sections in which the medial and lateral parts of the SNc

were clearly separated by the medial terminal nucleus of the

accessory optic tract were selected. The number of TH-IR

cells calculated from these levels from bregma has been

previously shown to accurately represent 6-OHDA-induced

degeneration of the SNc and VTA neurons (Carman et al.,

1991; Gordon et al., 1997). No attempt was made to

estimate the total TH-IR neurons number in three dimen-

sions. The images were assigned code names, and the FGF-

2 and TH-IR cells were counted by an individual blind to

the code assignment. The cell counts from the areas sampled

in each hemisphere of each brain region were summed and

divided by the total area examined.

Design and procedures

The timing of the treatments and experimental mani-

pulations in Experiments 2 and 3 is outlined in Figs. 1B

and C.

Experiment 2

Seven days after the last amphetamine injection of each

dose regimen, rats received unilateral 6-OHDA infusions

into the MFB. An additional group of rats given compa-

rable saline injections before the 6-OHDA lesions received

postlesion exercise treatment (exercise, n = 8) to compare

efficacy of amphetamine pretreatment to the well-estab-

lished beneficial effects of postinjury exercise intervention

(Tillerson et al., 2001, 2002, 2003). Postlesion exercise

consisted of walking or running on the floor of the

laboratory while inside commercially available transparent,

ventilated, shatter-resistant plastic balls (30 cm diameter)

that permitted a 360j range of motion (Jumbo Kritter

Krawler, Lee’s Aquarium and Pet Products, San Marcos,

CA). If rats initially did not move voluntarily when placed

inside the balls, the experimenter moved the ball in

random directions. For those rats that moved voluntarily,

the dimensions of the space available for exercise sessions

(about 4 m2) did not allow for the maintenance of higher

velocities over longer periods of time. Furthermore, the

speed of movement of each animal varied within each

exercise session. Typically, the rats were more energetic at

the beginning of the session, thus spending most time

running, after which they would switch to walking or even

stop moving. In the latter event, the experimenter would

immediately move the ball to ensure constant movement of

each animal throughout the 30-min sessions. Care was

taken to reproduce the various speeds and patterns of

movement displayed by rats that ran voluntarily. In addi-

tion, side-to-side and front-to-back rocking motions of the

ball were used on all rats intermittently throughout each

exercise session in an attempt to impose balance chal-

lenges and thus serving to strengthen the muscles of the

limbs. The exercise sessions began 4–5 h after lesioning

and continued for the next 14 days. The first session lasted

for 15 min, the next 7 for 30 min, and the final 7 for 15

min/day. Rats were tested once a week for forelimb use in

the cylinder test and for turning behavior in the novel

environment, starting 7 days after lesioning. The preoper-

ative test for asymmetry of forelimb use in the cylinder test

was conducted before the initiation of prelesion amphet-

amine treatments. Rats were killed and their brains were

taken for DA, DOPAC, and HVA tissue assays 28 days

after lesioning.

Experiment 3

Seven days after the last amphetamine or saline injection,

rats received unilateral 6-OHDA infusions into the MFB

Fig. 2. Experiment 1: Asymmetry of limb-use (mean F SEM) in the

cylinder test assessed 1, 3, 7, and 14 days after intrastriatal 6-OHDA lesions

was prevented by the esc/amph treatment [ F(1,109) = 13.51, P < 0.01].

The saline-treated rats housed in cages with free access to running wheels

(data not shown) did not differ from the saline-treated rats that did not run

[F(1,59) = 0.17, ns]; thus, these rats were combined to form the sal group.

I.A. Moroz et al. / Experimental Neurology 189 (2004) 78–93 83

followed immediately by bilateral intrastriatal guide cannulae

implantation.Microdialysis was performed at two time points

after lesioning—between 11 and 14 days and again between

25 and 28 days. Each rat was tested over 2 days at each time

point (either on days 11 and 12 and subsequently on days 25

and 26, or on days 13 and 14 and subsequently on days 27–

28, as determined by the number of microdialysis boxes

available). On the evening preceding each microdialysis

session, rats were moved to the microdialysis chambers

where the probes were inserted and perfused overnight at a

slow rate of < 0.25 Al/min. Dialysate sampling began the

following morning when half of the animals from each

treatment condition were dialyzed on either the lesioned or

the intact side. The same evening, a probe was inserted into

the other striata, and dialysate sampling of the opposite side

began the next morning. Half of the animals from each

treatment condition were dialyzed on days 11 and 12, and

half on days 13 and 14; similarly for the later time point after

lesioning, half of the animals from each treatment was

dialyzed on days 25 and 26, and 27 and 28 at the later time

point. The day after the microdialysis for both hemispheres

was completed, the rats were administered behavioral tests.

Following the last behavioral test conducted after micro-

dialysis at 28 days postlesion, the rats were killed and their

brains were removed and cut into anterior and posterior

portions. The anterior portion was taken for tissue analysis

of DA and its metabolites; sections from the posterior portion

were processed for TH immunoreactivity.

Statistical analyses

Behavioral data in Experiment 2 were analyzed by

mixed-factor ANOVAs with group (sal, esc/amph, 3 amph,

exercise) as the between factor and day (d7, d14, d21, and

d28) as the within factor. Pretest data for the forelimb

asymmetry test were analyzed with one-way ANOVA.

Tissue levels of DA, DOPAC, and HVA in Experiment 2

were analyzed by mixed-factor ANOVAs with group (sal,

esc/amph, 3 inj/amph, exercise) as the between factor and

side [nonlesioned (NL), lesioned (L)] as the within factor.

Behavioral data in Experiment 3 were analyzed using one-

way ANOVAs as required; post hoc comparisons were

made using the Fisher’s PLSC test (P < 0.05). Data from

two animals (one from the sal group and one from the esc/

amph group) were excluded from all analyses in Experi-

ment 3 due to lack of lesion as indicated by postmortem

analysis of tissue levels of DA and its metabolites. Rats

that performed fewer than five landings and fewer than 10

wall movements during the cylinder tests (criterion less

stringent than the typical 10 landings and 20 wall move-

ments criterion recommended by Schallert and Tillerson,

1999, employed here to compensate for general lower

levels of motor activity in Wistar than in Long–Evans

rats) and those that did not display tight turns of less than

30 cm in diameter (Fornaguera et al., 1994) during the

asymmetry of turning tests were not included in the

analyses. This resulted in slightly different degrees of

freedom for the F values on the two behavioral tests

conducted at two different time points after lesioning in

Experiment 3. Since none of these animals was inactive

during both behavioral tests, thus providing a measure of

their behavioral performance on at least one of the tests,

they were all included in the subsequent neurochemical

and cell counts analyses. Neurochemical analyses of tissue

and extracellular levels of DA, DOPAC, and HVA and

estimated numbers of TH-IR cells per squared millimeter

for each brain region in Experiment 3 were conducted

using mixed-factor ANOVAs with drug (sal, esc/amph) as

the between factor and side [nonlesioned (NL), lesioned

(L)] as the within factor. Tests for simple main effects were

used to determine the source of the significant Group �Side interactions; Bonferroni correction was used to keep

the overall error rate at 0.05.

Results

Behavior

Exposure to the 2-week escalating-dose regimen of

amphetamine before intrastriatal (Experiment 1) or intra-

MFB infusions of 6-OHDA (Experiments 2 and 3) pre-

vented the preferential use of the ipsilateral forelimb for

vertical exploration (Figs. 2–4A) and ipsilateral turning

(Figs. 3B and 4B) after 6-OHDA lesions. Remarkably, the

esc/amph groups from all three experiments displayed no

asymmetry of limb use or turning when compared to the sal

group (Figs. 2–4). In addition, in Experiment 2, the esc/

amph group, but not the 3 amph group, did not differ from

the postlesion exercise group (Fig. 3). In Experiment 1, rats

Fig. 3. Experiment 2: Mean (FSEM) asymmetry of (A) limb use and (B) spontaneous turning tested 7, 14, 21, and 28 days after 6-OHDA lesions of the MFB.

ANOVAs revealed a significant main effect of group for the limb-use asymmetry test [F(3,54) = 3.1, P < 0.05] and a similar tendency for the turning asymmetry

test [F(3,54) = 2.50, P = 0.09]. The esc/amph and the exercise groups displayed no asymmetry of limb use and did not differ from each other; both differed

significantly from the sal group (*P < .05). There were no differences between the groups in forelimb asymmetry during the pretest [F(3,18) = 1.00, ns]. Note in

(B), a dashed line at 50% represents no asymmetry or an equal number of ipsilateral and contralateral turns.

I.A. Moroz et al. / Experimental Neurology 189 (2004) 78–9384

housed in cages with free access to running wheels before

lesioning showed high levels of motor activity, running an

average of 9.4 km/24 h, but their postlesion forelimb use

asymmetry was not different from that of the sal group.

In vivo microdialysis

Dopamine (DA)

Basal levels of DA from the intact and lesioned striata

of animals treated with the escalating-dose regimen of

amphetamine before intra-MFB infusions of 6-OHDA

(Experiment 3) and then tested using microdialysis be-

tween days 11–14 and 25–28 are shown in Figs. 5A and

D, respectively. It can be seen at both time points that

the esc/amph group showed normalization of basal DA

levels on the lesioned side, whereas the sal group had

significantly lower basal levels of DA in the striatum on

the lesioned side than on the intact side. This effect is

reflected in a significant Group � Side interaction ob-

served 11–14 days after lesioning.

DOPAC and HVA

Basal levels of both DOPAC and HVA measured on

days 11–14 and 25–28 after lesioning (Experiment 3) are

shown in Figs. 5B, C, E, and F, respectively. The

ANOVAs revealed a significant effect of side, but no

Group � Side interaction. It can be seen that levels of

both metabolites were significantly lower on the lesioned

side in both amph/esc and sal groups. See Fig. 5 legend for

results of statistical analyses.

Fig. 4. Experiment 3: Mean (F SEM) asymmetry of (A) limb use and (B) spontaneous turning, tested 11–14 (Test 1) and 25–28 (Test 2) days after 6-OHDA

lesions of the MFB. ANOVAs on the data revealed significant group effects [limb-use asymmetry, Test 2, F(1,8) = 6.05, P < 0.05; turning, Test 1, F(1,9) =

8.13, P < 0.05, Test 2, F(1,10) = 10.93, P < 0.05]. The esc/amph group displayed no asymmetry of limb use on Test 2 or turning on either Test 1 or 2. Note in

ipsilateral and contralateral turns.

I.A. Moroz et al. / Experimental Neurology 189 (2004) 78–93 85

Postmortem tissue analysis

The tissue levels of DA, DOPAC, and HVA in the dorsal

striatum and in SN observed 28 days after lesioning are

shown in Figs. 6(Experiment 2) and 7(Experiment 3). In all

groups, the postmortem levels of DA and its metabolites were

significantly depleted on the side of the lesion, indicating

extensive degeneration of terminals and cell bodies. It can be

seen in Figs. 6B, D, and F that the 3 amph and the esc/amph

group appear to have higher levels of DA and its metabolites

in the nonlesioned SN. One-way ANOVAs conducted on data

expressed as the percentage of DA, DOPAC, and HVA

remaining on the lesioned side [(amount remaining on the

lesioned side / amount on the nonlesioned side) � 100%]

revealed no significant differences between the groups on any

of these measures.

TH immunoreactivity in SNc and VTA

The number of TH-positive cells remaining on the

lesion and no-lesion side in the SNc and VTA of rats

treated with the escalating-dose regimen of amphetamine

before intra-MFB infusions of 6-OHDA is shown in

(B), a dashed line at 50% represents no asymmetry or an equal number of

Fig. 8 (Experiment 3). It can be seen that 28 days after

lesioning, the number of TH-IR cells was reduced on the

side of the lesion in both the SNc and VTA. It is readily

apparent, however, that the esc/amph group had a greater

number of TH-IR cells remaining on the side of the lesion

than the sal group. This effect is reflected in a significant

Group � Side interaction. See Fig. 8 legend for results of

the statistical analyses.

Discussion

The purpose of this investigation was to determine

whether exposure to a sensitizing regimen of amphetamine

before 6-OHDA lesions of the nigrostriatal DA system

would facilitate behavioral sparing after such lesions. The

present experiments, conducted independently in separate

laboratories, using different strains of rats and different

placements of 6-OHDA infusions within regions of the

nigrostriatal pathway, show that exposure to the 2-week

escalating-dose regimen of amphetamine before lesioning

Fig. 5. Experiment 3: Mean (F SEM) basal concentrations of extracellular DA, DOPAC, and HVA in six dialysate samples taken at 20 min intervals on the

lesioned (L) and nonlesioned (NL) side of the striatum on days 10–14 (A, C, E) and 24–28 (B, D, F) after 6-OHDA lesions of theMFB. ANOVAs (Drug � Side)

yielded the following significant effects on Test 1: DA, side, F(1,10) = 17.06, P < 0.05, Drug � Side interaction, F(1,10) = 6.29, P < 0.05; DOPAC, side,

F(1,10) = 52.87, P < 0.05; HVA, side, F(1,10) = 80.48, P < 0.05, and on Test 2: DA, side, F(1,10) = 7.36, P < 0.05; DOPAC, side, F(1,9) = 28.86, P < 0.05;

HVA, side, F(1,10) = 48.41, P < 0.05. Paired sample t tests conducted to analyze simple main effects revealed that 11–14 days after lesioning, DA levels in the

dialysate were lower on the lesioned than on the nonlesioned side only in the sal group (P < 0.05, Bonferroni corrected).

I.A. Moroz et al. / Experimental Neurology 189 (2004) 78–9386

prevented the asymmetries of forelimb use and turning

behavior seen in rats given saline during the same period.

Furthermore, it was shown in Experiment 2 that exposure to

three nonescalating injections of amphetamine before

lesioning did not lead to behavioral sparing. The prophy-

lactic effect of the escalating-dose regimen was comparable

to the effect of postlesion exercise as seen here in Experi-

ment 2 and as reported previously (Tillerson et al., 2001,

2002, 2003). Note, however, that the effects seen here were

obtained with a novel and less intense exercise program than

the ones used by Tillerson et al.

The major strength of the present investigation is

derived from the fact that our primary findings of behav-

ioral sparing after prelesion treatment with the escalating

doses of amphetamine are consistent in all experiments,

despite methodological differences, such as the site of 6-

OHDA administration: intrastriatal (Experiment 1) versus

intra-MFB (Experiments 2 and 3). Even though the

Fig. 6. Experiment 2: Mean (FSEM) DA, DOPAC, and HVA (Ag/mg protein) in striatal (A, C, E) and SN (B, D, F) tissue punches taken postmortem from the

lesioned (L) and nonlesioned (NL) sides of the brain at 28 days after 6-OHDA lesion of the MFB. ANOVAs revealed significant effect of side [striatum: DA,

F(1,18) = 114, 91, P < 0.0001; DOPAC, F(1,18) = 81.13, P < 0.0001; HVA, F(1,18) = 67.40, P < 0.0001; SN: DA, F(1,18) = 50.27, P < 0.0001; DOPAC,

F(1,18) = 13.98, P < 0.01; HVA, F(1,18) = 11.40, P < 0.001]. There was no effect of prelesion treatment with any regimen of amphetamine on 6-OHDA-

induced depletions of DA and its metabolites on the side of the lesion.

I.A. Moroz et al. / Experimental Neurology 189 (2004) 78–93 87

behavioral and neurochemical consequences of intrastriatal

administration of 6-OHDA are not as pronounced and

devastating as those obtained after intra-MFB administra-

tion of 6-OHDA (for a review, see Schwarting and

Huston, 1996), intrastriatal 6-OHDA lesions have been

proposed by many (i.e., Kirik et al., 1998; Przedborski et

al., 1995; Sauer and Oertel, 1994) to be particularly useful

in modeling early Parkinson’s disease and for testing

protective, preventive, restorative, and symptomatic treat-

ments. In a study of dose-dependent neurotoxic effects of

6-OHDA infusions into the striatum, the dose utilized in

Experiment 1 of the present investigation was found to

induce a lesion characterized by robust drug-induced

rotation, changes in radioactively labeled mazindol bind-

ing in the striatum consistent with 70% dopamine dener-

vation, and 65% reduction in Nissl-stained neurons in

SNc, all of which were stable for at least 12 weeks

(Przedborski et al., 1995). Thus, our finding of beneficial

effects of the escalating-dose regimen of amphetamine

given before both intrastriatal and intra-MFB 6-OHDA

I.A. Moroz et al. / Experimental Neurology 189 (2004) 78–9388

insult has significant implications for understanding of

mechanisms involved in early and more advanced stages

of nigrostriatal degeneration.

Fig. 8. Experiment 3: TH-IR. Mean (FSEM) number of TH-IR cells per

square millimeter in the SNc (A) and VTA (B), 28 days after 6-OHDA lesion

of theMFB. ANOVAs (Drug� Side) revealed significant main effect of drug

[SNc, F(1,10) = 7.25, P = .02; VTA, F(1,10) = 5.22, P = 0.04], side [SNc,

F(1,10) = 53.26, P < 0.0001; VTA, F(1,10) = 196.17, P < 0.0001], and Drug

� Side interaction [SNc, F(1,10) = 4, 11, P = 0.07; VTA, F(1,10) = 6.46, P =

0.03]. Unpaired t tests conducted to analyze simple main effects revealed that

the number of TH-IR cells remaining on the side of the lesion was higher in

esc/amph group (*P < 0.05; Bonferroni corrected).

Fig. 7. Experiment 3:Mean (FSEM)DA,DOPAC, andHVA (Ag/mg protein)

in striatal tissue punches taken postmortem from the lesioned (L) and

nonlesioned (NL) sides of the brain at 28 days after 6-OHDA lesion of the

MFB. ANOVAs revealed significant effect of side [DA, F(1,10) = 74, 36, P <

0.001; DOPAC, F(1,10) = 86.97, P < 0.0001; HVA, F(1,10) = 56.87, P <

0.0001]. There was no effect of prelesion treatment with the escalating-dose

regimen of amphetamine on 6-OHDA-induced depletions of DA and its

metabolites on the side of the lesion.

I.A. Moroz et al. / Experimental Neurology 189 (2004) 78–93 89

Acutely, amphetamine, unlike other stimulants such as

cocaine, induces DA release in both cell body and terminal

regions through its actions at the dopamine transporter (DAT;

Povlock and Schenk, 1997; Seiden et al., 1993). The

importance of DAT in the acute effects of amphetamine on

dopamine release has raised previously the possibility that

adaptations in DAT function might underlie the development

of sensitization after repeated drug exposure. Similarly, in

light of findings showing that DAT plays a crucial role in the

neurodegenerative responses to dopaminergic neurotoxins,

and that processes that inhibit DAT activity can ameliorate

this neurotoxicity (for a review, see Fleming et al., in press;

Miller et al., 1999), it is tempting to speculate that the

behavioral sparing observed in the group exposed to the 2-

week escalating-dose regimen of amphetamine before

lesioning might have occurred as an effect of sustained

down-regulation of DAT resulting from the repeated expo-

sure of this group to amphetamine before lesioning. Inter-

estingly, however, unlike cocaine (Letchworth et al., 1997,

2001; Pilotte et al., 1994), repeated amphetamine adminis-

tration has been reported not to affect striatal DA uptake or

DAT binding (Allard et al., 1990; Mintz et al., 1994; Persico

et al., 1993), and on the contrary modest increases in DAT

mRNA have been observed (Lu and Wolf, 1997; Shilling et

al., 1997). It can be noted, as well, that in studies done in

vitro in PC12 cells, amphetamine has been found both to

sensitize dopamine release and to cause neurite outgrowth,

but not to have any residual effects on either dopamine

uptake or binding to the transporter (Kantor et al., 2002; Park

et al., 2002). Furthermore, unlike the findings showing

reduced nigrostriatal neurodegeneration when neurotoxins

were administered in the presence of DAT-uptake inhibitors

(Clarke and Reuben, 1995; Ricaurte et al., 1985) or in DAT

knockout mice (Gainetdinov et al., 1997; Sanghera et al.,

1997), the results of Experiments 2 and 3 revealed no

neuroprotection of striatal or SN tissue levels of DA or its

metabolites in the group of rats exposed to the escalating-

dose regimen of amphetamine administered 7 days before

lesioning. Thus, the behavioral sparing observed in that

group cannot be attributed to a different degree of striatal

tissue DA depletion.

In Experiment 3, using in vivo microdialysis, it was

found that behavioral sparing in rats exposed to the esca-

lating-dose regimen of amphetamine before lesioning was

accompanied by normalization of basal extracellular levels

of DA in terminal striatal regions observed as early as 11–

14 days after lesioning. Specifically, the concentration of

DA in dialysate from the striatum ipsilateral to the 6-OHDA

lesion was as high as that from the contralateral striatum in

rats treated with escalating doses of amphetamine before

lesioning but was significantly lower in rats treated with

saline during the same period. At the same time, significant

reduction of the basal levels of DOPAC and HVA in the

ipsilateral striatum, as well as depletion of striatal tissue

levels of DA, clearly indicated the presence of lesions in

both groups. The finding that behavioral sparing was not

accompanied by higher tissue levels of DA in Experiments 2

and 3 is in agreement with earlier microdialysis studies

showing that the gradual process of behavioral recovery

from partial 6-OHDA lesions is accompanied by normali-

zation of extracellular DA levels, but not tissue DA levels in

the striatum (Robinson and Whishaw, 1988; Robinson et al.,

1994). In particular, extracellular DA levels in the striatum

were reduced 4 days after the lesion when ipsilateral turning

predominated but were normalized 3–4 weeks later when

turning was no longer asymmetric. The significance of this

finding lies in the fact that even though a number of

presynaptic compensatory adaptations within the DA neu-

rons, such as increased DA metabolism, synthesis, and

release, proposed to play a role in behavioral recovery have

been found to be maximal within 3 days following such

lesions, behavioral recovery was not complete until 3–4

weeks after lesioning (Altar et al., 1994). Thus, the gradual

normalization of extracellular DA demonstrated by Robin-

son et al. (1994) seemed more critical for behavioral

recovery. The present findings of behavioral sparing and

normalization of extracellular DA being evident as early as

11–14 days after lesioning in rats treated with an escalating-

dose regimen of amphetamine before lesioning suggest that

exposure to this regimen may accelerate the development of

compensatory changes in the DA neurons that typically

accompany behavioral recovery. The present results are

particularly striking since, unlike in Robinson et al.

(1994), the lesions in Experiments 2 and 3 were made in

the MFB and not in the SN. In contrast to SN lesions, after

which behavioral recovery has been reported to occur

spontaneously (Emmi et al., 1996, 1997; Robinson and

Whishaw, 1988; Robinson et al., 1994), MFB lesions are

known to produce more extensive striatal DA depletions

(Costall et al., 1976), greater losses of TH-IR neurons in the

SN (Carman et al., 1991; Perese et al., 1989), and more

devastating behavioral deficits from which animals never

recover (Carman et al., 1991; Kirik et al., 1998; Lee et al.,

1996) unless interventions such as forced use of the im-

paired side of the body (Tillerson et al., 2001, 2002) or

wheel running (Tillerson et al., 2003) are employed.

Further, the results of Experiment 3 revealed that rats

exposed to the escalating-dose regimen of amphetamine

before lesioning had reduced lesion-induced loss of TH-IR

cells in the ipsilateral DA cell body regions, SNc and VTA,

compared to rats exposed to saline, indicating a partial

neuroprotective effect of prelesion treatment with escalating

doses of amphetamine. It is important to note, however, that

at the same time, these rats had comparably large depletions

of striatal tissue DA (Experiment 2 and 3) and SN tissue DA

(Experiment 3) as rats exposed to saline during the same

period. Another possible explanation for the reduced loss of

TH-IR cells that we observed 28 days after lesioning is that

it may reflect the return of the dopaminergic phenotype

observed 32 weeks after lesioning by Bowenkamp et al.

(1996) and Finkelstein et al. (2000). Although the functional

significance of this reduced loss of TH-IR cells is unknown

I.A. Moroz et al. / Experimental Neurology 189 (2004) 78–9390

at the present time, it may reflect compensatory changes in

the functioning of the remaining DA cells. As mentioned

earlier, the enduring enhancement of function within the

remaining DA neurons after partial lesions of the nigros-

triatal DA system is reminiscent of the enhanced DA

response seen in sensitization to amphetamine. Specifically,

behavioral sensitization is accompanied by augmented ex-

tracellular DA overflow in striatal regions in response to

subsequent challenges, such as exposure to drugs or stres-

sors, in the absence of changes in tissue levels of DA

(Robinson and Becker, 1986). Importantly, the events that

lead to enhanced DA functioning are initiated by the actions

of psychostimulants, such as amphetamine, in the DA cell

body regions (Cador et al., 1995; Kalivas and Weber, 1988,

Vezina, 1993; Vezina and Stewart, 1990) and may include,

among other factors, sustained increase in astrocytic FGF-2

expression in these regions (Flores and Stewart, 2000a,b;

Flores et al., 1998). Thus, possibly the reduced loss of TH-

IR cells in the SNc and VTA of rats exposed to the prelesion

treatment with escalating doses of amphetamine reflects a

local protection of these cells as a result of the ability of this

drug treatment to induce increases in the expression of one

or more neurotrophic factors such as FGF-2.

The idea that manipulations given before brain injury

can be protective is not unprecedented. Numerous studies

have shown that exogenous delivery of FGF-2, BDNF, or

GDNF before 6-OHDA lesions prevents the development

of lesion-induced behavioral deficits and protects against

loss of DA neurons and striatal DA innervation (Altar et

al., 1994; Choi-Lundberg et al., 1998; Mandel et al., 1997;

Shults et al., 1996, 2000). Recently, it was found that

forced use of one forelimb for 7 days before 6-OHDA

lesions led to sparing of limb use and attenuation of DA

loss in striatal tissue (Cohen et al., 2003). Interestingly,

forced use of one forelimb in itself increases FGF-2-IR in

the SNc and VTA in both hemispheres (unpublished

observations; Moroz et al., 2002) and GDNF in the

striatum contralateral to the overused forelimb (Cohen et

al., 2003). In the present experiment, exposure to the

escalating-dose regimen of amphetamine known to in-

crease the expression of FGF-2 in both the DA cell bodies

and terminals (Flores and Stewart, 2000b) led to behav-

ioral sparing after 6-OHDA lesions, whereas the exposure

to three injections of amphetamine, found previously to

increase FGF-2 expression only in the cell body regions of

DA neurons (Flores et al., 1998), did not. This finding

appears to suggest that increased expression of FGF-2, and

possibly other neurotrophic factors, in the terminal regions

of DA neurons may be more critical for behavioral

recovery. Support for this idea comes from studies show-

ing that preservation of normal motor functions in the 6-

OHDA lesion model requires intrastriatal, but not nigral,

administration of GDNF (Kirik et al., 2000; Shults et al.,

1996; Sullivan et al., 1998). We emphasize, however, that

the direct involvement of neurotrophic factors in the

beneficial effects of prelesion exposure to the escalating-

dose regimen of amphetamine or prelesion forced use of

one limb remains to be demonstrated.

Another explanation for the behavioral sparing induced by

previous exposure to the escalating-dose regimen of amphet-

amine is its effect on the dendritic morphology of postsyn-

aptic neurons (Li et al., 2003; Robinson and Kolb, 1997,

1999). Such morphological changes could represent a reor-

ganization of synaptic inputs onto these neurons. Conse-

quently, the prophylactic effects of exposure to the escalating-

dose regimen of amphetamine before 6-OHDA lesions of the

nigrostriatal DA neurons found in the present experiments

could be mediated by the amphetamine-induced increases in

neurotrophic factors (Flores and Stewart, 2000b) and the

enhanced synaptic transmission between the surviving DA

input neurons and their striatal and cortical targets.

Interestingly, postinjury administration of amphetamine

has been shown to enhance functional recovery in patients

suffering from stroke and in animals following cortical

lesions or ischemia (Feeney, 1997; Gladstone and Black,

2000; Goldstein, 2000). Multiple mechanisms have been

proposed to underlie this therapeutic effect of amphetamine,

including neuritogenesis, synaptogenesis (Stroemer et al.,

1998), and enhancement of noradrenergic transmission

(Feeney, 1997; Gladstone and Black, 2000). Importantly,

behavioral recovery observed in ischemic animals treated

with amphetamine correlates with increased expression of

markers of neuronal remodeling, such as growth associated

protein-43 (GAP-43) and synaptophysin (Stroemer et al.,

1995). As argued earlier, modifications of neuronal archi-

tecture and synaptic connectivity are likely to be mediated

via actions of neurotrophic factors. In fact, there is evidence

that exogenous administration of FGF-2 after the onset of

ischemia enhances behavioral recovery without reducing the

infarct volume (Kawamata et al., 1996). Instead, FGF-2

administration is associated with a selective increase in

GAP-43 expression (Kawamata et al., 1997).

In summary, we report that exposure to a 2-week

escalating-dose regimen of amphetamine before unilateral

6-OHDA lesions prevents asymmetries of forelimb use and

turning behavior and leads to normalization of the basal

extracellular DA in striatal dialysate on the side of a

unilateral 6-OHDA lesion in the absence of significant

protection of striatal tissue DA levels. Although the mech-

anisms mediating this effect remain to be elucidated, pos-

sibly this prelesion exposure to the escalating doses of

amphetamine leads to behavioral sparing after subsequent

6-OHDA lesions via acceleration of the development of

compensatory changes in the DA neurons that typically

accompany behavioral recovery. Furthermore, the endoge-

nous increases in the expression of neurotrophic growth

factors previously observed after exposure to the 2-week

escalating-dose regimen of amphetamine may play a key

role in the enhanced responsiveness of the DA system to a

subsequent 6-OHDA insult and the resulting behavioral

sparing. Although the findings do not speak directly to the

therapeutic use of amphetamine to facilitate recovery after

I.A. Moroz et al. / Experimental Neurology 189 (2004) 78–93 91

injury, they point to mechanisms that may be involved. For

example, in agreement with epidemiological data, emerging

evidence from animal models of neurogenerative diseases,

including Parkinson’s disease (PD), strongly suggest that

experiential interventions, such as exercise, enriched envi-

ronment housing, and dietary restriction, offer substantial

protection against the behavioral and neurochemical deficits

observed in these models (see Bezard et al., 2003; and

reviews by Mattson, 2000; Smith and Zigmond, 2003).

Interestingly, this protection has been demonstrated to be

due, at least in part, to the ability of these interventions to

increase availability of neurotrophic factors, such as BDNF,

GNDF, and FGF-2, in the areas affected by the injury. Along

similar lines, epidemiological data reporting an apparent

protective effect of cigarette smoking on the risk of PD have

been extended by experimental findings from animal mod-

els of PD suggesting that regional increases in neurotrophic

factors, BDNF and FGF-2, serve as a possible mechanism

by which (�)-nicotine protects from experimental parkin-

sonism (Belluardo et al., 1998; Blum et al., 1996; Maggio et

al., 1997). Thus, with the progress made in early detection

of neurodegenerative diseases, experiential and pharmaco-

logical interventions known to increase levels of neuro-

trophic factors in the brain may be used prophylactically to

slow down or even prevent the neurodegenerative cascade

and the consequent motor dysfunction associated with PD.

Acknowledgments

We thank Susan Ajersch and Heshmat Rajabi for their

assistance with behavioral testing and tissue processing in

Experiments 2 and 3. We also thank Gabriela Redwine for

editorial help. This project was supported by grants to JS from

Canadian Institutes of Health Research and Le Fonds

Quebecois de la recherche sur la nature et les technologies

(FRNTQ) and by the National Institutes of Health grant to TS.

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