unilateral striatal dopamine depletion: time-dependent effects on cortical function and behavioural...

Unilateral striatal dopamine depletion: time-dependenteffects on cortical function and behavioural correlates

Heinz Steiner* and Stephen T. KitaiDepartment of Anatomy and Neurobiology, University of Tennessee, College of Medicine, Memphis, TN 38163, USA

Keywords: basal ganglia, c-fos, cortex, nigrostriatal, rat, striatum, zif 268

Abstract

Previously, we showed that unilateral blockade of D1 dopamine receptors in the striatum inhibits immediate-early geneexpression bilaterally throughout large parts of the cortex, including sensory-evoked expression in the barrel cortex. To further

investigate this dopamine regulation of cortical function, we examined the effects of dopamine depletion on cortical gene

regulation and behavioural correlates. Two days after unilateral infusion of 6-hydroxydopamine into the midbrain, rats displayed a(to some degree) bilateral reduction in cortical zif 268 expression that was more pronounced on the lesioned side. This decrease

was found across motor, somatosensory, insular and piriform, but not cingulate, cortex, similar to the effects of blockade of

striatal D1 receptors. Furthermore, whisker stimulation-evoked c-fos and zif 268 expression in the barrel cortex ipsilateral to the

lesion was also attenuated by acute dopamine depletion. These cortical de®cits were accompanied by a breakdown ofspontaneous behaviours in an open-®eld test. In contrast, 21 days after dopamine depletion, both basal and sensory-evoked

gene expression in the cortex were near-normal. This cortical recovery was paralleled by recovery in locomotion and in sensory-

guided behaviour (scanning) related to the hemisphere contralateral to the lesion, but not in scanning by the dopamine-depletedhemisphere. Our results suggest that striatal dopamine exerts a widespread facilitatory in¯uence on cortical function that is

necessary, but not suf®cient, for normal behaviour. Moreover, the mechanisms mediating this cortical facilitation appear to be

subject to substantial neuroplasticity after dopamine perturbation.

Introduction

Interactions between the basal ganglia and the cerebral cortex are

critical for the generation of goal-directed behaviour. Dopamine in

the striatum regulates information ¯ow in cortico-basal ganglia±

cortical circuits. Striatal dopamine has been implicated in functions

as diverse as movement initiation, response selection, habit learning,

cognition and reward, and is associated with various basal ganglia/

cortical disorders, including Parkinson's disease, schizophrenia and

drug addiction (Albin et al., 1989; Alexander et al., 1990; Carlsson &

Carlsson, 1990; DeLong, 1990; Graybiel, 1995; Robbins et al., 1998;

Schultz et al., 1998; Redgrave et al., 1999; Sarter & Bruno, 1999;

Hollerman et al., 2000). Current models of basal ganglia±cortical

circuits propose, for example, that dopamine hypofunction or

hyperfunction in the striatum results in underactive or overactive

thalamocortical inputs and, consequently, in hypokinetic or hyper-

kinetic disorders, respectively (Penney & Young, 1983; Albin et al.,

1989; Alexander et al., 1990; DeLong, 1990). Results of various

functional studies principally support these models. Thus, dopamine

agonist treatments that produce behavioural activation also result in

increased cortical activation, as indicated by functional markers such

as the expression of immediate-early genes in the cortex (Paul et al.,

1992; Dilts et al., 1993; Daunais & McGinty, 1994; Steiner & Gerfen,

1994; Wang & McGinty, 1995; LaHoste et al., 1996; Badiani et al.,

1998). However, in these studies, the observed changes in cortical

gene regulation were generally widespread and not limited to the

frontal/premotor areas emphasized as cortical targets of basal ganglia

output in the models (Alexander et al., 1986; Alexander & Crutcher,

1990; Alexander et al., 1990). Interpretation of these ®ndings is

dif®cult, however, because these studies used systemic drug

treatments, thus precluding conclusions regarding the site of drug

action.

Recently, we showed that local manipulation of basal ganglia

output by intrastriatal drug administration produced similar wide-

spread cortical effects. Thus, blocking striatal dopamine action by

intrastriatal infusion of an opioid agonist attenuated immediate-early

gene expression in the cortex (Steiner & Gerfen, 1995), indicating a

role for striatal dopamine receptors in such cortical gene regulation.

A more detailed investigation of the cortical regions affected and the

dopamine receptor subtypes involved demonstrated that unilateral

blockade of striatal D1-type receptors inhibited immediate-early gene

expression induced by the D1/D2 receptor agonist apomorphine

bilaterally throughout motor, somatosensory, insular and piriform

cortex (Steiner & Kitai, 2000b). Furthermore, blocking striatal D1

receptors also inhibited whisker stimulation-evoked immediate-early

gene expression in the barrel cortex (Steiner & Kitai, 2000b). These

®ndings indicate that stimulation of striatal D1 receptors facilitates

cortical function.

In the present study, we further investigated this dopamine

regulation of cortical function by assessing the effects of dopamine

depletion on gene expression in the cortex and behavioural correlates.

The transcription factors/immediate-early genes zif 268 and c-fos

Correspondence: Dr Heinz Steiner, at *present address belowE-mail: steinerh@®nchcms.edu

*Present address: Department of Cellular and Molecular Pharmacology, FinchUniversity of Health Sciences/The Chicago Medical School, 3333 Green BayRoad, North Chicago, IL 60064, USA

Received 12 March 2001, revised 20 August 2001, accepted 24 August 2001

European Journal of Neuroscience, Vol. 14, pp. 1390±1404, 2001 ã Federation of European Neuroscience Societies

were used as functional markers (e.g. Sharp et al., 1993; Chaudhuri,

1997; Herdegen & Leah, 1998). In the ®rst experiment, we mapped

time-dependent effects of a 6-hydroxydopamine (6-OHDA) lesion on

`basal' expression of zif 268 in various cortical regions. In the second

experiment, 6-OHDA effects on whisker stimulation-evoked c-fos

and zif 268 expression in the barrel cortex were examined. These

cortical gene regulation effects were compared with behavioural

changes after dopamine depletion, especially with an index of

whisker utilization (thigmotactic scanning). Our results show that

acute unilateral dopamine depletion attenuated basal and sensory-

evoked immediate-early gene expression in the cortex, similar to

unilateral blockade of striatal D1 receptors. However, these effects

were transient and as animals regained near-normal cortical gene

expression they recovered from some, but not all, behavioural de®cits

produced by the lesion. A preliminary account of these results has

been presented in abstract form (Steiner & Kitai, 2000a).

Materials and methods

Subjects

A total of 56 male Sprague-Dawley rats (170±210 g; Sasco, St. Louis,

MO, USA) were used in this study, 28 in Experiment 1 and 28 in

Experiment 2. In both experiments, the animals were assigned to one

of four groups (n = 5±8 each) that received either an infusion of

6-OHDA or vehicle into the midbrain, and were tested either 2 or

21 days after the infusion. The rats were housed in groups of two to

three under standard laboratory conditions. They had free access to

food and water, and were maintained under a 12-h light : 12-h dark

cycle (lights on at 07.00 h). The experiments were carried out

between 13.00 and 16.00 h. The procedures used in this study were

approved by the Animal Care and Use Committee of the University

of Tennessee, Memphis.

Dopamine depletion

The rats were anaesthetized with equithesin (3.5 mL/kg) and placed

in a David Kopf stereotaxic frame. The neurotoxin 6-OHDA

(6-OHDA HBr, Sigma, St. Louis, MO, USA; 16 mg in 2 mL of

0.9% NaCl/0.02% ascorbic acid), or vehicle, was infused with a pump

(rate 0.5 mL/min) into the area of the rostral border of the right

substantia nigra. The coordinates used for the tip of the infusion

cannula were (relative to the interaural line): A, +4.5; L, 1.5; V, +2.0

with the incisor bar set at ±2.5 mm (Paxinos & Watson, 1998). All

rats received an injection of desipramine (25 mg/kg, i.p.; Sigma) 30±

45 min before the infusion.

Behavioural test

Two days (`acute') or 21 days after the 6-OHDA or vehicle infusion,

behavioural effects were examined during a 10-min test session in a

novel open ®eld (60 3 60 3 40 cm, with lines dividing the ¯oor into

3 3 3 squares). The behaviour was video-taped and assessed from

the tapes by an experimenter who was unaware of the treatment. The

behavioural analysis started 30 s after the animal was placed into

the centre of the open ®eld. The following behavioural items

were measured: distance traveled (number of lines crossed with

all four feet) and turning towards the left or right side (number of

half turns with a diameter < 20 cm). As a measure of whisker

utilization, we assessed thigmotactic scanning (Steiner et al., 1986)

with the left or right side (measured as the number of lines crossed

while walking along the wall with the snout within 5 cm of the wall)

(see Fig. 2D).

Whisker stimulation

In Experiment 2, we tested whisker stimulation-evoked immediate-

early gene expression in the barrel cortex ipsilateral to the dopamine

depletion at 2 or 21 days after the 6-OHDA infusion. Thus, following

the open-®eld test, one mystacial whisker (C2 on the left side of the

snout) was stimulated using procedures described previously in detail

(Melzer & Steiner, 1997; Steiner & Kitai, 2000b). In short, 30 min

prior to the start of the stimulation a steel ®lament (length 6 mm,

diameter 0.3 mm) was glued to whisker C2 (centre of ®lament

» 8 mm from the skin) under light Metofane anaesthesia. The distal

end of whisker C2 was clipped. All other mystacial whiskers on both

sides of the face were clipped close to the skin. The rat was then

allowed to recover. Whisker C2 was stimulated by exposing the rat to

a pulsating magnetic ®eld (Van der Loos stimulator; Melzer et al.,

1985). The magnetic ®eld (mean strength 10.8 mT, RMS) was

produced by a copper coil powered by a solid-state relay (George

Dold, Research Services Branch, NIMH; based on the design by

Melzer et al., 1985). With the longitudinal axis of the magnetic ®eld

orientated horizontally, the ®eld pulses de¯ect whiskers mostly in

rostrocaudal directions. A pulse rate of » 8 Hz was chosen in order to

mimic the whisking frequency of a whisking (`snif®ng') rat. The

awake rat was placed into the acrylic stimulation chamber (cylinder,

15 cm in length, 14 cm in diameter) inside the coil and stimulated for

15 min.

Tissue preparation

Immediately following the open-®eld test, or 10 min after whisker

stimulation, the rat was killed with CO2. The brain was rapidly

removed, frozen in isopentane cooled on dry ice, and stored at ±20 °C

until cryostat sectioning. Coronal sections (12 mm) through striatum

and midbrain or, for the stimulated brains, tangential sections through

the somatosensory cortex followed by coronal sections through

striatum and midbrain, were collected. The sections were thaw-

mounted onto glass slides twice coated with gelatin, dried on a

hotplate and stored at ±20 °C. In preparation for in situ hybridization

histochemistry, the sections were, at room temperature, ®xed in 4%

paraformaldehyde/0.9% saline for 10 min, incubated in a fresh

solution of 0.25% acetic anhydride in 0.1 M triethanolamine/0.9%

saline (pH 8.0) for 10 min, dehydrated, defatted for 2 3 5 min in

chloroform, rehydrated and air-dried. The slides were then stored at

±20 °C until hybridization. Every sixth tangential section through the

somatosensory cortex was ®xed and stained for cytochrome oxidase

activity to locate barrel C2 in layer IV (Melzer & Steiner, 1997). In

addition, sets of striatal sections were processed for tyrosine

hydroxylase immunohistochemistry (Steiner et al., 1999) to assess

the loss of dopamine terminals.

In situ hybridization histochemistry

Oligonucleotide probes (48-mers; Life Technologies, Baltimore, MD,

USA) were labelled with [35S]-dATP (Steiner & Kitai, 2000b). The

probes had the following sequence: c-fos, complementary to bases

1227±1274, GenBank accession number X06769; zif 268, bases 352±

399, M18416; enkephalin, bases 436±483, M28263; tyrosine

hydroxylase, bases 1441±1488, M10244. Labelled probe (» 3 3

106 c.p.m.) in 100 mL of hybridization buffer was added to each

slide. The sections were coverslipped and incubated at 37 °C

overnight. After incubation, the slides were ®rst rinsed in four

washes of 13 saline citrate (150 mM sodium chloride, 15 mM sodium

citrate). Then they were washed three times 20 min each in 23 saline

citrate/50% formamide at 40 °C, followed by two washes 30 min

each in 13 saline citrate at room temperature. After a brief water

Dopamine depletion and cortical function 1391

ã 2001 Federation of European Neuroscience Societies, European Journal of Neuroscience, 14, 1390±1404

rinse, the sections were air-dried and then apposed to X-ray ®lm

(BioMax MR-2, Kodak) for 3 days to 3 weeks.

Analysis of autoradiograms

Enkephalin expression was measured in coronal sections from a

rostral striatal level (`rostral', at » 10.5 mm rostral to the interaural

line; Paxinos & Watson, 1998) and a mid-striatal level (`middle',

9.5 mm), and also monitored at two more caudal striatal levels (8.2±

7.6 mm). Basal zif 268 expression was analysed in sections from the

rostral and middle striatal levels, as well as in coronal sections

collected at the level of the barrel ®eld (`caudal', 7.2±6.7 mm).

Stimulation-evoked c-fos and zif 268 expression was investigated in

tangential sections through the right somatosensory cortex that were

adjacent to sections containing barrel C2 (layer IV), as determined by

cytochrome oxidase histochemistry. Tyrosine hydroxylase immunor-

eactivity was assessed in rostral and middle striatal sections, and

tyrosine hydroxylase mRNA expression was determined at several

rostrocaudal levels through the substantia nigra (4.3±3.0 mm).

Gene expression was measured in the following areas (Paxinos &

Watson, 1998). Rostral and middle striatal levels: striatum, nucleus

accumbens, olfactory tubercle, total cortex, cingulate, `sensorimotor'

(including M2, M1, S1, S2, insular regions), piriform cortex. Caudal

level: total cortex, retrosplenial, motor, hindlimb/forelimb, `somato-

sensory' (rest S1, S2), insular, piriform cortex. In stimulated animals,

gene expression was measured in barrel C2 and in the surrounding

barrels (C1, C3, B2, D2).

Levels of gene expression were determined by densitometry on

®lm autoradiograms, using a Macintosh-based image analysis system

(NIH Image, Wayne Rasband, NIMH). The `mean density' values

presented are background-corrected: for coronal sections, mean

density over gray matter minus mean density over white matter; for

tangential sections, mean density in C2 minus averaged mean density

in surrounding barrels. The illustrations of ®lm autoradiograms

displayed in Figs 1, 3 and 6 are computer-generated images, and are

contrast-enhanced where necessary. Maximal hybridization signal is

black.

Statistics

Treatment effects on gene expression and behaviour were determined

with three-factor [main effects: lesion, time, hemisphere/side (with-

in)] or two-factor ANOVAs (main effects: lesion, time) using the

Statistica software package (Statistica 4.1 for Macintosh, StatSoft,

Tulsa, OK, USA). Differences between individual groups were

described with Newman±Keuls post hoc tests.

noninfused6-OHDA

noninfusedvehicle

striatum

2 days 21 daysV 6V 6

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6-OHDA 6-OHDA 6-OHDA

A C

B

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tyrosine hydroxylase enkephalin

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FIG. 1. Effects of unilateral 6-OHDA lesion on tyrosine hydroxylase and enkephalin expression 2 days (`acute') and 21 days after the lesion. (A) Examples ofcoronal sections from the rostral and middle striatum stained for tyrosine hydroxylase immunoreactivity are shown for animals killed 2 or 21 days after the6-OHDA infusion into the right midbrain. (B) Illustrations of ®lm autoradiograms depict tyrosine hydroxylase mRNA expression in sections from threerostrocaudal levels [4.3±3.4 mm rostral to the interaural line (Paxinos & Watson, 1998)] through the midbrain for such animals. (C) Examples of ®lmautoradiograms show enkephalin expression in sections from the rostral and middle striatum. The arrows point to the lateral striatal zone that displayed thestrongest increase in enkephalin expression 2 days after the lesion. (D) Mean density values (mean 6 SEM, arbitrary units) for enkephalin expressionmeasured across the total striatum at the middle level in the noninfused and infused hemispheres are given for rats that received an infusion of vehicle (V) or6-OHDA (6) into the right midbrain (n = 5±8). **P < 0.01 vs. noninfused or as indicated; aaP < 0.01 vs. acute. S, striatum; c, nucleus accumbens core; sh,nucleus accumbens shell; SNc, substantia nigra pars compacta; VTA, ventral tegmental area.

1392 H. Steiner and S. T. Kitai


Results

Dopamine depletion characterized by tyrosine hydroxylaseimmunoreactivity and mRNA, and enkephalin expression:2 days vs. 21 days

Twenty-one days after unilateral 6-OHDA infusion, tyrosine

hydroxylase immunoreactivity in the striatum (Fig. 1A) and mRNA

in the substantia nigra (Fig. 1B) were undetectable on the side of the

infusion in all animals. Enkephalin expression was increased

throughout the striatum ipsilateral to the infusion, as compared to

the contralateral striatum or to vehicle-infused controls (Fig. 1C;

Fig. 1D shows numerical data for the middle striatal level from

animals in Experiment 1). These results indicate a near-total

dopamine depletion at this time point (Li et al., 1990; Nisenbaum

et al., 1996). In contrast, 2 days after 6-OHDA administration, both

tyrosine hydroxylase immunoreactivity and mRNA were only partly

eliminated. In the substantia nigra, tyrosine hydroxylase mRNA was

generally mostly reduced in rostral and medial parts and less or little

in lateral and caudal parts (Fig. 1B). In the striatum, immuno-

reactivity was preferentially reduced in medial areas (Fig. 1A). At

this time point, changes in enkephalin mRNA levels poorly matched

those in tyrosine hydroxylase distribution. Thus, a small but

statistically signi®cant increase in enkephalin expression was seen

across the striatum (Fig. 1D), but this increase was most robust in

lateral parts (Fig. 1C). A dissociation between enkephalin expression

and tyrosine hydroxylase levels was also apparent in the nucleus

accumbens. Thus, minimal or no changes in enkephalin expression

were noted at any time (Fig. 1C; numerical data not shown), despite

an apparently near-total loss of tyrosine hydroxylase immunoreac-

tivity at 2 days (core) and 21 days (core and shell) (Fig. 1A), and of

tyrosine hydroxylase mRNA in the ventral tegmental area at 21 days

(Fig. 1B).

Open-®eld behaviour

Figure 2 depicts lesion effects on open-®eld behaviour in Experiment

1. Locomotor activity (distance traveled) did not differ between the

vehicle-infused groups tested 2 days or 21 days after the infusion

(Fig. 2A). Two days after unilateral 6-OHDA administration, the rats

showed very little forward locomotion, but displayed occasional

turning towards the side of the lesion. This was re¯ected by an almost

complete lack of line crossings (1% of vehicle controls) (Fig. 2A) and

a moderate number of ipsiversive half turns (Fig. 2B). The rats tested

21 days after the lesion still showed a reduced amount of locomotion

(42% of controls), but their crossing rate was statistically signi®cantly

increased compared to the 2-day group (Fig. 2A). The amount of

FIG. 2. Open-®eld behaviour 2 days (acute) and 21 days after the 6-OHDA infusion into the right midbrain. (A) The distance traveled (number of crossings,mean 6 SEM) during the 10-min test, given as the total (left) and for 2-min blocks (right), is shown for rats that received a vehicle (V) or a 6-OHDAinfusion (6) (n = 5±7). (B) Turning behaviour (total number of net half turns) is depicted for these animals. Positive values indicate turning ipsiversive to thelesion. (C) Scanning (number of crossings inside a 5-cm-wide zone along the wall, indicated by the broken line in D) is shown for the sides of the facefunctionally related to the noninfused and vehicle (V)- or 6-OHDA (6)-infused hemispheres. (D) Schematic illustration of the dominant behaviouralasymmetries seen in the open ®eld 21 days after unilateral dopamine depletion, turning ipsiversive to the lesion (left) and scanning with the whisker siderelated to the noninfused hemisphere (i.e. the side ipsilateral to the 6-OHDA lesion) (right). **P < 0.01 as indicated; aaP < 0.01, aP < 0.05, vs. acute.



turning ipsiversive to the lesion was somewhat higher at 21 days than

at 2 days (Fig. 2B).

The animals that received a vehicle infusion into the midbrain did

not display signi®cant asymmetries in thigmotactic scanning, either

2 days or 21 days after the infusion (Fig. 2C). The behavioural

breakdown 2 days after the 6-OHDA infusion also resulted in an

almost complete elimination of scanning at this time point (Fig. 2C).

The animals tested 21 days after the 6-OHDA lesion showed a

scanning asymmetry. Thus, scanning with the side of the face

functionally related to the dopamine-depleted hemisphere (i.e.

scanning with the left side) was not or minimally increased compared

to 2-day animals. In contrast, scanning with the side associated with

the noninfused hemisphere (i.e. scanning with the right whiskers) had

almost completely recovered (Fig. 2C). Principally, the same time-

dependent effects of dopamine depletion on scanning behaviour were

found in Experiment 2 (Fig. 6C); the other behavioural effects in

these latter animals were also similar (not shown).

Reduced basal zif 268 expression in the cortex after acute,but not prolonged, unilateral dopamine depletion

Effects of dopamine depletion on basal zif 268 expression are shown

in Figs 3±5 and Table 1. Acute dopamine depletion (2-day group)

zif 268 expression in total cortex

noninfused6-OHDA

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zif 268 expression after "acute" dopamine depletion (2 days)rostral middle caudal

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FIG. 3. Expression of zif 268 mRNA in the cortex, striatum and nucleus accumbens after unilateral dopamine depletion. (A) Illustrations of ®lmautoradiograms depicting zif 268 expression in coronal sections from rostral (left), middle (middle) and caudal (right) striatal levels are shown for rats killed2 days after the 6-OHDA infusion into the right midbrain. The arrows point to zones with increased zif 268 expression in the lateral striatum ipsilateral to thelesion. (B) Mean density values (mean 6 SEM) for zif 268 expression measured across the total cortex in middle and caudal sections are given for rats thatwere killed 2 or 21 days after the infusion (n = 5±8). The results are shown as absolute values for the noninfused and vehicle (V)- or 6-OHDA (6)-infusedhemispheres (left), and as relative values, either expressed as percentage of the noninfused side (% NIF) (middle) or as percentage of corresponding values invehicle-infused controls (% C) (right). **P < 0.01, *P < 0.05 vs. noninfused or as indicated; aaP < 0.01 vs. acute; ccP < 0.01, cP < 0.05 vs. vehicle-infusedcontrols.



produced a statistically signi®cant decrease in basal zif 268 mRNA

levels in the ipsilateral cortex, as compared to the noninfused side and

to vehicle-infused controls, on all rostrocaudal levels examined

(Fig. 3A and B). Moreover, a statistically signi®cant reduction in zif

268 expression was also found in the cortex contralateral to the

6-OHDA infusion (noninfused side), although this effect was less

robust. In contrast, at 21 days after the lesion, zif 268 mRNA levels in

the dopamine-depleted and noninfused hemispheres were close to the

levels in vehicle-infused control animals (Fig. 3B). However, there

were also statistically signi®cant effects of the infusion per se (lower

values in vehicle-infused hemisphere compared to noninfused

hemisphere) and general time effects (lower levels at 2 days, e.g. in

noninfused hemisphere of 2-day vehicle-infused animals compared to

corresponding 21-day animals). Some of these effects may be related

to damage produced by lowering the infusion cannula to the ventral

midbrain (Dragunow & Robertson, 1988; Jacobs et al., 1994). To

eliminate the contribution of such effects, we also analysed relative

zif 268 mRNA levels (values expressed as percentage of those in the

noninfused hemisphere, or of those in the corresponding vehicle-

infused controls). This analysis con®rmed a signi®cant, to some

degree bilateral, effect of 6-OHDA on zif 268 expression in the cortex

at 2 days after the neurotoxin infusion, and a subsequent recovery of

this effect (Figs 3B and 5, Table 1).

Our regional analysis showed that this 6-OHDA-induced decrease

in zif 268 mRNA levels was, to a variable degree, present in all

cortical areas examined, with the exception of the cingulate cortex

(Table 1, Figs 4 and 5). The ipsilateral decrease was very robust

across retrosplenial, motor, somatosensory, insular and piriform

cortical areas. The more moderate contralateral reduction was most

robust in the retrosplenial and the somatosensory cortex (Table 1,

Fig. 4C). Notably, the distinct bilateral decrease in the retrosplenial

area was contrasted by a lack of an effect in the rostrally adjacent

cingulate cortex (Fig. 4). Thus, zif 268 mRNA levels in the cingulate

cortex at both rostrocaudal levels examined were unchanged 2 and

21 days after dopamine depletion (Figs 4 and 5A).

Effects on basal zif 268 expression in the striatum andnucleus accumbens

Effects of 6-OHDA on zif 268 expression in striatal areas on three

rostrocaudal levels are depicted in Fig. 3A. Figure 5 compares the

effects in the cortex (middle level) (Fig. 5A±C) with those in the

striatum (middle level) (Fig. 5D and E) and in the nucleus accumbens

(rostral level) (Fig. 5F). When measured across the total striatum, zif

268 mRNA levels were statistically signi®cantly decreased in both

hemispheres at 2 days after the 6-OHDA infusion (Fig. 5D).

However, on the lesioned side a more pronounced decrease medially

was contrasted by a statistically signi®cant increase in a restricted

zone in the most lateral striatum (Figs 3A and 5E). At 21 days after

the lesion, zif 268 mRNA levels in the lateral striatum were not

signi®cantly different from controls. Similar to the medial striatum, in

the nucleus accumbens, zif 268 expression was signi®cantly

decreased bilaterally, at both time points (Fig. 5F). Again, this effect

was somewhat more pronounced on the dopamine-depleted side,

especially at 21 days after the lesion (Fig. 5F). Similar effects were

seen for both core and shell of nucleus accumbens (data not shown).

Changes in zif 268 expression in the olfactory tubercle were similar to

those in the nucleus accumbens (data not shown).

Attenuation of whisker stimulation-evoked immediate-earlygene expression in the somatosensory cortex after acutedopamine depletion

In Experiment 2, we assessed sensory stimulation-evoked immediate-

early gene expression in the dopamine-depleted hemisphere follow-

ing the open-®eld test. These rats received 15-min stimulation of

whisker C2 on the side of the face contralateral to the 6-OHDA

lesion, and c-fos and zif 268 mRNA levels were measured in the

stimulated barrel C2. Acute dopamine depletion produced a pro-

nounced attenuation of whisker stimulation-evoked gene expression

in the barrel cortex ipsilateral to the lesion (Figs 6A and B, and 7).

Thus, for both immediate-early genes, stimulation-evoked mRNA

levels in barrel C2 were signi®cantly reduced compared to those in

the vehicle-infused animals, at 2 days after 6-OHDA infusion.

Especially, c-fos mRNA levels were hardly above background in

most of these animals (Fig. 7B). In contrast, 21 days after the lesion,

whisker stimulation-evoked c-fos and zif 268 expression in barrel C2

did not differ from that in the vehicle controls (Figs 6B and 7A).

Therefore, whisker stimulation-evoked immediate-early gene expres-

sion in the barrel cortex had recovered at a time when the same rats

failed to show signi®cantly improved utilization of these whiskers

(i.e. scanning with the side functionally related to the dopamine-

depleted hemisphere; Fig. 6C).

Using enkephalin expression as a marker, we examined whether

there was a relationship between dopamine depletion-induced

changes in gene expression in the striatum and changes in whisker

stimulation-evoked immediate-early gene expression in the barrel

cortex after the 6-OHDA lesion. Stimulation-evoked expression of

TABLE 1. Expression of zif 268 mRNA in the cortex after dopamine depletion

Cortical area

Expression of zif 268 mRNA

At 2 days At 21 days

Noninfused 6-OHDA Noninfused 6-OHDA

Retrosplenial 78.3 6 5.6³³ 58.5 6 5.3**³³ 96.6 6 6.5²² 91.8 6 8.0²²

Motor 82.2 6 3.7 49.8 6 7.1**³³ 94.6 6 4.6 94.7 6 6.2²²

Hindlimb/forelimb 90.2 6 8.0 47.2 6 4.9**³³ 100.4 6 7.1 90.6 6 7.3²²

Somatosensory 84.1 6 7.0³ 61.0 6 3.7**³³ 103.1 6 8.3² 99.4 6 8.5²²

Insular 84.8 6 2.9 58.3 6 4.9**³³ 91.0 6 7.8 104.0 6 7.9²²

Piriform 93.0 6 4.1 67.7 6 6.4**³³ 96.1 6 9.3 118.8 6 10.4²²

Total cortex 85.2 6 5.1³ 61.0 6 4.0**³³ 98.5 6 7.6²² 99.6 6 7.8²²

Mean density values (mean 6 SEM, in percentage of values in the vehicle-infused controls) measured in various cortical regions on the noninfused and 6-OHDA-infused sides in coronal sections collected at the level of the barrel ®eld (`caudal' level) are given for animals that were killed 2 days or 21 days after 6-OHDAinfusion into the right midbrain (n = 5±8). **P < 0.01 vs. noninfused side; ²²P < 0.01, ²P < 0.05, vs. acute (2 days); ³³P < 0.01, ³P < 0.05 vs. vehicle-infusedcontrols.



c-fos and zif 268 in the stimulated barrels is highly correlated

(Fig. 7A), and thus a similar relationship with enkephalin expression

was seen for the two genes (Fig. 7B and C). Two days after the

6-OHDA infusion, there was a signi®cant negative correlation

between the dopamine depletion-induced increase in enkephalin

expression in the striatum and stimulation-evoked zif 268 expression

in the ipsilateral barrel C2; a similar tendency was seen for c-fos

expression. In contrast, there was no relationship between striatal

enkephalin mRNA levels and such immediate-early gene expression

in the barrel cortex 21 days after the lesion (Fig. 7B and C).

Relationship between neuronal plasticity in the dopamine-depleted striatum and whisker utilization by the contralateralhemisphere

In order to investigate whether there was an association between

neuronal plasticity in the striatum, as exempli®ed by increased

enkephalin expression, and behavioural changes after dopamine

depletion, we examined whether changes in enkephalin expression

were related to changes in whisker use (scanning) 21 days after the

lesion. Our results show that there was a signi®cant positive

correlation between striatal enkephalin mRNA levels ipsilateral to

the lesion and the amount of scanning with the whiskers associated

with the noninfused hemisphere (Fig. 8A). In contrast, there was no

relationship between enkephalin expression and scanning with the

whiskers associated with the dopamine-depleted hemisphere

(Fig. 8B).

Discussion

The purpose of the present study was to determine whether a lesion of

ascending dopamine projections would affect cortical function as

indicated by changes in immediate-early gene expression, and

whether there was a relationship between such changes in cortical

gene regulation and behavioural alterations after dopamine depletion.

Our most signi®cant ®ndings are the following. (i) Acute unilateral

dopamine depletion produced a decrease in basal zif 268 mRNA

levels throughout large parts of the cortex. This decrease was to some

degree bilateral. (ii) Sensory stimulation-evoked immediate-early

gene responses in the barrel cortex ipsilateral to the dopamine

depletion were largely abolished in such animals. (iii) These effects

were time-dependent, as cortical gene expression recovered by

21 days after the lesion. (iv) Inhibition and subsequent recovery of

cortical gene expression were paralleled by a breakdown and partial

recovery of spontaneous behaviour. However, there was a dis-

sociation between gene expression and behaviour related to the

dopamine-depleted hemisphere. Although whisker stimulation-

evoked gene expression in the ipsilateral barrel cortex was near-

normal at 21 days after the lesion, rats did not recover whisker

utilization by this hemisphere. These results suggest that dopamine

exerts a (to some degree) bilateral facilitatory in¯uence on cortical

function that is necessary but not suf®cient for normal behaviour.

Enkephalin and immediate-early gene expression in thestriatum as markers for dopamine depletion and striatalneuron function

Increased enkephalin expression in striatopallidal neurons is one of

the best established changes in striatal gene regulation after unilateral

depletion of nigrostriatal dopamine. Both enkephalin mRNA and

peptide levels have been shown to be elevated (Young et al., 1986;

Voorn et al., 1987; Normand et al., 1988; Gerfen et al., 1990; Engber

et al., 1992; for review, see Steiner & Gerfen, 1998). Increased

expression can be seen within 2±3 days, reaches near-maximal levels

between 7 and 21 days after the lesion (e.g. Nisenbaum et al., 1994),

and is associated with a near-total (> 90%) loss of dopamine tissue

content at that time (Li et al., 1990; Nisenbaum et al., 1996). In our

study, at 21 days after unilateral 6-OHDA infusion, rats displayed

increased enkephalin expression and undetectable levels of tyrosine

hydroxylase immunoreactivity and mRNA in the ipsilateral striatum

and midbrain, respectively, thus indicating near-total dopamine

depletion in these animals.

Early events after 6-OHDA administration are less well studied.

Degeneration of ascending dopamine ®bres occurs over a time period

noninfusedvehicle

noninfused6-OHDA

B cingulate (middle)

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FIG. 4. Regional selectivity in the effects of dopamine depletion on theexpression of zif 268 mRNA in the cortex. (A±C) Mean density values(mean 6 SEM) for expression in the cingulate cortex at (A) the rostral and(B) middle striatal levels and (C) in the retrosplenial cortex at the caudallevel measured in the noninfused and vehicle (V)- or 6-OHDA (6)-infusedhemispheres are shown for rats that were killed 2 or 21 days after theinfusion (n = 5±8). **P < 0.01 vs. noninfused or as indicated; aaP < 0.01vs. acute.



of several hours to several days after the neurotoxin infusion during

which some tyrosine hydroxylase immunoreactivity is still present

(e.g. Zahm, 1991; for review, see Schwarting & Huston, 1996a). In

contrast, impulse ¯ow-dependent dopamine release in the striatum

appears to cease within 20±40 min of 6-OHDA infusion

(Svenningsson et al., 1999). This early loss of dopamine release is

followed closely by increased expression of c-fos and zif 268 in the

striatum (Cole & Di Figlia, 1994; Brog & Zahm, 1995; Svenningsson

et al., 1999; Schuller & Marshall, 2000), which occurs predominantly

in enkephalin/D2 receptor-expressing (striatopallidal) neurons

(Svenningsson et al., 1999; Schuller & Marshall, 2000). As shown

here and before (Svenningsson et al., 1999; Schuller & Marshall,

2000), this immediate-early gene response is most robust in the lateral

striatum, similar to gene induction after acute D2 receptor blockade

(e.g. Steiner & Gerfen, 1999). Such increased immediate-early gene

expression is thought to re¯ect increased activity in these neurons due

to a loss of stimulation of inhibitory D2 receptors. Current models of

basal ganglia function predict that increased activity in striatopallidal

neurons together with reduced activity in striatonigral neurons after

dopamine depletion produce diminished thalamocortical activation

and bradykinesia (e.g. Albin et al., 1989; DeLong, 1990).

Consistently, in our study, basal and sensory-evoked immediate-

early gene expression in the cortex were attenuated at this early time

point.

The early strong immediate-early gene response in the striatum

subsequently subsides to some degree (Schuller & Marshall, 2000),

and 3 weeks after a 6-OHDA lesion, net zif 268 mRNA levels in the

striatum re¯ect a minor increase in expression in striatopallidal

neurons in conjunction with a more pronounced decrease in

expression in striatonigral neurons (Gerfen et al., 1995). Together,

these dopamine release and gene regulation studies indicate that

striatal neuron function is altered within a short time period after

6-OHDA infusion and that these initial effects are subsequently

modi®ed (see also below).

Acute dopamine depletion results in attenuated immediate-early gene expression in the cortex

Earlier studies demonstrated that acute treatments with various

dopamine agonists produce increased expression of immediate-early

genes throughout most of the cortex (Paul et al., 1992; Dilts et al.,

1993; Daunais & McGinty, 1994; Steiner & Gerfen, 1994; Wang &

McGinty, 1995; LaHoste et al., 1996; Badiani et al., 1998; Berke

et al., 1998; Steiner & Kitai, 2000b). Consistently, our present

®ndings show that acute dopamine depletion results in decreased zif

268 mRNA levels in the cortex. The unilateral 6-OHDA lesion

produced robust decreases throughout large parts of the ipsilateral

cortex that reached 40±50% in some areas. In addition, smaller

reductions (10±20%) were seen in some contralateral cortical areas.

Although widespread, this lesion effect was regionally selective, as

attenuated zif 268 expression was found across retrosplenial, motor,

somatosensory, insular and piriform cortex, but not in the cingulate

cortex. Furthermore, sensory-evoked responses (i.e. whisker stimu-

A cingulate cortex B sensorimotor cortex C piriform cortex

D total striatum E lateral striatum F nucl. accumbens

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mea

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(% C

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dens

ity (%

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mea

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(% C

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dens

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FIG. 5. Differential effects of dopamine depletion on zif 268 expression in cortical and striatal regions. (A±F) Mean density values (mean 6 SEM) expressedas percentage of corresponding values in vehicle-infused controls (% C) for (A) cingulate, (B) sensorimotor and (C) piriform cortex, and for (D) the total and(E) lateral striatum, all at the middle striatal level, and (F) the nucleus accumbens at the rostral level measured in the noninfused and 6-OHDA (6)-infusedhemispheres are given for rats that were killed 2 or 21 days after the 6-OHDA infusion (n = 5±8). **P < 0.01, *P < 0.05 vs. noninfused; aaP < 0.01 vs.acute; ccP < 0.01, cP < 0.05 vs. vehicle-infused controls.



lation-evoked c-fos and zif 268 expression) in the ipsilateral barrel

cortex were also greatly reduced in such animals.

Our ®ndings of to some degree bilateral effects of unilateral

dopamine depletion on cortical gene expression are consistent with

results of previous studies. Unilateral 6-OHDA lesions have been

found to produce changes in the expression of neuropeptides,

glutamic acid decarboxylase and glutamate receptors in fronto-

parietal cortical areas that were partly bilateral (Lindefors et al.,

1989; Lindefors et al., 1990; Rodriguez-Puertas et al., 1999). For

example, metabotropic glutamate receptor subtype 3 mRNA levels

were increased ipsilaterally, whereas subtype 4 mRNA levels were

increased bilaterally 4 weeks after a unilateral 6-OHDA lesion

(Rodriguez-Puertas et al., 1999). Moreover, D1 receptor agonist

treatments after unilateral dopamine depletion produced widespread

induction of immediate-early genes in the cortex of both hemispheres

(Steiner & Gerfen, 1996; Berke et al., 1998; Rodriguez-Puertas et al.,

1999).

Regional and laminar patterns of changes in cortical gene

expression after dopamine agonist (see above) or antagonist

(Steiner & Kitai, 2000b) treatments, as well as after dopamine

depletion (see above; present results) poorly match dopamine

innervation (Bjorklund & Lindvall, 1984; Berger et al., 1991) or

dopamine receptor localization (Mansour et al., 1990; Bouthenet

et al., 1991; Mengod et al., 1991; Gaspar et al., 1995; Defagot et al.,

1997; Ciliax et al., 2000) in the rat cortex, suggesting that such gene

regulation is the effect of altered cortical inputs rather than local

dopamine action (LaHoste et al., 1996). Our previous results showed

that acute blockade of striatal D1 receptors by intrastriatal infusion of

a D1 receptor antagonist attenuates basal and apomorphine-induced

immediate-early gene expression in the cortex, indicating a

facilitatory role for striatal D1 receptors in cortical gene regulation

(Steiner & Kitai, 2000b). The present results are consistent with such

a role for striatal D1 receptors. Notably, the present lesion effects

shared several characteristics with those of blockade of striatal D1

receptors (Steiner & Kitai, 2000b). For example, in both studies,

`basal' and sensory-evoked gene expression were inhibited; all

cortical areas examined were affected with the exception of the

cingulate cortex (see also LaHoste et al., 1996, for similar effects of

dopamine agonists); the effects were to various degrees bilateral, but

were more robust ipsilaterally. We thus speculate that, in the present

study, the attenuated cortical immediate-early gene expression after

acute dopamine depletion re¯ects a loss of dopamine action in the

striatum and ensuing functional alterations in basal ganglia±cortical

circuits. The correlation between increased striatal enkephalin

expression (marker for dopamine depletion, see above) and reduced

whisker stimulation-evoked gene expression in the barrel cortex

observed 2 days after 6-OHDA infusion is consistent with this notion.

However, despite these similarities between the effects of striatal

D1 receptor blockade and those of the 6-OHDA infusion, we can not

exclude the possibility that other effects of the 6-OHDA lesion

contributed to the observed neuronal and behavioural changes. For

example, the neurotoxin infusion may also have produced nonspeci®c

injury, the effects of which dissipated over time. Moreover, although

the vast majority of mesotelencephalic dopamine projections termin-

ates in the striatum, several other forebrain structures also receive

dopamine inputs from the midbrain (Fallon & Moore, 1978; Fallon

et al., 1978; Bjorklund & Lindvall, 1984). A loss of such extrastriatal

dopamine terminals may thus have contributed to the lesion effects.

Regarding the effects on gene expression in the contralateral cortex, it

can be argued that they could be due to lesion of the small crossed

nigrostriatal projection (Fass & Butcher, 1981; Fallon et al., 1983;

Pritzel et al., 1983), or to diffusion of the neurotoxin across the

midline and resulting injury to dopamine neurons in the contralateral

hemisphere. Although we did not observe evidence for a contralateral

lesion by inspecting tyrosine hydroxylase immunoreactivity in the

c-fosB zif 268

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vehi

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FIG. 6. Whisker stimulation-evoked expression of c-fos and zif 268 mRNAsin the barrel cortex after dopamine depletion. (A) Illustrations of ®lmautoradiograms show c-fos (left) and zif 268 expression (middle) in barrel C2in tangential sections through layer IV of the barrel cortex ipsilateral to thevehicle or 6-OHDA infusion in animals that had the contralateral whisker C2stimulated 2 days after the infusion (acute). Barrel C2 was located bycytochrome oxidase (CO) activity in adjacent sections (right); surroundingbarrels C1, C3, B2 and D2 are outlined. (B) Mean density values (mean6 SEM) for stimulation-evoked c-fos and zif 268 expression in barrel C2 areshown for animals that received an infusion of vehicle (V) or 6-OHDA (6)into the ipsilateral midbrain and were stimulated 2 or 21 days after theinfusion and killed thereafter (n = 5±8). Values presented are the differencebetween the C2 value and averaged values measured in the surroundingbarrels. (C) Scanning (mean 6 SEM) during a 10-min open-®eld test prior towhisker stimulation is shown for the sides of the face functionally related tothe noninfused and vehicle (V)- or 6-OHDA (6)-infused hemispheres in theseanimals. Note the dissociation between stimulation-evoked gene expressionin the barrel cortex ipsilateral to the dopamine depletion (recovered) andactive utilization (scanning) of the whiskers functionally related to thedopamine-depleted hemisphere (not recovered) 21 days after the lesion.**P < 0.01, *P < 0.05 as indicated; aaP < 0.01 vs. acute.



striatum and mRNA in the midbrain, these markers may not be

sensitive enough for more subtle effects. However, zif 268 expression

in the lateral striatum (the most sensitive marker we assessed, see

above) tended to be decreased in the contralateral striatum, and not

increased as seen after dopamine depletion. Future studies will have

to ascertain the role of striatal dopamine.

Parallel changes in cortical immediate-early gene expressionand some behaviours after dopamine depletion

The decrease in cortical gene expression after dopamine depletion

was transient. Both basal zif 268 expression and sensory-evoked c-fos

and zif 268 expression were back to control levels 21 days after the

lesion. Parallel functional recovery was seen for some, but not all,

behaviours when rats were tested in a novel open ®eld. Numerous

studies have shown that unilateral dopamine depletion produces early

bradykinesia/akinesia followed by some recovery, and persisting

behavioural asymmetries including ipsiversive turning and a loss of

responsiveness to sensory stimulation on the contralateral body

surface (sensorimotor `neglect') (Ungerstedt, 1971; Marshall et al.,

1974; Ljungberg & Ungerstedt, 1976; Carli et al., 1985; Brown &

Robbins, 1989; for review, see Schwarting & Huston, 1996b). The

degree of early bradykinesia is directly related to the extent of

dopamine depletion in the striatum (Fornaguera et al., 1994). In the

present study, rats tested 2 days after 6-OHDA infusion showed very

little forward locomotion and scanning, but some turning. In contrast,

rats tested 21 days after the lesion displayed signi®cantly more

locomotion, but retained turning and scanning asymmetries. Thus, the

recovery in cortical immediate-early gene expression was paralleled

by a partial recovery from akinesia.

An important question raised by these parallel changes in

immediate-early gene expression and behaviour is to what degree

the observed effects on gene expression were in¯uenced by changes

in behavioural activity (sensorimotor feedback). Thus, did the acute

decrease and subsequent recovery in cortical zif 268 mRNA levels

merely re¯ect acute akinesia and subsequent behavioural recovery in

the open-®eld test? An in¯uence of behavioural activities on some

cortical gene expression is likely. For example, sensory input

produced by whisking seems to affect gene expression in the

somatosensory cortex (Steiner & Gerfen, 1994). However, several

observations argue against behavioural activation, sensorimotor

feedback and/or nonspeci®c arousing inputs being the principal

determinants of changes in cortical gene expression in our studies.

First, while possible, it is not obvious how such effects could account

for the distinct regional differences found. For example, after acute

dopamine depletion, the insular cortex was affected to a similar

degree as sensory and motor areas, whereas the cingulate cortex was

not affected. Also, in the contralateral cortex, in contrast to the

unaffected cingulate regions, the most robust decrease was observed

in the caudally adjacent retrosplenial cortex. Second, rats displayed

signi®cantly lower zif 268 mRNA levels ipsilateral than contralateral

to the lesion across all affected cortical areas, despite an almost

complete cessation of behaviour. Third, a dissociation between

cortical gene regulation and behavioural activity was also found in

our previous study. Thus, intrastriatal infusion of a low dose of the

D1 antagonist SCH-23390 produced a bilateral partial attenuation of

apomorphine-induced gene expression across the cortex without

causing noticeable behavioural inhibition (Steiner & Kitai, 2000b).

These ®ndings suggest that at least part of the present changes in

cortical gene expression were a direct consequence of dopamine

depletion and ensuing neuronal changes rather than the result of

behavioural changes.

Barrel cortex function and whisker use after dopaminedepletion

In order to directly compare whisker-evoked responses in the barrel

cortex with whisker-guided behaviour after the 6-OHDA lesion, we

assessed the amount of scanning during exploration of the open ®eld.

When rats travel along the wall of such a ®eld, they generally

maintain a certain distance to the wall that allows them to touch the

wall with their mystacial whiskers (`scanning'). The importance of

whisker sensory input for this behaviour is demonstrated by the

®nding that scanning is lateralized after clipping the mystacial

whiskers on one side; rats then follow the wall preferentially with the

whisker-intact side orientated towards the wall (Steiner et al., 1986;

J

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zif 268 mRNA (% of V)

c-fo

s m

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enkephalin mRNA (% of intact) enkephalin mRNA (% of intact)c-

fos

mR

NA

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68 m

RN

A (

% o

f V)

A CB

r = 0.00

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r = 0.16

r = -0.60

r = 0.93***

2 daysV

621 days

V

6

FIG. 7. Correlation between enkephalin expression in the dopamine-depleted striatum and whisker stimulation-evoked immediate-early gene expression in theipsilateral barrel cortex 2 days, but not 21 days, after dopamine depletion. (A) Correlation between stimulation-evoked expression of c-fos and zif 268 (meandensity values, expressed as percentage of those in vehicle controls, % of V) in barrel C2 in individual animals that received an infusion of vehicle (V) or6-OHDA (6) and were stimulated either 2 or 21 days after the infusion. (B,C) Relationship between mid-striatal enkephalin mRNA levels (expressed aspercentage of those in the noninfused hemisphere, % of intact) and stimulation-evoked c-fos (B) and zif 268 expression (C) in the ipsilateral barrel C2 isshown for 6-OHDA-infused rats (6) stimulated 2 or 21 days after the lesion. ***P < 0.001, *P < 0.05.



Milani et al., 1989). Likely a re¯ection of the contralateral

sensorimotor `neglect', rats with unilateral dopamine depletion also

show lateralized scanning; they mostly follow the wall or edge of the

®eld with the side ipsilateral to the lesion (Steiner et al., 1988;

Fornaguera et al., 1993; Sullivan et al., 1994). Several reports showed

that such scanning is a sensitive behavioural index for compromised

dopamine function (Mattioli et al., 1992; Fornaguera et al., 1993;

Emmi et al., 1997).

In our study, 2 days after the lesion both scanning and whisker

stimulation-evoked immediate-early gene expression in the barrel

cortex were largely abolished. In contrast, 21 days after dopamine

depletion, such sensory-evoked cortical gene expression had also

recovered. At this time, rats displayed near-normal amounts of

scanning with the whiskers functionally related to the noninfused

hemisphere. However, minimal or no recovery was seen for scanning

related to the dopamine-depleted hemisphere, con®rming earlier

®ndings (see above). Therefore, these rats showed a de®cit in active

whisker use at a time when passive stimulation of these whiskers

elicited near-normal immediate-early gene responses in the barrel

cortex. These results thus show a dissociation between cortical

function, as indicated by immediate-early gene expression, and

behaviour for the dopamine-depleted hemisphere.

We also investigated whether there was a relationship between

changes in such scanning behaviour and neuroplasticity in the

dopamine-depleted striatum. After dopamine lesions various time-

dependent neuronal changes have been identi®ed in the basal ganglia,

including changes in the expression of receptors, enzymes and

neuropeptides, some of which probably contribute to recovery of

function (Schwarting & Huston, 1996a; for review). Consistently,

early alterations in neuronal ®ring followed by `normalization' have

been reported for several basal ganglia nuclei or their thalamic targets

(e.g. Schultz & Ungerstedt, 1978; Nisenbaum et al., 1986; MacLeod

et al., 1990; Ni et al., 2000), but not for others (e.g. Nisenbaum et al.,

1988; Vila et al., 2000). In our study, 21 days after dopamine

depletion recovery of scanning with the whiskers functionally related

to the hemisphere opposite to dopamine depletion was positively

correlated with the increase in enkephalin expression in the

dopamine-depleted striatum. In contrast, no relationship was seen

between enkephalin expression and scanning by the dopamine-

depleted hemisphere (or sensory-evoked immediate-early gene

expression in that hemisphere). It is unclear whether increased

enkephalin peptide function in the lesioned striatum was causally

related to such recovery of scanning by the noninfused hemisphere.

However, these ®ndings are consistent with neuroadaptive changes in

the dopamine-depleted striatum contributing to recovery of function

of the contralateral hemisphere, and thus with interhemispheric

effects of striatal output. On the other hand, such neuronal changes

are insuf®cient for recovery of behaviour related to the ipsilateral

hemisphere.

Potential pathways mediating the cortical facilitation

Together, our present and previous (Steiner & Kitai, 2000b) results

suggest a widespread, to some degree bilateral, facilitatory in¯uence

of dopamine on cortical function mediated by D1 receptors in the

striatum (Fig. 9). It is currently unknown which of several possible

anatomical pathways could convey this basal ganglia output to the

cortex. Current models of basal ganglia function are insuf®cient to

explain this effect (Alexander et al., 1986; Alexander & Crutcher,

1990; Alexander et al., 1990; but see Middleton & Strick, 2000). For

one, largely based on primate anatomy, these models emphasize basal

ganglia output directed towards frontal/premotor/motor cortical areas,

although some cortical regions outside the frontal lobe have also been

shown to be targets of basal ganglia output in primates (cf. Middleton

& Strick, 1996). Also, these models do not address interhemispheric

interactions. However, crossed pallidothalamic and nigrothalamic

projections have been described in primates and rats (for review, see

Parent & Hazrati, 1995).

In the rat, basal ganglia±cortical circuits are mostly routed through

the ventromedial and intralaminar/midline thalamic nuclei (e.g. Faull

& Mehler, 1978; Beckstead et al., 1979; Gerfen et al., 1982; for

reviews, see Groenewegen & Berendse, 1994; Gerfen & Wilson,

1996) that have widespread access to the cortex (Herkenham, 1979;

Herkenham, 1980; Arbuthnott et al., 1990). Widespread projections

from midline thalamic nuclei to the cortex have also been shown in

ϑ

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of V

)

enkephalin mRNA (% of intact)

enkephalin mRNA (% of intact)

scan

ning

(%

of V

)

21 daysnoninfused

6-OHDA

FIG. 8. Correlation between enkephalin expression in the dopamine-depletedstriatum and whisker utilization (scanning) by the noninfused hemisphere,but not by the 6-OHDA-infused hemisphere, 21 days after dopaminedepletion. (A and B) Striatal enkephalin expression (mean density values,expressed as percentage of those in the noninfused hemisphere, % of intact)at the middle striatal level is compared to the amount of scanning(expressed as percentage of that in vehicle-infused controls, % of V) withthe whisker side related to the noninfused hemisphere (A) and that relatedto the dopamine-depleted hemisphere (6-OHDA) (B) in individual animalswith dopamine lesions (21 days, two experiments pooled). Note thatremoving the outlier at 140/174 in A improves the positive correlation tor = 0.83 (P < 0.001). *P < 0.05.



the monkey (e.g. Bentivoglio et al., 2000). Interestingly, a recent

tracing study in the rat showed that neurons of the ventromedial

thalamic nucleus send axon collaterals to both the whisker motor and

the barrel cortex, thus providing a potential anatomical route by

which basal ganglia output could directly in¯uence both of these

cortical areas (Jones et al., 2000).

However, it is also possible that pathways not classically

considered part of basal ganglia±cortical circuits contribute to such

basal ganglia regulation of cortical function. For example, it has been

shown that unilateral dopamine depletion produces bilateral changes

in gene expression in the reticular nucleus of the thalamus (Delfs

et al., 1996), a structure that also receives inputs from the basal

ganglia and is of central importance for the regulation of cortical

activation. Therefore, several candidate anatomical pathways exist

that could mediate the widespread facilitatory in¯uence of striatal

dopamine on cortical function suggested by our studies. Future

experimentation will have to elucidate the possible contributions of

these pathways.

Functional signi®cance: a hypothesis

Regardless of the speci®c connections involved, the present time-

dependent effects on cortical gene regulation in comparison with the

associated behavioural effects may offer some indications as to the

functional organization of this basal ganglia±cortical input. Our

results suggest that this input provides rather general, bilateral

cortical facilitation and is likely superimposed upon and acts in

concert with the speci®c (e.g. motor) basal ganglia±cortical circuitry

(Fig. 9). For example, after unilateral dopamine depletion, the early

bilateral behavioural breakdown was followed by complete recovery

in behaviour (scanning) dependent on speci®c sensorimotor functions

of the contralateral hemisphere, but not in scanning related to the

hemisphere ipsilateral to the lesion. Similarly, early bilateral de®cits

and some subsequent recovery for the `intact' side were found for

skilled forelimb reaching movements after unilateral 6-OHDA lesions

(e.g. Miklyaeva et al., 1994; Whishaw et al., 1997). Whilst it is clear

that activity in sensorimotor basal ganglia-cortical circuits is

extensively altered in the dopamine-depleted hemisphere (e.g.

Albin et al., 1989; Levy et al., 1997; Obeso et al., 1997), our

®ndings suggest that the bilateral facilitatory cortical input is only

transiently suppressed in both hemispheres, perhaps because of

remaining cortical input from the unlesioned basal ganglia and/or

neuroadaptive changes following the lesion (see above). Thus, after

an initial period of general cortical failure re¯ected by the general

behavioural breakdown, recovery of the widespread cortical input

seems to allow recovery of contralateral cortical function and

dependent behaviours, and probably `mixed' behaviours to some

degree (e.g. locomotion), but not (or considerably less) of `ipsilateral'

behaviours that are still affected by compromised function in the

ipsilateral speci®c sensorimotor circuits.

Our results thus suggest dual basal ganglia±cortical inputs

(unilateral speci®c and bilateral general, facilitatory) that seem to

arise in the same striatal areas (Fig. 9; Steiner & Kitai, 2000b). Both

seem to be necessary for normal cortical function and behaviour. This

concept of dual basal ganglia±cortical inputs may provide an

improved framework for investigating and better understanding

how the basal ganglia in general, and dopamine in particular, regulate

cortical function.

Conclusions

The results of our studies suggest that stimulation of D1 dopamine

receptors in the striatum provides bilateral facilitation of cortical

function that is necessary for normal behaviour. This facilitation

affects many cortical regions, which may explain in part the diversity

of functions and disorders associated with dopamine.

Acknowledgements

This work was supported by the National Parkinson Foundation and USPHSGrants DA11261 (H.S.) and NS26473 (S.T.K.). We thank Weiwen Sun forexcellent technical assistance and Pastor Couceyro for helpful discussions.

Abbreviation

6-OHDA, 6-hydroxydopamine.

whiskerC2

barrelC2

D1

specific (e.g., motor)

widespread facilitatory

striatum

++

++

+

+

++

++

FIG. 9. Schematic illustration of thewidespread, to some degree bilateral,facilitatory input to the cortex arising in thestriatum, as indicated by our ®ndings. Thiscortical facilitation is dependent on D1receptors in the striatum (Steiner & Kitai,2000b). It is presently unknown which ofseveral possible anatomical circuits from thestriatum to the cortex mediates this facilitation(see Discussion). However, our results suggestthat the facilitatory circuits (solid arrows) are,partly or completely, separate from andsuperimposed upon the speci®c (e.g. motor;broken arrows) basal ganglia±cortical circuitsthat target the frontal lobe. The whisker-to-barrel pathway from the left whisker C2 to theright barrel C2 is indicated by a dotted line.



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unilateral striatal dopamine depletion: time-dependent effects on cortical function and behavioural...

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