Stimulatory role of dopamine on fibroblast growth factor-2 expression in rat striatum: Dopamine regulates FGF2 expression in rat brain

Journal of Neurochemistry, 2001, 76, 990±997

Stimulatory role of dopamine on ®broblast growth factor-2

expression in rat striatum

Mila Roceri,*,² Raffaella Molteni,* Fabio Fumagalli,* Giorgio Racagni,*,² Massimo Gennarelli,²Giovanni U. Corsini,³ Roberto Maggio³ and Marco A. Riva*

*Centre for Neuropharmacology, Department of Pharmacological Sciences, University of Milan, Milan, Italy

²IRCCS San Giovanni di Dio-Fatebenefratelli, Brescia, Italy

³Department of Neuroscience, Pharmacology Section, University of Pisa, Pisa, Italy

Abstract

We have previously shown that systemic injection of

(±)nicotine produces a selective up-regulation of ®broblast

growth factor (FGF)-2 mRNA levels in rat striatum. Because

(±)nicotine can increase striatal release of dopamine and

glutamate, in the present study we have investigated the

contribution of these neurotransmitters in the modulation of

FGF-2 expression. We found that coinjection of dopaminergic

D1 (SCH23390) or D2 (haloperidol) receptor antagonists

prevents nicotine-induced elevation of FGF-2 expression.

However, injection of the NMDA receptor antagonist MK-801

produced a signi®cant increment of FGF-2 mRNA and protein

levels in rat striatum similar to the effect produced by

(±)nicotine alone. Interestingly this effect of MK-801 could

also be prevented by D1 or D2 receptor antagonists,

suggesting that an elevation of dopamine levels may be

required for the regulation of the trophic molecule. Accordingly

we found that the non-selective dopaminergic agonist

apomorphine can similarly increase striatal FGF-2 mRNA

levels. Despite the observation that both D1 and D2 receptors

appear to contribute to the modulation of FGF-2 expression,

only a direct activation of D2 receptors, through quinpirole

administration, was able to mimic the effect of apomorphine.

On the basis of FGF-2 neurotrophic activity, these results

suggest that direct or indirect activation of dopaminergic

system can be neuroprotective and might reduce cell

vulnerability in degenerative disorders.

Keywords: gene expression, glutamate, neuroprotection,

neurotrophic factor, Parkinson's disease.

J. Neurochem. (2001) 76, 990±997.

Basic ®broblast growth factor (FGF)-2 represents the

prototype member of a family of polypeptide growth

factors with different biological activities on central

and peripheral nervous systems (Baird 1994; Bikfalvi et al.

1997). FGF-2 supports the survival and maturation of

several neuronal phenotypes (Walicke 1988), determines

the fate of CNS progenitor cells (Vescovi et al. 1993) and

acts on astrocytes and oligodendrocytes (Bikfalvi et al.

1997). Moreover, FGF-2 participates in a cascade of

neurotrophic events contributing to neuronal repair and

cell survival. For example, this neurotrophic peptide

rescues cholinergic neurones following ®mbria fornix

transection (Anderson et al. 1988), prevents thalamic

degeneration after cortical infarction (Yamada et al. 1991)

and protects dopaminergic neurones from the toxic

activity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

(MPTP) (Otto and Unsicker 1990). In vitro FGF-2 exerts a

remarkable neuroprotective activity in different models of

excitotoxic cell death (Mattson et al. 1989; Freese et al.

1992).

The expression of FGF-2 increases in response to

neuronal activation (Riva et al. 1992; Van Der Wal et al.

1994) or as a consequence of cell damage occurring

following kainate injection (Riva et al. 1994) or brain

injury (Gomez-Pinilla et al. 1992; Logan et al. 1992). The

observation that the levels of FGF-2 are elevated in different

990 q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 990±997

Received May 18, 2000; revised manuscript received September 5,

2000; accepted September 22, 2000.

Address correspondence and reprint requests to Dr Marco A. Riva,

Center of Neuropharmacology, Department of Pharmacological

Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy.

E-mail: [email protected]

Abbreviations used: DDC, diethyldithiocarbamate; FGF, ®broblast

growth factor; GAPDH, glyceraldehyde 3 phosphate dehydrogenase;

MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; nAchR, nicotinic

acethylcoline receptor.

situations suggests that this increase may enhance the

resistance of neurones to potentially lethal damage. Indeed,

it has been observed that blockade of FGF-2 activity by

neutralizing antibodies retards functional recovery from

motor cortex injury in rats (Rowntree and Kolb 1997). On

this basis, the possibility of modulating the expression of

FGF-2 in a very speci®c way within the CNS might

represent a potential strategy for the treatment of acute and

chronic degenerative diseases, which may be characterized

by an increased vulnerability of speci®c cell phenotypes to

damage.

We have previously shown that acute intermittent

injection of (±)nicotine can protect dopaminergic neurones

from the neurodegenerative effect induced by diethyldithio-

carbamate (DDC) 1 MPTP in mice or metamphetamine in

mice and rats (Maggio et al. 1998). This neuroprotective

effect was accompanied by an up-regulation of FGF-2

mRNA in striatum. Because nicotinic acetylcholine receptor

subtypes are present in various brain regions (Galzi and

Changeux 1995) and play important roles in the modulation

of neurotransmitter release (Marshall et al. 1997; Wonnacott

1997), we hypothesized that other neurotransmitters may be

involved in the modulation of FGF-2. Hence we compared

the effect of (±)nicotine with that of the NMDA receptor

antagonist MK-801, which is also known to modulate

neurotransmitter release by blocking glutamate NMDA

receptors on GABAergic neurones (Olney et al. 1991;

Olney and Farber 1995).

Our data demonstrate that there is a close similarity in the

regulation of FGF-2 mRNA and protein by (±)nicotine and

MK-801 and highlight a major role played by dopamine in

these regulatory mechanisms.

Materials and methods

Materials

General reagents were purchased from Sigma-Aldrich (Milan,

Italy), whereas molecular biology reagents were obtained from

Ambion (Austin, TX, USA), New England Biolabs (Beverly, MA,

USA) and Promega Italy (Milan, Italy). (±)Nicotine, mecamyl-

amine and SCH 23390 were purchased from Sigma; haloperidol,

MK-801, apomorphine, quinpirole and SKF38393 were obtained

from Sigma/RBI (Milan, Italy).

Animal treatment

Male Sprague Dawley rats (Charles River, Calco, Italy) weighing

250±300 g were used throughout the experiments. Animals

received three subcutaneous injections of (±)nicotine (1 mg/kg)

every 30 min or a single intraperitoneal dose of the following

drugs: mecamylamine (1 mg/kg); MK-801 (1 mg/kg); haloperidol

(1 mg/kg); SCH 23390 (1 mg/kg); apomorphine (1.5 mg/kg);

quinpirole (2 mg/kg) and SKF 38393 (10 mg/kg). In the combined

treatments, dopaminergic antagonists (SCH 23390 or haloperidol)

were injected 30 min before administration of (±)nicotine, MK-801

or apomorphine. Animals were killed by decapitation 6 h after the

last injection of the drug(s). The brain regions were rapidly

dissected, frozen on dry ice and stored at 2 708C for further

analysis.

RNA preparation

Rat brain tissue was homogenized in 4 m guanidinium isothio-

cyanate (containing 25 mm sodium citrate pH 7.5, 0.5% sarcosyl

and 0.1% 2-mercaptoethanol) and total RNA was isolated by

phenol/chloroform extraction (Chomczynski and Sacchi 1987).

Quanti®cation was carried out by spectrophotometric analysis and

RNA aliquots were re-precipitated in ethanol for RNase protection

assay.

cRNA probes and RNase protection assay

A transcription kit (MAXI script, Ambion) was used to generate

cRNA probes and 32P-CTP was used as a radiolabelled nucleotide.

Plasmid RObFGF503 containing a 1016-bp portion of the rat

FGF-2 cDNA and pTRI-GAPDH-Rat (Ambion) containing a

portion of rat glyceraldehyde 3-phosphate dehydrogenase

(GAPDH) cDNA were employed in the RNase protection assay.

The cRNA probes and the relative protected fragment (p.f.) were as

follows: FGF-2 � 524, p.f. � 477; GAPDH � 376, p.f. � 316.

The RNase protection assay was performed on a 10-mg sample of

total RNA as described previously (Riva et al. 1996). Brie¯y, after

ethanol precipitation, total RNA was dissolved in 20 mL of

hybridization solution (80% formamide, 40 mm PIPES pH 6.4,

400 mm sodium acetate pH 6.4 and 1 mm EDTA) containing

150 000 cpm of 32P-labelled FGF-2 cRNA probe (speci®c activity

. 108 cpm/mg) or 50 000 cpm of 32P-labelled GAPDH probe. After

being heated at 858C for 10 min, the cRNA probes were allowed to

hybridize the endogenous RNAs at 458C overnight. At the end of

hybridization, the solution was diluted with 200 mL of RNase

digestion buffer (300 mm NaCl, 10 mm Tris HCl pH 7.4, 5 mm

EDTA pH 7.4) containing a 1/400 dilution of an RNase cocktail

(1 mg/mL RNase A and 20 U/mL RNase T1) and incubated for

30 min at 308C. Proteinase K (10 mg) and SDS (10 mL of 20%

stock solution) were then added to the sample and the mixture was

incubated at 378C for an additional 15 min. At the end of

incubation, the sample was extracted with phenol/chloroform and

ethanol-precipitated. The pellet, containing the RNA±RNA

hybrids, was dried and resuspended in loading buffer (80%

formamide, 0.1% xylene cyanol, 0.1% bromophenol blue, 2 mm

EDTA), boiled at 958C for 5 min and separated on 5%

polyacrylamide gel under denaturing conditions (7 m urea).

Western blot analysis

Rat striata were homogenized on ice in Tris HCl buffer (pH 7.5)

containing the following: glycerol (10%), NaCl (150 mm), Triton

X-100 (1%), EDTA (5 mm), EGTA (1 mm), vanadate (1 mm),

ZnCl2 (1 mm), NaF (10 mm), leupeptin (10 mg/mL), PMSF

(100 mm), aprotinin (10 mg/mL). Samples were clari®ed by

centrifugation at 15 000 g for 15 min at 48C. Following protein

determination (BCA protein assay reagent kit: Pierce; Rockford, IL,

USA), samples (25 mg per lane) were run on an SDS-12%

polyacrylamide gel under reducing conditions, and proteins were

then electrophoretically transferred to a polyvinylidene di¯uoride

(PVDF) membrane (Biorad, Segrate, Italy). Membranes were

incubated with a 1 : 250 dilution of anti-FGF-2 monoclonal

antibody (Transduction Laboratories, Lexington, KY, USA)

followed by anti-mouse IgG peroxidase conjugate (Sigma-Aldrich).

Dopamine regulates FGF-2 expression in rat brain 991

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 990±997

Tubulin was used as internal standard. Anti-b tubulin mono-

clonal antibody (Sigma-Aldrich) was used at 1 : 1000 dilution,

followed by anti-mouse IgG peroxidase conjugate (Sigma).

Immuno-complexes were visualized by chemiluminescence utiliz-

ing the ECL western blotting kit (Amersham Life Science) in

accordance with the manufacturer's instructions.

Quanti®cation of RNA or proteins and statistical analysis

The levels of mRNA were calculated by measuring the peak

densitometric area of the autoradiography analysed with an LKB

laser densitometer. In order to ensure that the autoradiographic

bands were in the linear range of intensity different exposure times

were used. GAPDH was employed as the internal standard for the

RNase protection assay: its expression was not regulated by any

pharmacological treatment. The data represent the ratio between

FGF-2 and GAPDH levels. The mean value of the control group

within a single experiment was set to 100 and the data of animals

injected with different drugs were expressed as a `percentage' of

control animals. FGF-2 protein isoform levels, quanti®ed by

computer analysis as the ratio between each immunoreactive

band and the level of tubulin, are expressed as a percentage of

saline-injected animals.

One-way analysis of variance (anova), followed by Dunnett's

test for multiple comparison if required, was used for statistical

analysis. Experimental differences were considered signi®cant at

least when p , 0.05.

Results

Subcutaneous injection of (±)nicotine produced a signi®cant

and rather selective increase of FGF-2 mRNA levels in rat

striatum (179%, *p , 0.001). As summarized in Fig. 1, a

signi®cant elevation of FGF-2 was also observed in parietal

and entorhinal cortex (*p , 0.05), whereas the alkaloid did

not affect its expression in frontal cortex, hippocampus,

thalamus or hypothalamus. The induction of FGF-2 in

striatum appears to be transient, as 24 h after (±)nicotine

injection its mRNA expression had returned to basal levels

(data not shown). The speci®city of the (±)nicotine effect

was determined by pre-treatment with the non-competitive

nAChR receptor antagonist mecamylamine. As shown in

Table 1, mecamylamine per se did not change the levels

of FGF-2 mRNA, but prevented its induction in response to

(±)nicotine.

It is well established that (±)nicotine, by acting on pre-

synaptic receptors, increases the release of different

neurotransmitters, including dopamine and glutamate

(Marshall et al. 1997; Wonnacott 1997). On this basis, we

investigated the contribution of these neurotransmitters in

the regulation of striatal FGF-2 expression. As shown in

Fig. 2, we found that (±)nicotine-induced elevation of

FGF-2 mRNA was antagonized by injection of dopamine

Fig. 1 Levels of FGF-2 mRNA in different rat brain structures 6 h

after systemic injection of (±)nicotine (1 mg/kg, three times). The

results, expressed as percentage difference of saline-injected rats,

represent the means ^ SEM of at least six independent determina-

tions, as measured by the RNase protection assay, of the following

brain structures: STR, striatum; HIP, hippocampus; PC, parietal

cortex; EC, entorhinal cortex; THA, thalamus; HYP, hypothalamus.

*p , 0.05 and **p , 0.001 versus saline-injected rats (one-way

ANOVA).

Table 1 Antagonism of mecamylamine on (±)nicotine-induced

upregulation of FGF-2 mRNA levels in rat striatum

Group

FGF-2 mRNA

(% of control levels)

Saline 100� ^ 9

Mecamylamine 85� ^ 12

(±)Nicotine 166� ^ 13*

Meca 1 Nico 101� ^ 13

The results, expressed as the percentage of vehicle-injected rats,

represent the means ^SEM of at least ®ve independent determina-

tions. Mecamylamine was injected 30 min before (±)nicotine and the

animals were killed 6 h after the injection of the alkaloid. *p , 0.01

versus saline-injected animals (one-way ANOVA with Dunnett's test).

Fig. 2 RNase protection assay analysis of FGF-2 mRNA levels in

rat striatum following injection of (±)nicotine or MK-801, a NMDA

receptor antagonist. Arrows indicate the protected fragments for

FGF-2 or GAPDH, used as internal standard, in the following

experimental groups: 1, saline; 2, nicotine; 3, MK-801; 4, nicotine

1 MK-801. The lane marked P represents an aliquot of the hybridi-

zation solution with the original cRNA probes. A signi®cant increase

in the mRNA levels of FGF-2 is evident after exposure to both

drugs. The autoradiographic ®lm was exposed at 2 708C with an

intensifying screen for 5 h (GAPDH) or 18 h (FGF-2).

992 M. Roceri et al.


D1 (SCH 23390) or D2 (haloperidol) receptor antagonists.

Systemic injection of MK-801, a non-competitive antagonist

of the glutamate NMDA receptor, per se increased the levels

of FGF-2 mRNA in striatum (*p , 0.001). When

(±)nicotine was co-administered with MK-801, we observed

an up-regulation of FGF-2 expression that was not

signi®cantly different from the effect produced by the two

drugs alone (p . 0.05 versus nicotine alone) (Figs 2 and 3).

The rise of striatal FGF-2 mRNA was paralleled by a

signi®cant increase in its protein levels. FGF-2 mRNA is

translated into different protein isoforms, which have

different subcellular localizations (Florkiewicz and Sommer

1989; Prats et al. 1989; Delrieu 2000). As shown in Fig. 4,

injection of (±)nicotine or MK-801 increased the 18

(*p , 0.05) and 21/22 kDa (*p , 0.05), but not the 24

kDa, FGF-2 isoforms in rat striatum.

Because the effects on FGF-2 expression produced by

MK-801 and (±)nicotine in striatum were similar, we

investigated whether dopamine could also contribute to the

effects produced by the NMDA receptor antagonist. It is

indeed known that systemic injection of MK-801 can

increase dopamine release in several brain structures,

including striatum (Miller and Abercrombie 1996). As

depicted in Fig. 5, SCH 23390 and haloperidol antagonized

the up-regulation of FGF-2 after MK-801 injection,

suggesting that an increased activity of the dopaminergic

system may contribute to its modulation in this brain region.

In order to further investigate the role of dopamine in the

regulation of FGF-2 gene expression we treated rats with the

Fig. 3 Determination of FGF-2 mRNA levels in rat striatum in

response to systemic injection of (±)nicotine: modulation by the glu-

tamate NMDA receptor antagonist MK-801, or the dopamine D1

(SCH 23390) and D2 (haloperidol) receptor antagonists. Receptor

antagonists were injected 30 min prior to (±)nicotine administration

and the animals were killed 6 h later. The data, expressed as the

percentage of saline-injected rats, represent the means ^SEM of at

least ®ve independent determinations. *p , 0.01 and **p , 0.001

versus saline-injected rats (one-way ANOVA with Dunnett's test).Fig. 4 Detection of FGF-2 protein isoforms by western blot analysis

in rat striatum of rats treated with saline (open bars) (±)nicotine

(dark bars) or MK-801 (hatched bars) and killed 6 h after drug injec-

tion. (a) Three immunoreactive bands (18, 21/22 and 24 kDa) are

evident from the analysis; tubulin was used as the internal standard.

(b) The quantitative results (FGF-2/tubulin) are expressed as the

percentage of saline-injected rats, and represent the means ^SEM

of four independent determinations. *p , 0.05 versus saline-injected

rats (one-way ANOVA).

Fig. 5 Modulation of MK-801-induced elevation of FGF-2 mRNA

levels in rat striatum by dopamine D1 (SCH 23390) or D2 (haloperi-

dol) receptor antagonists. SCH23390 or haloperidol were injected

30 min prior to MK-801 and the animals were killed 6 h later. The

data, expressed as the percentage of saline-injected rats, represent

the means ^SEM of at least ®ve independent determinations.

*p , 0.01 (one-way ANOVA with Dunnett's test).



non-selective dopamine receptor agonist apomorphine. As

summarized in Table 2, the mRNA levels of the trophic

factor were signi®cantly increased 6 h after the systemic

injection of the dopamine agonist (*p , 0.05), an effect that

was prevented by D1 or D2 receptor antagonists. Even

though both dopaminergic receptor subtypes appear to be

involved in the regulation of striatal FGF-2 following

injection of apomorphine or dopamine-releasing agents

(nicotine and MK-801), we found that direct stimulation

of D2 (but not D1) receptors was suf®cient to up-regulate

striatal FGF-2 expression. As shown in Fig. 6, quinpirole,

but not SKF 38393, increased FGF-2 mRNA levels in

striatum (196%, *p , 0.01) although the injection of the

D1 receptor agonist produced a slight, but not signi®cant,

potentiation of quinpirole effects (1131%, p � 0.59).

Discussion

In the present paper we demonstrated that the expression of

the neurotrophic molecule FGF-2 can be regulated in rat

striatum by a dopamine-dependent mechanism. This con-

clusion is supported by the effects produced by direct

dopaminergic agents as well as by drugs that modulate

dopaminergic neurotransmission.

The observation that (±)nicotine up-regulates striatal

FGF-2 expression is in good agreement with recent data

produced by Belluardo and coworkers even though, by using

in situ hybridization, they reported a signi®cant induction

of FGF-2 in response to (±)nicotine or ABT-594, an agonist

of nAChR receptor with preferential selectivity for a4b2

nAChR subtype, in brain regions that were not affected in

our experimental paradigm (Belluardo et al. 1998; Belluardo

et al. 1999). By contrast, it has been shown that chronic

continuous (±)nicotine treatment by minipump implantation

signi®cantly and dose-dependently reduces FGF-2 mRNA

levels within the neostriatum and the substantia nigra (Blum

et al. 1996). The difference between acute and chronic

treatment with (±)nicotine could be related to the desensi-

tization of nAChR, which may occur after repeated agonist

administration (Galzi and Changeux 1995).

Nicotinic AChR are present on the soma and terminals of

mesolimbic and mesostriatal neurones (Clarke et al. 1985),

and their stimulation induces the release of dopamine in

several brain structures (Marshall et al. 1997; Wonnacott

1997; Hildebrand et al. 1999). However (±)nicotine-induced

release of dopamine is larger in striatum than in cortical

structures (Marshall et al. 1997), and this may contribute to

the induction of c-fos expression in response to (±)nicotine

(Kiba and Jayaraman 1994). Moreover (±)nicotine can

increase the release of glutamate, which in turn might

regulate the expression of trophic molecules. The levels of

FGF-2 are in fact increased by glutamate in primary culture

of astroglial cells (Pechan et al. 1993) as well as by systemic

injection of kainic acid (Riva et al. 1994). However our

experiments, using the NMDA receptor antagonist MK-801

to antagonize the effect of (±)nicotine on FGF-2, did not

clarify this issue. Injection of MK-801 produced a

signi®cant increase in FGF-2 mRNA (see Fig. 2) and

protein (Fig. 3) in rat striatum, an effect that was not

signi®cantly different from the co-administration of

(±)nicotine and MK-801, suggesting that common events

might contribute to the activity of the two drugs on FGF-2

expression. The apparent discrepancy between the effects

produced by glutamate agonists and MK-801 may be

explained on the basis of the `disinhibition theory' proposed

by Olney and coworkers (Olney et al. 1991; Olney and

Fig. 6 Regulation of FGF-2 mRNA levels in rat striatum in response

to systemic injection dopaminergic receptor agonists. SKF 38393

(D1 agonist, SKF), quinpirole (D-2 agonist, QUI) or a combination of

the two drugs were injected in rats and the animals were killed 6 h

later. The data, expressed as the percentage of saline-injected rats,

represent the means ^SEM of ®ve to eight independent deter-

minations. *p , 0.01 versus saline-injected rats (one-way ANOVA with

Dunnett's test).

Table 2 Apomorphine increases FGF-2 mRNA levels in rat striatum:

modulation by dopaminergic receptor antagonists

Drug treatment

FGF-2 mRNA levels

(% of control levels)

Saline 100� ^ 10

SCH 23390 99� ^ 12

Haloperidol 110� ^ 4

Apomorphine 142� ^ 7*

Apomorphine 1 SCH 23390 115� ^ 6

Apomorphine 1 Haloperidol 101� ^ 8

The results, expressed as the percentage of vehicle-injected rats,

represent the means ^SEM of at least ®ve independent determina-

tions, the animals being killed 6 h after apomorphine injection.

*p , 0.05 versus saline-injected animals (one-way ANOVA with

Dunnett's test).



Farber 1995). According to this hypothesis, MK-801 can

block NMDA glutamate receptors on GABAergic neurones

thereby releasing the inhibitory control on the ®ring of

monoaminergic neurones, including dopamine. It has been

reported that systemic injection of MK-801 can increase

spontaneous dopamine release in striatum and potentiate

amphetamine-induced dopamine release suggesting that

NMDA receptors may exert a tonic inhibitory in¯uence

upon striatal dopamine ef¯ux (Miller and Abercrombie

1996).

We show that there is a close similarity between

(±)nicotine and MK-801 in the regulation of FGF-2

mRNA and protein. A selective increment of the 18 and

21/22 kDa FGF-2 isoforms were observed after acute

injection of these two drugs. Although little is known

regarding the contribution of each isoform to FGF-2 activity

(Florkiewicz and Sommer 1989; Prats et al. 1989; Delrieu,

2000), our data suggest that, in vivo, they may undergo

different regulatory mechanisms.

Although (±)nicotine and MK-801 act through different

membrane receptors, the effects of both agents on FGF-2

mRNA levels were signi®cantly reduced by antagonists for

dopamine D1 or D2 receptors, suggesting that dopamine

contributes to the elevation of striatal FGF-2 gene expres-

sion. These results suggest that there is some cooperation

between dopamine D1 and D2 receptors in regulating FGF-2

levels. This possibility is further supported by the observa-

tion that the non-selective dopaminergic agonist apomorhine

up-regulates striatal FGF-2 mRNA levels, an effect

antagonized by D1 as well as D2 receptor antagonists.

The possibility that these two receptors may interact

functionally at striatal levels has already been put forward.

D1 and D2 receptors can colocalize in striatal neurones

(Aizman et al. 2000), thus supporting the possibility that

intracellular events set in motion by their stimulation may

integrate in determining transcriptional events. Accordingly

D1 and D2 receptor agonists can synergize in enhancing

arachidonic acid release in cells transfected with both

receptor subtypes (Piomelli et al. 1991). Although both

dopamine receptors (D1 and D2) appear to participate in the

regulation of striatal FGF-2 expression, only the selective

D2 receptor agonist quinpirole was capable of inducing

FGF-2 expression in striatum. Conversely SKF 38393, a D1

receptor agonist, was not effective. We may assume that at

high receptor occupancy, as occurs with pharmacological

stimulation, activation of the D2 receptor is suf®cient to

increase FGF-2 expression and does not require concomitant

stimulation of D1 receptors. In fact quinpirole-induced

elevation of FGF-2 mRNA levels was not antagonized by

SCH 23390, a selective D1 receptor antagonist (data not

shown). On these bases, we may hypothesize that D1

receptors might come into play in the regulation of FGF-2

when a more `physiological' tuning of dopamine neuro-

transmission is occurring. However we cannot rule out the

possibility that striatal expression of FGF-2 is regulated

through different systems, which in turn may co-operate

with D1 and D2 dopamine receptors. An alternative

explanation of the rather selective activity of quinpirole on

FGF-2 mRNA levels may relate to its af®nity for D3

dopamine receptors. Future experiments with the use of

speci®c receptor agonists or antagonists will examine this

possibility.

Our results can be viewed in light of potential

neuroprotective activity of drugs able to modulate in a

very selective fashion the expression of FGF-2. A large

number of growth factors, including FGF-2, IGF-1 and 2,

TGF-a, BDNF, NT-4/5 and GDNF, stimulate dopaminergic

neurone survival or differentiation in cell culture systems

(Hefti 1994). The importance of FGF-2 is suggested by the

observation that adult dopaminergic neurones express this

trophic factor (Bean et al. 1991; Cintra et al. 1991), which is

anterogradely transported by rat nigrostriatal dopaminergic

neurones (McGeer et al. 1992). The expression of the FGF

receptors, FGFR-1 and FGFR-2, in the substantia nigra and

the striatum (Wanaka et al. 1990; Asai et al. 1993), and the

depletion of FGF-2, in the substantia nigra of Parkinsonians

(Tooyama et al. 1992), strongly suggest a role for this

trophic polypeptide in the regulation of dopaminergic

neurones. Furthermore FGF-2 is highly neuroprotective in

experimental Parkinsonism, as it promotes the recovery of

the dopaminergic function in adult mice treated with MPTP

(Otto and Unsicker 1990).

There is now substantial evidence that (±)nicotine exerts

a protective role against the degeneration of several

neuronal phenotypes, although the exact mechanisms

through which it occurs remain to be fully clari®ed (Akaike

et al. 1994; Marin et al. 1994). For example, acute

intermittent administration of (±)nicotine partially protects

dopaminergic neurones from MPTP-induced degeneration

(Janson et al. 1988). We recently demonstrated that

DDC-induced enhancement of MPTP toxicity in mice and

metamphetamine-induced neurotoxicity in rats and mice can

be prevented by (±)nicotine administered with the same

paradigm used in the present study (Maggio et al. 1998).

Such treatment can transiently enhance the expression of

FGF-2 mRNA and protein in the striatum and may

contribute to the neuroprotective effects of the alkaloid.

The observation that a direct stimulation of the dopamine

D2 receptors increases the expression of FGF-2 in the

striatum may also be of interest in light of a possible use of

dopaminergic agonist in the treatment of Parkinson's

disease. For example pramipexol, a novel highly selective

D2/D3 receptor agonist, is effective in early Parkinson's

disease monotherapy (Shannon et al. 1997). Moreover this

compound, as well as other D2 receptor agonists, can be

neuroprotective although the exact mechanism of this effect

remains to be fully established (O'Neill et al. 1998;

Takashima et al. 1999). Despite the fact that the use of



dopaminergic agonists is aimed at re-establishing dopamine

transmission, an increased expression of FGF-2 may

reduce cellular vulnerability thus opposing the ongoing

degenerative process.

The levels of FGF-2 increase with speci®c temporal and

spatial patterns in response to seizures, brain damage or

activation of speci®c neurotransmitter receptors (Gomez-

Pinilla et al. 1992; Logan et al. 1992; Riva et al. 1992; Riva

et al. 1994; Van Der Wal et al. 1994; Riva et al. 1996).

Although the functional relevance of these events remains to

be fully established, it has been demonstrated that blockade

of endogenous FGF-2 expression by neutralizing antibodies

retards recovery from motor cortex injury in rats (Rowntree

and Kolb 1997). This suggests that the induction of FGF-2

in response to cell injury or neuronal activation can be

neuroprotective for speci®c cellular phenotypes.

Although technological improvements may lead to a

direct use of trophic factors in degenerative diseases, a

speci®c modulation of their CNS expression by pharmaco-

logical agents could represent an alternative strategy for the

treatment and prevention of these disorders which, to some

extent, may be characterized by a reduced activity of

endogenous protective systems.

Acknowledgements

We wish to thank Dr A. Baird for the generous gift of an FGF-2

cDNA probe. Special thanks to Drs M. Armogida and B. Begni

for contributing to part of this study.

This work has been carried out under a research contract with

NEFAC, Pomezia, Italy, within the Neurobiological Systems

National Research Plan of the Ministero dell'UniversitaÁ e della

Ricerca Scienti®ca e Tecnologica.

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Stimulatory role of dopamine on fibroblast growth factor-2 expression in rat striatum: Dopamine regulates FGF2 expression in rat brain

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