Chronic continuous infusion of (−)nicotine reduces basic fibroblast growth factor messenger RNA levels in the ventral midbrain of the intact but not of the 6-hydroxydopamine-lesioned rat

Neuroscience Vol. 70, No. 1, pp. 169-177, 1996

Pergamon

0306-4522(95)00364-9

Elsevier ScienceLtd Copyright © 1995 IBRO Printed in Great Britain. All rights ~served 0306-4522/96 $9.50 + 0.00

CHRONIC C O N T I N U O U S INFUSION OF ( - ) N I C O T I N E REDUCES BASIC FIBROBLAST GROWTH FACTOR MESSENGER R N A LEVELS IN THE VENTRAL MIDBRAIN OF THE INTACT BUT NOT OF THE 6-HYDROXYDOPAMINE-LESIONED RAT M. B L U M , * G. W U , t G. M U D 0 , ~ ' ~ N. B E L L U A R D O , f : ~ K. A N D E R S S O N , * L. F. A G N A T I § and K. F U X E f ¶ *Fishberg Research Center of Neurobiology, Mount Sinai School of Medicine, New York, NY, U.S.A. 1"Department of Neuroscience, Karolinska Institute, 171 77 Stockholm, Sweden §Department of Human Physiology, University of Modena, Modena, Italy Abstract--A negative correlation has been found between smoking and Parkinson's disease. There is evidence to Suggest that this correlation appears to be associated with a neuroprotective role of nicotine for dopamine neurons at least in relation to mechanical injury. However, 1-methyl-4-phenyl-l,2,3,6,-tetrahydropyridine (MPTP) neurotoxicity to dopamine neurons is enhanced by chronic continuous (-)nicotine. More recently, basic fibroblast growth factor has been found to possess neurotrophic activities for many nerve cells including the dopamine cells/n vivo and/n vitro. Therefore, it is of interest to explore a possible effect of nicotine on basic fibroblast growth factor expression in the ventral midbrain of intact and 6-hydroxydopamine-lesioned rats and how treatment with nicotine can alter the 6-hydroxydopamine-induced injury of the nigral dopamine nerve cells as evaluated by dopamine biochemistry. In the present paper, an analysis of the effects of chronic continuous infusion of (-)nicotine via minipumps was carried out on basic fibroblast growth factor expression in the rental midbrain of the intact male rat and of the 6-hydroxydopamine lesioned rat. A quantitative messenger RNA protection assay analysis was used as well as an immunocytochemical analysis in the substantia nigra. Our findings give evidence that a two-week continuous infusion with (-)nicotine in the intact rat leads to substantial and dose-related (0.03-0.3 mg/kg per h) reductions of basic fibroblast growth factor messenger RNA levels in the ventral midbrain. These changes are not associated with changes in neuronal and glial basic fibroblast growth factor immunoreactivity in this region with the antibodies used. However, a one-week continuous infusion with (-)nicotine (0.125mg/kg per h) failed to significantly alter the basic fibroblast growth factor messenger RNA levels in the ventral midbrain of solvent and 6-hydroxydopamine-injected rats and thus also the 6-hydroxydopamine-induced increase of basic fibroblast growth factor messenger RNA levels in the ventral midbrain of the lesioned side observed at this time-interval and known to be of astroglial origin [Chadi G. et al. (1994) Neuroscience 61, 891-910]. In agreement, the 6-hydroxydopamine-induced depletion of dopamine in the neostriatum was unaltered by the nicotine treatment (0.125 mg/kg per h). Thus, chronic continuous (-)nicotine treatment may lead to a reduced basic fibroblast growth factor trophic tone in the ventral midbrain of the intact but not of the 6-hydroxydopamine-lesioned rat neither on the lesioned nor on the unlesioned side of the ventral midbraln. It seems possible that chronic nicotine treatment mainly reduces basic fibroblast growth factor messenger RNA levels of neuronal origin, since the astroglial messenger RNA levels dominate after the 6-hydroxydopamine-induced lesions. Key words: substantia nigra, dopamine, 3,4-dihydroxy-phenyl acetic acid, homovanillic acid, RNAse

protection assay, immunohistochemistry.

:~Present address: Institute of Human Physiology, University of Catania, Catania, Italy. ¶To whom correspondence should be addressed. Abbreviations: bFGF, basic fibroblast growth factor; DA, dopamine; DAB, diaminobenzidine; DOPAC, 3,4dihydroxyphenyl acetic acid; EDTA, ethylenediamine tetra-acetate; HPLC, high-performance liquid chromatography; HRP, horseradish peroxidase; HVA, homovanillic acid; IR, immunoreactive/immunoreactivity; MPTP, l-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine; 6OHDA, 6-hydroxydopamine; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulphate; SET, SDSEDTA-Tris-HC1; TE, Tris-HCI-EDTA.

The incidence of Parkinson's disease has been found to be reduced in smokers compared with nonsmokers.: The question that has been raised is whether nicotine is involved in the reported negative correlation between smoking and Parkinson's disease. It certainly seems possible that nicotine could simply enhance the ongoing compensation processes in terms of dopamine (DA) release in the remaining D A terminals in the neostriatnm of parkinsonian patients. 21 There is presently no evidence that treatment with nicotine in Parkinson's disease 169

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M. Blum et al.

prevents the onset of the disease or delays the development o f the symptoms. In support of a neuroprotective role of nicotine, however, is the morphological, neurochemical and physiological evidence obtained for ( - ) n i c o t i n e administered via chronic continuous infusion in the mechanical injury model of D A n e u r o n s . 16.19,22,23,24,26,36 In these experiments, the implantation of osmotic pumps for nicotine infusion was performed immediately after the lesion, and in addition, ( - ) n i c o t i n e was given four times i.p. with 30-min time-intervals at a dose of 0.5 mg/kg i.p. immediately following the lesion to allow for rapid increases in serum nicotine levels in relation to the lesion. Based on these observations it seems possible that nicotine may exert trophic actions against mechanically induced injury by favouring the synthesis of dopaminergic neurotrophic factors, possibly leading to increased survival of lesioned D A neurons. Nevertheless, in another model of Parkinson's disease, chronic continuous ( - ) n i c o t i n e treatment increases 1methyl-4-phenyl- 1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity to D A n e u r o n s : ,2°,25 It, therefore, becomes of interest to study a possible modulatory effect of nicotine on D A neurotrophic factors in intact rats and in different types of models for Parkinson's disease. Basic fibroblast growth factor ( b F G F ) has been shown to promote the survival of many nerve cell populations including the D A nerve cells in the substantia nigra both in vitro and in vivo probably via activation of astroglia. 7,11A3,34,33,37 It is released from injured neurons and/or from cells during brain damage, 34'35 suggesting a significant role in injury. b F G F may also act as an intracellular nuclear factor controlling gene expression 9:7,39 since inter alia it lacks a signal sequence present in proteins known to be secreted. Also 6-hydroxydopamine-(6-OHDA) induced degeneration of nigrostriatal D A neurons is associated with a marked temporal and spatial increase of astroglial b F G F synthesis. 9 Such data are not yet available for mechanical injury to the nigrostriatal D A neurons. All the above reported data suggested a possible correlation at the substantia nigra level between the neuroprotective effects of nicotine and enhancement of nigral b F G F mechanisms. To verify this hypothesis in the present paper we have analysed the effects of a chronic continuous infusion of ( - ) n i c o t i n e on b F G F m R N A levels and immunoreactivity within the ventral midbrain rich in D A nerve cells of intact and 6-OHDA-lesioned rats using a quantitative m R N A protection assay analysis and b F G F immunocytochemistry. The ability of such a treatment to alter the vulnerability of the nigrostriatal D A system to 6 - O H D A neurotoxicity has also been analysed. EXPERIMENTAL PROCEDURES

Specific pathogen-free adult Sprague-Dawley rats (B&K Universal Sweden) 150-250 g body weight were used in the

present study. The rats were kept under controlled temperature and humidity conditions with standardized lighting (lights on at 6.00 a.m. and off at 8.00 p.m.) and had free access to food pellets and tap water. The nicotine treatment protocol was as follows: Alzet minipumps (model 2002, SMA, London, U.K.) were implanted subcutaneously in the neck under anaesthesia with halothane (1.5-3%, Trofiel, Switzerland). The minipumps were filled with (-)nicotine hydrogen(+)tartrate (BDH Chemicals, Poule, U.K.) in amounts calculated to produce doses of 0.03, 0.125 and 0.3 mg/kg per h. In the case of 6-OHDA-lesioned rats (unilateral nigral microinjections, see below) the nicotinecontaining minipumps were implanted immediately after the nigral 6-OHDA injection (for bFGF mRNA level) or one week earlier (for DA and DA metabolite levels). In previous experiments in this laboratory, after infusion of 0.125 mg/kg per h, serum nicotine levels reached a similar level as found in smokers (50-65 ng/ml). Sham-operated animals were implanted with minipumps containing 0.9% sodium chloride instead of nicotine. Quantitative basic fibroblast growth factor mRNA protection assay After two weeks of continuous infusion of (-)nicotine the rats were rapidly decapitated and the ventral midbrain containing the substantia nigra and the ventral tegmental area were rapidly dissected out, frozen and taken for bFGF mRNA analysis. In the case of unilateral 6-OHDA-induced nigral lesions only a one week continuous (-)nicotine infusion was made, since the 6-OHDA-indueed increase in astroglial bFGF mRNA in the substantia nigra disappeared after this time-interval.9 RNA was isolated from the tissue samples and the levels of bFGF mRNA were determined by a quantitative ribonuclease protection assay essentially as previously described) ,6 Briefly, the tissue was homogenized in 0.5 ml of cold RNAse-free AT buffer [10 mM Tris-HC1, pH8, 3mM CaCI2, 2mM MgC12, 0.5raM dithiothreitol, 0.3 M sucrose, 0.15% Triton X-100] and layered over 0.4 ml of a 0.4 M sucrose cushion (in AT buffer) and centrifuged at ~2500g for 10min at 4°C. The supernatant was removed, transferred to an eppendorf tube and 50 #1 of 10 × SET buffer [1 × SET: 1% sodium dodecyl sulphate (SDS), 5 mM EDTA, I0 mM Tris-HC1, pH 8], and 5 #1 of proteinase K [10mg/ml] was added. After incubation at 45°C for 1 h, the samples were extracted with phenol chloroform, precipitated in ethanol and reconstituted in TE [I0 mM Tris-HCl, pH 7.5, 1 mM EDTA]. To ensure that the RNA samples were not degraded and to determine the concentration of RNA, two aliquots were taken from each sample for agarose gel electrophoresis and spectrophotometry at A260. For the nuclease protection assay a Sma I/Xho I fragment (nucleotides 525-1004) from a rat bFGF cDNA clone provided by Dr S. Shimasaki, 38 which includes most of the coding region for the mature bFGF peptide, was subcloned into the vector Bluescript/SK + (Stratagene). Unlabelled sense and high-specific activity (~ 1 × 109 c.p.m.//tg) 32p-labelled antisense RNA sequences were transcribed from the bFGF/Bluescript subclone according to the manufacturers recommendations. A standard curve was set up that consisted of known amounts of synthetic sense strand bFGF RNA ranging from 3 to 30 amol. The standards and known amounts of cytoplasmic RNA (5#g) isolated from the tissue samples were hybridized [40% formamide, 0.6 M NaCI, 4mM EDTA, 40mM Tris-HC1, pH7.4, 16mg/ml yeast RNA] with ~ 600 amol of bFGF antisense 32p-labelled RNA probe for ~ 1 6 h at 68°C followed by S1 nuclease digestion (500-700 IU; Pharmacia, Piscataway, NJ). Samples were then phenol-chloroform extracted, precipitated, reconstituted in TE and run on a 5% nondenaturing polyacrylamide gel. The gels were dried, exposed to a phosphosimaging screen and the amount of radioactivity in the protected

Nicotine and b F G F mRNA in the ventral midbrain RNA-RNA hybrids was counted. Attomoles of bFGF mRNA in the RNA samples were then determined by linear regression analysis of the standard curve. Student's unpaired t-test or one way ANOVA with the protected LSD test was used for the statistical analysis.

lmmunohistochemistry of basic fibroblast growth factor immunoreactivity Nine specific pathogen free male intact Sprague-Dawley rats (150g body wt) were used. Chronic continuous (-)nicotine infusion was given at a dose of 0.125 mg/kg per h (for details, see above). Previous work has demonstrated that chronic continuous infusion of this dose resulted in a protective action against mechanical injury of DA neurons when combined with acute intermittent nicotine treatment. 16'23 After a two-week (-)nicotine infusion the rats were perfused under deep barbiturate anaesthesia (pentobarbital, ACO, 60 mg/kg i.p,) with 0.9% saline followed by perfusion fixation with 0.16 M phosphate buffer (pH 6.9) containing 4% paraformaldehyde and 0.2% picric acid. 4° Post-fixation time was 90 min after which the brains were transferred into a 10% sucrose-containing phosphate buffer (0.1 M). Sections 14 # m thick were incubated with a mouse monoclonal antibody (bFM-I) raised against a synthetic 14-16 amino acid long b F G F peptide (provided by Dr K. Nishikawa at the Kanazawa Medical University, Japan) and shown to recognize the conformation of the b F G F essential to biological activity32 or a rabbit polyclonal antibody against an N-terminal (residues 1-24) synthetic peptide of bovine b F G F TM (generously provided by Dr A. Baird at the Whittier Institute for Diabetes and Endocrinology, Scripps Memorial Hospital, U.S.A.) overnight at 4°C in a moist chamber. These two antibodies can probably recognize different epitopes of the bFGF molecule which may help explain the different pattern of localization of bFGFimmunoreactivity (-IR), related, e.g., to the different masking of parts of the b F G F molecule at different locations. The primary monoclonal and polyclonal antibodies were diluted 1:1500 and 1:1000 in a phosphate-buffered saline (PBS) containing 0.3% Triton-X 100, respectively, These antibodies do not cross-react with acidic fibroblast growth factor. ~8'32 It was also confirmed by immunoblot analysis that the two anti-bFGF antisera used show little crossreactivity with acidic F G F ) ,t° The sections were washed and incubated with a biotinylated anti-mouse or antirabbit antiserum (1:100) (Amersham, U.K.) for 2 h at room temperature. After two rinses in PBS at room temperature the sections were subsequently incubated with horseradish peroxidase (HRP) streptavidin complex (1 : I00) (Amersham, U.K.) for 1 h. The sections were then rinsed in Tris-HC1 buffer (50 mM pH 7.4) and reacted in the same buffer containing 0.005% H202 and 0.02% diaminobenzidine (DAB). The sections were dehydrated and coverslipped with Entellan. In control experiments the sections were incubated with a b F G F antiserum which had been absorbed with 50/~g of the human recombinant bFGF (50 pg/ml antibody diluted l:10).

Computer-assisted morphometry and microdensitometry The monoclonal bFGF antibody only showed glial bFGF-IR profiles, while the polyclonal bFGF antibody showed both neuronal and glial bFGF-IR profiles (Fig. 2). Image analysis of the immunoreactivity obtained with the monoclonal antibody was used to measure the glial profiles and the polyclonal antibody was used to measure the neuronal profiles since the glial bFGF-IR profiles could be removed by a skip function. Thus, a size-threshold (circle diameter >6.5/~m) for accepting positive profiles was set. Morphometrical and microdensitometrical analysis of bFGF-IR nerve cell bodies and glial profiles within the substantia nigra were made in coronal sections of the rat brain after staining with the avidin-biotin peroxidase system (see above). Neuronal bFGF-IR profiles represent cyto-

171

plasmic profiles, while the astroglial profiles represent nuclear profiles. The image was acquired by a television camera from the sampled regions in the substantia nigra section and the profiles were evaluated after shading correction and discrimination. Together with the density of immunoreactivity profiles also the specific immunoreactive area was determined which depends on the size and density of the bFGF-IR profiles. The specific mean gray value of the bFGF-IR profiles was determined by subtraction of the mean gray value of adjacent gray matter (unspecific value). This gives an index of the intensity (concentration of the antigen in the neuronal and glial bFGF-IR profiles analysed) (for details see Refs 8,43). Pars compacta plus pars reticulata were analysed in the case of glial bFGF-IR since it was difficult to establish the border between zona compacta and zona reticulata in this glial bFGF staining (Fig. 2B). Only pars compacta was analysed in the case of neuronal bFGF-IR since the intention was to analyse mainly DA nerve cells (Fig. 2A). The area of specific immunoreactivity profiles (spArea), the number of specific immunoreactive profiles (n), and the mean gray value of specific immunoreactivity profiles (spMGV) are presented as means+S.E.M. For sampled fields, see Fig. 2. The Mann-Whitney U-test was used (non-parametrical procedures), since ratios are involved in evaluating the specific mean gray value. These procedures have been shown to produce reliable semiquantitative evaluations of the above parameters. 43

6-Hydroxydopamine lesion experiments A total of 70 male Sprague-Dawley rats (200 g) were used. Experiment 1. To test if continuous (--)nicotine infusion (0.125 mg/kg per h) could modulate the increase in nigral astroglial b F G F mRNA levels induced by nigral 6-OHDAinduced lesions as demonstrated at the one-week timeinterval after the lesion.9 Continuous (-)nicotine infusion was started immediately after the nigral 6-OHDA lesion (8 #g/4/t 1), using the same high dose as used by Chadi et al.9 For further details, see above and Fig. 3. The values obtained were expressed as ng/g of tissue wet weight and presented as Medians with the interquartile ranges. Non-parametrical statistic was performed with the Kruskal-Wallis distribution-free test followed by the Mann-Whitney U-test. Experiment 2. After a one-week continuous (-)nicotine infusion a partial 6-OHDA-induced lesion of the nigrostriatal DA system was made by means of intranigral injections of 6-OHDA (0.25/~g/4#1, Sigma, U.S.A.), dissolved in 0.9% saline containing ascorbic acid (0.2 mg/ml) on the left side for 4min. 4°'4~ The coordinates were - 4 . 4 mm, lateral 1.2 mm, ventral 7.8 mm according to the atlas of K6nig and Klippel. 29 Sham-operated rats instead received the vehicle alone. After a two-week continuous (-)nicotine infusion and one week of 6-OHDA lesion or solvent injection the rats were killed for biochemical analysis, involving high-performance liquid chromatography (HPLC) determination of neostriatal DA, 3,4-dihydroxyphenyl acetic acid (DOPAC) and homovanillic acid (HVA). 28,31 RESULTS

Intact rats Changes of basic fibroblast growth factor mRNA expression in the ventral midbrain after a two-week continuous (-)nicotine infusion. As seen in Table 1, c o n t i n u o u s nicotine infusion for two weeks at a dose o f 0 . 1 2 5 m g / k g per h was f o u n d in three separate experiments to p r o d u c e a reduction o f b F G F m R N A levels within the ventral m i d b r a i n , resulting in a n

172

M. Blum et al. Table 1. Effects of chronic continuous infusion with (-)nicotine on basic fibroblast growth factor mRNA levels in the ventral midbrain of the intact male rat as evaluated by RNAse protection assay Treatment Control Nicotine

Ventral midbrain amol/#g RNA Experiment 1 Experiment 2 Experiment 3 3.50 + 0.32 (8) 1.79 _ 0.37** (8)

3.10 + 0.33 (8) 1.24 _+0.35** (8)

3.67 + 0.23 (7) 2.55 ___0.27* (7)

Results from three independent experiments are shown. The overall effects of the treatment obtained by pooling the results from the individual experiments are as follows: 3.40+0.19 (23; control) vs 1'.73+0.23'** (23; nicotine). Means + S.E.M. are shown. Number of rats within parentheses. The Student's unpaired t-test was used. *P < 0.05; **P < 0.01; ***P < 0.001. overall reduction of 50% of the b F G F mRNA levels in this region containing the substantia nigra and the ventral tegmental area. In one experiment the effects of chronic continuous infusion of ( - ) n i c o t i n e was also analysed on b F G F mRNA levels within the dorsal hippocampal formation and the neostriatum. However, chronic continuous ( - ) n i c o t i n e infusion over two weeks in a dose of 0.125 mg/kg per h failed to modulate the b F G F mRNA levels both in the dorsal hippocampal formation (7.84+0.58 and 8.1 + 1.29 amol/#g RNA in control and ( - ) n i c o t i n e treated rats, respectively) and in the neostriatum (9.34+0.76 and 10.19+0.95amol/#g RNA in control and (-)nicotine-treated rats, respectively). The results from the dose-response experiment are shown in Fig. 1. Chronic infusion o f ( - ) n i c o t i n e over two weeks in the dose range used (0.03-0.3 mg/kg per h) resulted in a significant and marked dose-related disappearance of b F G F m R N A levels in the ventral midbrain.

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large numbers of b F G F - I R neuronal profiles were observed with the polyclonal b F G F antiserum against an N-terminal (residues 1-24) synthetic peptide of bovine b F G F ~8 in the zona compacta of the substantia nigra, with scattered neuronal profiles also found in the zona reticulata, while the monoclonal mouse antibody against a synthetic b F G F peptide 32 failed to show such neuronal immunoreactivity profiles (Fig. 2). The neuronal immunoreactivity was predominantly cytoplasmic and restricted to the perikarya and the larger dendritic proximal processes. Numerous astroglial nuclear b F G F profiles were found both within the zona reticulata and the zona compacta of the substantia nigra with the above antisera. As seen in Fig. 2 and Table 2, chronic continuous ( - ) n i c o t i n e infusion at a dose of 0.125 mg/kg per h neither altered the neuronal nor the astroglial b F G F - I R as seen from the specific immunoreactivity area, the numbers of specific immunoreactivity profiles and from the specific mean gray values. No immunostaining with the two b F G F antisera was found after incubation of the antibodies with human recombinant b F G F (50/~g/ml diluted antibody 1: 10).

6-Hydroxydopamine-lesioned rats 2-

Experiment h Effects of a one-week continuous (-)nicotine infusion on 6-hydroxydopamine induced changes in the basic fibroblast growth factor mRNA levels in the substantia nigra. A s seen in Fig. 3, a

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Changes of nigral basic fibroblast growth factor immunoreactivity after a two-week continuous (-)nicotine infusion. In line with previous findings17

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Fig. 1. Effects of chronic continuous infusion of different doses of (-)nicotine on the bFGF mRNA levels in the ventral midbrain as evaluated by mRNA protection assays. For details on treatment, see Experimental Procedures. Means ___S.E.M. are shown (n = 8 rats, except for the intermediate dose (0.125mg/kg per h, n =4)). One-way ANOVA with the protected LSD test was used. *P < 0.02; **P < 0.001 vs the control group.

6-OHDA-induced increase in nigral b F G F mRNA levels can be demonstrated on the lesioned side vs solvent injected side of sham-operated rats at the one-week time-interval in agreement with previous work 9 with no lesion-induced changes on the unlesioned contra lateral side. Continuous ( - ) n i c o t i n e treatment failed to significantly modulate the b F G F mRNA levels in the sham-injected and 6-OHDAlesioned rats, neither on the lesioned nor on the unlesioned side. Experiment 2: Effects of a two-week continuous

(--)nicotine infusion on a partial depletion of striatal levels of dopamine and its metabolites induced by

Nicotine and bFGF mRNA in the ventral midbrain

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Fig. 2. Photomicrographs of bFGF-immunoreactive nerve cell bodies and glial profiles within the substantia nigra. The polyclonal antibody showed both neuronal and glial bFGF-IR profiles (A, C, E), whereas the monoclonal antibody only showed glial bFGF-IR profiles (B, D, F). In A and B, the overview pictures show the sampling areas for image analysis of microdensitometric and morphometric parameters of the bFGF-IR. The area sampled for the polyclonal antibody is 0.100 + 0.0037 mm 2 (mean + S.E.M.) which covers only pars compacta (dashed line area in A) and the area for the monoclonal antibody is 0.289 mm 2 (0.517 x 0.560 mm) which covers both pars compacta and pars reticulata (solid line box in B). Enlargements of region shown in A and B are given in C and D, respectively, which are from control rats. The pictures in E and F have the same magnification as those in C and D but they are from nicotine-treated animals. Note that no difference can be seen between C and E or D and F. Scale bars = 200/~m. Scale bar in B applies also to A and scale bar in F applies also to C-E. SNC, pars compacta of the substantia nigra; SNR, pars reticulata of the substantia nigra.

6-hydroxydopamine. As seen in Table 3, the continuous ( - ) n i c o t i n e infusion starting one week before the 6-OHDA-induced lesion did not alter the reduction of the neostriatal DA, DOPAC and HVA levels induced by the lesion nor the lesion induced increase in neostriatal D A utilization. Negative results were also observed on the unlesioned side (Table 3). In intact and sham-operated rats a two-week continuous ( - ) n i c o t i n e infusion also failed to modulate the neostriatal DA, DOPAC and HVA levels on the two sides of the brain (Table 3).

DISCUSSION

The present results give clearcut evidence that a two-week continuous infusion of ( - ) n i c o t i n e in the intact male rat but not in the 6-OHDA-treated rat strongly and dose-dependently reduces b F G F m R N A levels selectively in the ventral midbrain containing the substantia nigra, and the ventral tegmental area rich in b F G F - I R DA containing nerve cell bodies. 3'1°A7 Nevertheless, the immunocytochemical analysis failed to demonstrate any

174

M. Blum et al. Table 2. Effects of chronic continuous (-)nicotine treatment on microdensitometric and morphometric parameters of the basic fibroblast growth factor immunoreactive neuronal and astroglial profiles in the substantia nigra Number of rats

spArea Fm 2

n

spMGV

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3905 _ 608 4609 _ 357

223 _ 37 221 ± 28

24 _ 1.4 27 ___3.0

4 5

4935 _ 641 6026 _ 432

58 + 5.8 62 + 2.6

30 _ 0.5 3 ! _ 3.8

Glial bFGF-IR Control Nicotine Neuronal bFGF-IR Control Nicotine

For details on treatment, see Experimental Procedures. Means + S.E.M. are shown. The Mann-Whitney U-test was used for the statistical analysis, spArea, specific immunoreactive area; n = number of immunoreactivity profiles; spMGV, specific mean gray value.

significant changes in n e u r o n a l a n d astroglial nigral b F G F - I R . However, it m u s t be realized t h a t the present b F G F antibodies with the i m m u n o c y t o chemical procedures used m a y n o t have seen all forms o f the b F G F protein. 14'15 If this aspect is n o t considered, there are several ways in which the present findings c a n be discussed such as reduction o f b F G F p r o t e i n turnover, a n d stabiliza t i o n o f the b F G F m R N A levels by the nicotine treatment, explaining the selective c h a n g e in b F G F m R N A levels vs b F G F p r o t e i n levels in the intact rat.

The fact t h a t n o reduction o f nigral b F G F m R N A levels was observed in the 6 - O H D A - t r e a t e d rats n o r in the sham-injected rat after c o n t i n u o u s one-week ( - ) n i c o t i n e t r e a t m e n t m a y be explained o n the basis t h a t nigral astroglial b F G F m R N A levels m a y dominate after s h a m or 6 - O H D A lesions o f the s u b s t a n t i a nigra. 9 Thus, astroglial b F G F m R N A levels m a y be less sensitive to d o w n r e g u l a t i o n by c o n t i n u o u s ( - ) n i c o t i n e treatment. F u r t h e r m o r e , diaschisis like p h e n o m e n a m a y occur, o n the unlesioned side, 12 m a k i n g it less sensitive to nicotine t r e a t m e n t with regard to reduction o f b F G F m R N A levels. Thus, n o

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Fig. 3. Effects of one week continuous (-)nicotine infusion on the bFGF mRNA levels in the ventral midbrain of rats unilaterally injected with 6-OHDA into the substantia nigra, b F G F mRNA was measured by a quantitative RNAse protection assay. The chronic continuous (-)nicotine infusion was carried out by subcutaneous implantation of Alzet minipumps, filled with (-)nicotine hydrogen(+)tartrate in amounts calculated to produce a dose of 0.125 mg/kg per h, immediately after unilateral nigral 6-OHDA injection had been made. The (-)nicotine infusion lasted for one week. Group: sham (the rat received a sham injection with the vehicle in one substantia nigra); sham/nit (plus the subcutaneous (-)nicotine infusion); lesion (the rat received a unilateral nigral injection of 8 #g 6-OHDA dissolved in 0.9% saline containing ascorbic acid); lesion/nit (plus the (-)nicotine infusion as above). Means ___S.E.M. are shown (n = 10 except for the lesion/nic group of unlesioned side n = 9). One-way ANOVA with Fisber's PLSD test was used. *P < 0.05 vs the sham of the same side.

Nicotine and bFGF mRNA in the ventral midbrain

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175

downregulation of b F G F mRNA levels was observed on the unlesioned side after a one week treatment with ( - ) n i c o t i n e after unilateral solvent or 6-OHDA injections. These results on b F G F mRNA levels are in agreement with the present observations that the depletion of striatal DA and its metabolites was unaltered by a two-week continuous treatment with nicotine, which was true also for the compensatory increase in DA metabolism found in the partially lesioned DA neurons. It seems possible that the maintained b F G F mRNA levels after the ( - ) n i c o t i n e treatment in 6-OHDA-lesioned rats may contribute to this observation. It will be of interest to evaluate the effect of MPTP on the nigral b F G F mRNA levels after this type of nicotine treatment, since MPTP induced DA neurotoxicity is enhanced by continuous ( - ) n i c o t i n e treatment. 4,25,27 In the intact animal the major action of chronic continuous nicotine infusion appears to be a downregulation of nicotinic cholinergic transmission including that in the DA cells.l This action of nicotine leads to a reduction in the excitability of the nigrostriatal and mesolimbic DA neurons and possibly to reduced burst firing. 19The higher the dose the greater may be the downregulation of transduction over the nicotinic receptors. It is conceivable that the reduction of impulse flow, especially of burst firing, will by itself lead to a reduction of b F G F gene expression within the DA nerve cell bodies in view of a possible reduced activation of protein kinase C and cAMP pathways as a response to reduced depolarization.39 Similar events may not take place in the astroglia explaining why 6-OHDA-induced and mechanically (sham) induced lesions of the DA neurons leading to increased astroglial b F G F mRNA levels9 do not respond to chronic nicotine with a downregulation of b F G F mRNA levels. In agreement, 6-OHDAinduced DA neurotoxicity was unaltered by (-)nicotine. Furthermore, diaschisis like phenomena in the substantia nigra on the contralateral side also may explain the failure to downregulate b F G F mRNA levels also in this side by continuous ( - ) n i c o t i n e treatment. Chronic continuous ( - ) n i c o t i n e infusion may also produce a reduction of b F G F protein metabolism, so that the reduction of b F G F mRNA levels does not lead to reduced b F G F protein levels. As a matter of fact, the reduced b F G F mRNA levels may even be the result of a negative feedback of the b F G F protein on its own transcription rate. It must also be considered that ( - ) n i c o t i n e possibly promotes the synthesis of another DA neurotrophic factor, so that a compensatory reduction of b F G F mRNA levels develops. However, chronic treatment with nicotine (1.18 mg/kg per day for 14 days) has not been found to change BDNF mRNA levels in the hippocampal formation. 3° A similar nicotine treatment protocol has also failed to significantly alter N G F mRNA levels in the hippocampus, the cerebral cortex and the striatum. 33

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Whatever the mechanism, the present analysis suggests a reduction o f b F G F trophic tone within cells o f the substantia nigra after chronic ( - ) n i c o t i n e treatment in the intact rats. It seems possible based on the present findings that mainly the neuronal b F G F tone is reduced, since no effects were found within the neostriatum, where the b F G F - I R is exclusively found within the astroglial cell populations 1° nor after 6 - O H D A - i n d u c e d lesions o f the D A nerve cells where astroglial b F G F tone may dominate. It must also be considered that a two-week continuous ( - ) n i c o t i n e treatment may alter the effects of b F G F on the injured D A nerve cells, e.g. by modulation o f F G F receptors.

CONCLUSION In conclusion, the present findings give evidence that chronic continuous infusion with ( - ) n i c o t i n e in the intact but not in the 6-OHDA-lesioned rat leads to substantial and dose-related reductions of b F G F m R N A levels which are selective for the ventral midbrain without any associated changes in the nigral astroglial and neuronal b F G F - I R . Acknowledgements--This work has been supported by a grant from the Verum Foundation, Germany. MG and BN were supported by Swedish Medical Research Council (MFR) and Italian National Research Council (CNR) Cooperation Agreement.

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