Chronic nicotine treatment counteracts dopamine D2 receptor upregulation induced by a partial meso-diencephalic hemitransection in the rat

BRAIN RESEARCH ELSEVIER

Brain Research 655 (1994) 25-32

Research report

Chronic nicotine treatment counteracts dopamine D 2 receptor upregulation induced by a partial meso-diencephalic hemitransection in the rat Ann Marie Janson *, Peter B. Hedlund, Kjell Fuxe, Gabriel von Euler Department of Neuroscience, Karolmska hzstitutet, S-171 77 Stockhohn. Sweden

Accepted 10 May 1994

Abstract

To further elucidate the previously demonstrated protective actions of nicotine on lesioned nigrostriatal dopamine (DA) systems (Janson and Moiler, Neuroscience, 57 (1993) 931-941), the present receptor binding experiments were carried out. Rats were partially hemitransected at the meso-dicncephalic junction and the effects of chronic continuous ( -)nicotine trcalment (osmotic pumps s.c., 0.125 mg/kg/h, 14 days) on [3H]N-propylnorapomorphine ([3H]NPA) and [3H]mcthylcarbamylcholinc ([3H]MCC) binding were investigated in striatal coronal sections to study the agonist binding sites of D A D , receptors and nicotinic cholinoceptors, respectively. In saline-treated but not in nicotine-treated rats, the lesion led to an increased B,,,~,~ w~luc of [3H]NPA binding. The B.... value of [3H]MCC binding was increased by nicotine treatment and decreased by the partial hemitransection. These results indicate that chronic nicotine treatment counteracts the lesion-induced upregulation of the high-affinity agonist binding site of the D A D e receptor, which may be explained by an increased presence of DA via a protective effect of nicotine on neostriatal DA terminals. This action of nicotine may be of interest in the treatment of ncurodcgencrativc diseases such as Parkinson's disease. Key words': Lesion; Nicotine; [3H]Methylcarbamylcholine; [3H]N-Propylnorapomorphinc: Protcction: Ncostriatum: Dopaminc

1. Introduction

Several early epidemiologic studies reported that the incidence of Parkinson's Disease (PD) was significantly lower in smokers as compared to non-smokers [15,25]. Although some studies have found contradictory results [37], the majority of epidemiologic studies confirms that a negative correlation exists between tobacco smoking and PD independent of other associated factors [4]. The etiology of PD is not known, but the disease largely depends on degeneration of the nigro-neostriatal dopamine (DA) neurons [32]. The protective actions by ( - ) n i c o t i n e against a mechanically induced retrograde and anterograde degeneration of this DAergic system [20] suggests that nicotine is involved

* Corresponding author. Fax: (46) (8) 216689. 0006-8993/94/$07.00 ~t;~1994 Elsevier Science B.V. All rights reserved SSDI 00(16-8993(94)00601-8

in the reported negative correlation between smoking and PD. Several histochemical and biochemical techniques have been employed to further analyze the putatively protective effect of chronic continuous nicotine treatment after a partial hemitransection at the meso-diencephalic junction [13,18,19,21,23,36]. The hemitransection allows comparisons between a lesioned and an intact side, since the latter has been shown not to be affected by the contralatera[ lesion [13,19,21,23]. This unilateral transection is only partial, which makes it possible to study compensatory mechanisms in surviving DA neurons [2,18]. In the present study we have investigated whether chronic, continuous nicotine treatment blocks the Icsion-induced upregulation of high-affinity DA D~ agonist binding in the rat neostriatum [2,10]. Furthermore, we have studied whether chronic nicotine treatment affects the disappearance of nicotinic cholinoceptors [21] caused by the lesion.

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2. Materials and methods 2.1. /ttffmal lreatment.s

Thirty-two male specific pathogen-free Sprague-Dawley rats (160 g body weight, B&Q Universal, Stockholm, Sweden) were used. The animals were kept under regular light conditions (lights on at 06.00 h and off at 18.00 h) and had free access to food pellets and tap water. The rats were anesthetized (chloral hydrate, 350 m g / k g ) and partially hemitransected at the right meso-diencephalic junction (K/3nig and Klippel level A3000) [27]. The operation, which spares the most medial portion of the ascending and descending pathways, has been described in detail [2,13,18,21,23]. In brief, the rats were m o u n t e d in a stereotaxic frame (David Kopf, Tujunga, CA, USA) and a knife (4 mm wide, 1 mm thick at the edge) was inserted at an angle 20 ° caudally to the frontal plane (Br - 1.0 ram, L + 1.0 ram, ventrocaudally 10.0 mm). W h e n reaching the skull base, the knife was retracted 2 m m and then moved laterally until reaching the bone. The extent of the lesion was histologically verified in sagittal brain sections (Fig. 1). Immediately following the lesion, osmotic minipumps (Alzet model 2(I02, Alza Corporation, Palo Alto. CA, USA) were implanted subcutaneously along the dorsal aspect of the neck between the scapellae. The p u m p s continuously released ( - ) n i c o t i n e - h y d r o g e n ( + )tartrate (0.125 m g / k g / h nicotine base dissolved in 0.9% saline, BDH Chemicals, Poole, UK) for 14 days. This treatment leads to a mean serum nicotine level of 50-65 n g / m l [13,21]. In addition to the chronic continuous administration of nicotine, four injections of ( - ) n i c o t i n e were given starting immediately following the lesion (intraperitoneal, 0.5 m g / k g , 30-min time intervals) to allow prompt high nicotine concentrations in the brain. Rats with a similar lesion receiving saline with an identical protocol served as control animals. Two weeks after the unilateral transection, the rats were decapitated using a guilliotine (Harvard A p p a r a t u s Ltd., Edenbridge, UK) without any prior anesthesia. After removal, the brains were rapidly frozen on dry-ice. Coronal 20-1zm-thick sections were made in a Leitz 1720 cryostat throughout the neostriatum (K6nig and Klippel level A8920-A7200) [27] and thaw-mounted on gelatin-coated slides. 2.2. [ ¢tt]N-Propylnorapornorphine binding

Cryostat sections were incubated under equilibrium conditions (I h, +23°C) with 0.15-13 nM (-)N-propyl-[3H]N-propylnorapo morphine ([3H]NPA, 2300 G B q / m m o l , NEN, Boston, MA, USA) in a 50 m M Tris buffer (pH 7.4, 23°C) containing 5 m M MgCI 2, 1 m M E D T A , 0.01% L-ascorbic acid and 0.05% bovine serum albumin. This agonist selectively labels high-affinity D 2 receptors in vitro [12]. Saturation curves with ten concentrations of the ligand were performed, and the binding in the presence of the selective D2 antagonist raclopride (10 /~M; K6hler et al., 1985, Astra, S6dert~lje, Sweden) was defined as non-specific. The incubation was stopped by washing the sections (3 × 5 min) in ice-cold Tris buffer, after which the slides were dried under a stream of cold air. The filter-wipe procedure was performed according to Hedlund et al. [17]. Each half of the dried sections was carefully wiped off with slightly moist G F / B filter papers ( W h a t m a n International, UK),

Fig. 1. Histological verification of the lesion (see Materials and methods). Composite photomicrographs of Cresyl violet-stained sagittal sections at three different medio-lateral levels (A = +3.4 mm, B = + 1.4 ram, C = +0.9 m m laterally to the sagittal midline of the brain) showing the extension of the partial meso-diencephalic hemitransection. *, lesion; CPu, caudate putamen; Th, thalamus; SN, substantia nigra; VTA, ventral tegmental area. Bar = 1 ram.

i ¸ i;iiiiiiiiii i ¸¸ iii~

~

A.M. Janson et aL / Brain Research 655 (1994) 25 32 allowing the intact and lesioned halves of the section to be analyzed separately. T h e radioactivity of the filters was determined by liquid scintillation spectrometry (Beckman LS 1800, Irvine, CA, USA). Bm~X values were expressed as f m o l / s e c t i o n instead of f m o l / m g of protein due to the regional distribution of the receptors within the section. The K d and Bin, × values were calculated using a computerassisted iterative non-linear regression analysis [30]. Data points having standard residual values with an absolute value > 2 were excluded from the analysis, as were curves with less than 8 points.

27

2.5. Statistics Statistical analysis comparing the lesioned and intact sides in each animal was performed with a two-tailed paired Student's t-test The effects of nicotine were analyzed using a two-tailed unpaired Student's t-test. Bonferroni's procedure was applied where more than one comparison was done in the same animals [28].

3. Results 2.3. Quantitatit,e receptor autoradiography

3.1. [ 3H]N-Propylnorapomorphine binding Slides from other rats (K6nig and Klippel level A7400) [27] were processed for receptor autoradiography and incubated under similar conditions with 1 nM of [3H]NPA in the presence or absence of 10 /xM raclopride. The dried sections were exposed to a 3H-sensitive film (3H-hyperfilm, A m e r s h a m , Buckinghamshire, U K ) for 3 months. The autoradiograms were analyzed using a computer-assisted image analyzer (Sistemi Avanzati, Milan, Italy) linked to a video camera (A.I.S.I., A n n Arbor, MI, USA) equipped with a macro-objective (Olympus). The computer software was developed by Imaging Research Inc. (Brock University, St. Catharines, ON, Canada). T h e entire neostriatum was divided into a medial and a lateral half, since the protective effects of ( - ) n i c o t i n e were previously demonstrated to be more prominent in the medial neostriatum [21], where less D 2 receptors are found [3,24], and the respective m e a n grey values were measured. The specific binding was calculated on the intact and lesioned sides as the m e a n value of four sections (the specific binding was approximately 6 5 - 7 0 % of the total binding). Using standard curves calculated from pre-fabricated [3HI-labeled polymer strips with 8 different known activities ( A m e r s h a m micro-scale, A m e r s h a m , Buckinghamshire, UK) the grey values could be converted to f m o l / m m 2. The protein content in a 20-/xm section has been determined to be 2.80 / x g / m m 2 and the values could therefore be recalculated to f m o l / m g protein [6,11]. T h e lesion induced a small but significant ipsilateral reduction in neostriatal area (see also Kultas-Ilinsky et al., 1990 [29]), both in saline-treated (6.3_+2.3% reduction, mean _+S.E.M., n = 8) and in nicotine-treated rats (6.5 _+ 3.7% reduction, n = 8). T h e results obtained in the present study were not corrected for this minor shrinkage.

2.4. [~H]Methylcarbamyleholine binding The entire binding experiment was carried out in the cold ( + 4°C). After rinsing the cryostat sections (2 × 5 min) in a 50 m M Tris buffer (pH 7.4,+4°C) containing 1 m M MgC12. 120 m M NaCI, 5.0 m M KCI and 2.0 m M CaCI 2, the sections were incubated under equilibrium conditions (1 h, +4°C) with 0.85-74 nM N-methyl-[3H]methyl carbamylcholine iodide ([3H]MCC, 3200 G B q / m m o L NEN, Boston, MA, USA). This agonist selectively labels nicotinic cholinoceptors [1,7]. Saturation curves with ten concentrations of the ligand were performed, and the binding in the presence of 10 /xM ( - )nicotinehydrogen-(+)tartrate was defined as non-specific. The incubation was stopped by washing the sections (3 x 1 min) in the same Tris buffer, after which the slides were dried u n d e r a stream of cold air. The filter-wipe procedure and the data analysis were performed as described for the [3H]NPA binding. In order to relate the effects of the partial hemitransection on the n u m b e r of nicotinic cholinoceptors to the estimated a m o u n t of D A terminals remaining, the lesion-induced effects on the Bmax value of [3H]MCC binding were compared to the lesion-induced effects on three different parameters for neostriatal D A terminals: tyrosine hydroxylase immunoreactivity [21], catecholamine levels [13] and extracellular dopamine levels [23].

In saline-treated rats, the partial hemitransection significantly increased the B .... value ( > 40%) of [3H]NPA binding in striatal sections, whereas the increase in K 0 value was not significant (Fig. 2). However, in rats chronically treated with nicotine no significant changes in the Bmax or K 0 values of [3H]NPA binding were observed (Fig. 2). The [3H]NPA binding on the lesioned side was significantly lower after nicotine treatment ( P < 0.05, Student's two-tailed unpaired t-test) than after saline-treatment, as illustrated by representative saturation curves in Fig. 3. In saline-treated rats, the partial hemitransection significantly increased (20-30%) the specific binding of [3H]NPA in both the medial and the lateral part of the neostriatum, as analyzed by quantitative receptor autoradiography at the rostrocaudal level A7400 [27] (Fig. 4). This increase in [3H]NPA binding is illustrated by a %

KD

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1

100

1

Bmax

50

0

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Nicotine

bame

Nicotine

Fig. 2. Effects of chronic continuous treatment with (-)nicotine (subcutaneous osmotic pumps, 0.125 m g / k g / h , 14 days) on changes in [3H]N-propy]norapomorphine ([3H]NPA) binding parameters induced by partial hemitransection at the meso-diencephalic junction. T h e K d and Bmax values on the lesioned side (11) are expressed as a percentage (mean + S.E.M., n = 6) of the intact side ( [] ). The absolute K d and Bmax values on the intact side were 3.9 nM and 11.4 f m o l / s e c t i o n in saline-treated rats and 3.4 nM and 9.9 f m o l / s e c t i o n in nicotine-treated rats, respectively. The saturation curves were obtained from filter-wiped coronal striatal sections incubated with 10 concentrations of [3H]NPA, using raclopride ( 1 0 / x M ) for the determination of non-specific binding. Statistical analysis was performed with a two-tailed paired Student's t-test (* P < 0.05) comparing the lesioned side with the unlesioned side and applying Bonferroni's procedure.

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Free ligand (nM) Fig. 3. Representative saturation curves with [3H]N-propylnorapomorphine ([3H]NPA) from the lesioned side of rats partially hemitransected at the meso-diencephalie junction and treated with saline ( [ ] ) or ( -)nicotine (II, osmotic pump s.c., 0.125 m g / k g / h , 14 days). The K d and Bmax values were 3.3 nM and 13.4 fmol/section in the saline-treated rat and 2.9 nM and 10.1 fmol/section in the nicotinetreated rat, respectively. The saturation curves were obtained from filter-wiped coronal striatal sections incubated with 10 concentrations of [3H]NPA, using raclopride (10/xM) for the determination of non-specific binding.

representative autoradiogram in Fig. 5. However, in nicotine-treated rats, the partial hemitransection significantly increased [3H]NPA binding only in the lateral

0

Saline

Nicotine

0

Saline

Nicotine

Fig. 4. Effects of chronic continuous treatment with (-~nicotine (subcutaneous osmotic pumps, 0.125 m g / k g / h , 14 days) tm changes in regional [3H]N-propylnorapomorphine ([3HJNPA) binding induced by partial hemitransection at the meso-diencephalic junction. The binding of [3H]NPA on the lesioned side (111) is expressed as a percentage (mean _+S.E.M., n = 6) of the intact side ( Q ). The absolute values were 697 (saline) and 600 (nicotine) f m o l / m g protein in the media[ neostriatum and 1010 (saline) and 928 (nicotine) f m o l / m g protein in the lateral neostriatum. The binding of [3H]NPA (t nM), using raclopride (10 # M ) for the determination of non-specific binding, was analyzed by quantitative autoradiography in coronal striatal sections. Statistical analysis was performed with a two-tailed paired Student's t-test (** P < 0.01, *** P < 0.002) comparing the lesioned side with the unlesioned side.

Fig. 5. Photomicrograph of total [3HlN-propylnorapomorphine ([3H]NPA) binding (1 nM) in a coronal section (K6nig and Klippel level A7400) [27] from a saline-treated rat partially hemitransected at the right meso-dieneephalic junction. The brighter right side of the section indicates the lesion-induced increase in [3H]NPA binding. Bar = 1 mm.

A.M. Janson et al. / Brain Research 655 (19941 25-32 Table 1 Effects of chronic continuous treatment with ( - ) n i c o t i n e (subcutaneous osmotic pumps, 0.125 m g / k g / h , 14 days) on [3H]methylcarbamylcholine ([3H]MCC) binding in striatal sections of rats partially hemitransected at the meso-diencephalic junction For details on treatment and procedures, see text. Means+S.E.M., n = 5-6. Statistical analysis performed with a two-tailed paired Student's t-test applying Bonferroni's procedure. * P < 0.05. Treatment Saline Intact side Lesion side

Bmax (fmol/section)

Kd (nM)

3.59 + 0.36-]

10.00 + 2.08

2.43 _+0.18 /

8.19 + 1.20

29

Specific bound [3H]MCC (fmol/section) 4 3 •

g° 0

S 20

..........

40

rl

60

80

Free ligand (nM)

Nicotine Intact side Lesion side

6.11 + 0.21q

9.38+ 1.76

4.09 + 0.403

7.75 +_ 1.48

part (20-30%) but not in the medial part (10-15%) of the neostriatum (Fig. 5) at the rostrocaudal level analyzed. The [3H]NPA binding on the lesioned side was significantly lower after nicotine treatment ( P < 0.05, Student's two-tailed unpaired t-test) than after salinetreatment in the medial part of the caudate-putamen, whereas no significant difference was found in the lateral part.

3.2. [*H]Methylcarbamylcholine binding The partial hemitransection significantly decreased ( > 30%) the Bma× value of [3H]MCC binding both in saline- and nicotine-treated rats (Table 1), and the decreases were not statistically different between the two groups (Table 2). No significant effects of the partial hemitransection were observed on the Ko value

Fig. 6. Representative saturation curves with [~tl]-methylcarbamylcholine ([3H]MCC) from the lesioned side of rats partially hemitransected at the meso-diencephalic junction and treated with saline ( [] ) or ( - )nicotine ( • ) (subcutaneous osmotic pump. I). 125 m g / k g / h , 14 days). The K d and Bm,,x values were 8.5 nM and 2.2 fmol/section in the saline-treated rat and 6.8 nM and 3.9 fmol/section in the nicotine-treated rat. The saturation curves were obtained from filterwiped coronal striatal sections incubated with 10 concentrations of [3H]MCC using ( - ) n i c o t i n e 10 ~tM) for the determination of nonspecific binding.

of [3H]MCC binding m either group. The chronic nicotine treatment by itself resulted in an increase in the Bm~,× value of [3H]MCC binding but did not affect the K d value (Table 1). The difference in [3H]MCC binding on the lesioned side after nicotine as compared to saline treatment is illustrated by representative saturation curves in Fig. 6. The partial hemitransection increased the calculated number of [3H]MCC binding sites per estimated amounts of D A terminals by 40-140% in saline-treated rats in contrast to 5 - 2 5 % in nicotine-treated rats (Table 2).

Table 2 Effects of chronic continuous treatment with ( - ) n i c o t i n e (subcutaneous osmotic pumps, 0.125 m g / k g / h , 14 days) on remaining [3H]methylcarbamylcholine ([3H]MCC) binding sites and DA nerve terminals ipsilateral to a partial hemitransection at the meso-diencephalic junction The left panel shows the mean number of [3H]MCC binding sites (A) and DA terminal parameters ( B - D ) on the lesioned side expressed as a percentage of the intact side. The right panel shows the ratios between remaining [3H]methylcarbamylcholine ([3H]MCC) binding sites (A) and DA nerve terminal parameters (B-D). Treatment

Saline Lesion side Nicotine Lesion side

Levels remaining in % of intact side

A in relation to B - D

A

B

C

D

A/B

A/C

A/D

[3H]MCC ~'

TH-Ii IR b

CA c

DA in vivo d

[3H]MCC

[3H]MCC

[~H]MCC

(B .... )

(area)

(levels)

(extracellular levels)

TH-Ii IR b

CA c

DA in vivo d

68%

48%

28%

29%

1.42

2.43

2.34

67%

63%

53%

63%

1.06

1.26

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a Bmax of [3H]MCC binding determined in sections by the filter wipe technique, for absolute values, see Table I. h Area of tyrosine hydroxylase-like immunoreactive (TH-li IR) nerve terminal profiles determined by semi-quantitative immunohistochemistry, for absolute values see Janson et al., 1988 [21]. c Catecholamine (CA) levels determined by quantitative histofluorimetry, for absolute values see Fuxe et al., 1990 [13]. a Bilateral intrastriatal microdialysis in the halothane-anesthetized rat, for absolute values see Janson et al., 1991 [23].

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4. Discussion In saline-treated rats the partial hemitransection caused an upregulation of the D A D 2 receptor agonist binding, in agreement with earlier studies where decreased striatal D A led to a D A D 2 receptor supersensitivity [2,10]. Thus, we found both an increase in [3H]NPA binding at 1 nM using quantitative receptor autoradiography and an increase in the Bmax value of [3H]NPA binding using the filter-wipe technique. Both the medial and lateral part of the neostriatum appeared to be similarly affected, at least at the rostrocaudal level analyzed, although the present lesion model preferentially cuts the lateral nigro-striatal dopamine projections [2,13,18,21]. The K a value of [3H]NPA binding was not significantly affected by the lesion. However, the great variance of the data may have hidden a small lesion-induced K d increase. The lack of significant effects of the partial hemitransection on [3H]NPA binding following chronic nicotine treatment, as evaluated in saturation analysis on striatal sections, indicates that nicotine counteracts the upregulation of the high-affinity agonist binding site of the D A D 2 receptor. This counteraction was also observed by quantitative receptor autoradiography in the medial, but not in the lateral part of the neostriatum, using a single concentration of [3H]NPA close to its K d value. The higher degree of counteraction of the lesion-induced D 2 receptor upregulation in the medial part of the neostriatum is in agreement with the finding that chronic nicotine treatment mainly protects DA nerve terminals in the medial and intermediate parts of the neostriatum, as evaluated by tyrosine hydroxylase immunoreactivity [21]. Thus, the sparing of DA terminals by nicotine appears to be sufficient to release enough dopamine to prevent DA D 2 receptor upregulation. However, it cannot be excluded that the present results are partly due to a direct modulation by chronic continuous nicotine administration of the high-affinity agonist binding site of the D A D 2 receptor [22] on the lesioned side. We have previously postulated that the putatively protective actions of chronic nicotine treatment against nigral DA nerve cell degeneration after partial hemitransection [13,19,21,23] is related to a nicotine-induced desensitization of nicotinic cholinoceptors located on DA neurons [9,21]. This functional desensitization is thought to be responsible for the increase in the number of nicotinic cholinoceptors induced by chronic continuous nicotine treatment, without affecting the K d value [31,38] as was also observed in the present study. Thus, the functional desensitization of the central nicotinic cholinoceptor by chronic nicotine treatment [34] may be due to a decreased ability to form cholinoceptors capable of opening their cation channels permeable for Na +, K + and Ca 2+ [8,35],

which could also explain the nicotine-induced reduction in burst firing of DA neurons on the lesioned side [14]. The partial hemitransection resulted in a significant reduction in the Bmax value of striatal [3H]MCC binding indicating a reduced total number of nicotinic cholinoceptors or at least a reduced number of the receptor subunits at which nicotine binds [40]. It seems likely that this reduction mainly reflects the disappearance of DA nerve terminals on which a large part of the nicotinic cholinoceptors may be located [5,9,16,21. 39]. However, this reduction remained the samc in nicotine-treated rats despite of the partial protection of striatal DA nerve terminals by chronic nicotine treatment [13,18,21,23]. The calculated amount of nicotinic cholinoceptors per estimated DA nerve terminal parameters on the lesioned side as compared with the intact side was almost unchanged in nicotine-treated rats but twice as high in saline-treated rats. This result may imply that the protective activity of chronic continuous nicotine treatment on DA nerve terminals is mainly related to a protection of DA nerve terminals lacking presynaptic nicotinic cholinoceptors. An alternative explanation is that after chronic continuous nicotine treatment the DA nerve terminals are initiating sprouting phenomena [13,21] and therefore may not express agonist binding nicotinic cholinoceptors. Alternatively, a different distribution of nicotinic cholinoceptors is seen on the nicotine-treated lesioned side of striatum. These results support the view that chronic continuous treatment with nicotine may have neuroprotective actions against degeneration of the nigro-neostriatal dopamine system, and may explain the negative correlation between tobacco smoking and PD [4]. Thus, chronic continuous nicotine treatment may be beneficial in the treatment of neurodegenerative diseases such as PD by reducing the progressive degeneration of nigro-neostriatal DA neurons [33]. Taken together, the present results demonstrate that chronic continuous nicotine treatment counteracts the lesion-induced D A D 2 receptor upregulation, which probably reflects an increased survival of DA nerve terminals.

Acknowledgements We thank Beth Andbjer, Ulla-Britt Finnman and Ulla Hasselrot for excellent technical assistance. This work was supported by grants from the German Research Council Smoking and Health, Karolinska Institutet Research Funds, Wiberg's, Bergvall's and Hierta's Foundations, the Swedish Society of Medicine, and the Swedish Medical Research Council (12X-10816-01 and 14X-10377-02).

A.M. Janson et al. /Brain Research 655 (1994) 25-32

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