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Research Report

A nitric oxide synthase inhibitor decreases 6-hydroxydopamine effects on tyrosine hydroxylase and neuronal nitric oxide synthase in the rat nigrostriatal pathway
Margarete Zanardo Gomes a , Rita Raisman-Vozari b,c,d , Elaine A. Del Bel a,
a

Department of Physiology, FORP, University of So Paulo, Av. Caf S/N, 14040-904 Ribeiro Preto, SP, Brazil Institut National de la Sant et de la Recherche Mdicale, Unit 679, Paris F-75013, France c Universit Pierre et Marie Curie-Paris6, Institut Fdratif de Recherche, de Neurosciences Unit Mixte de Recherche S679, Paris F-75013, France d Assistance PubliqueHpitaux de Paris, Groupe Piti-Salptrire, Paris, F-75013, France
b

A R T I C LE I N FO Article history: Accepted 31 January 2008 Available online 13 February 2008 Keywords: Dopamine
L-NOARG

AB S T R A C T There is evidence that nitric oxide plays a role in the neurotransmitter balance within the basal ganglia and in the pathology of Parkinson's disease. In the present work we investigated in striatal 6-hydroxydopamine (6-OHDA) lesioned rats the effects of a nitric oxide synthase (NOS) inhibitor, NG-nitro-L-arginine (L-NOARG), given systemically on both the dopaminergic (DA) neuronal loss and the neuronal NOS cell density. We analyzed the DA neuronal loss through tyrosine hydroxylase immunohistochemistry (TH). The nitrergic system was evaluated using an antibody against the neuronal NOS (nNOS) isoform. Treatment with the L-NOARG significantly reduced 6-OHDA-induced dopaminergic damage in the dorsal striatum, ventral substantia nigra and lateral globus pallidus, but had no effects in the dorsal substantia nigra and in the cingulate cortex. Furthermore, L-NOARG reduced 6OHDA-induced striatal increase, and substantia nigra compacta decrease, in the density of neuronal nitric oxide synthase positive cells. These results suggest that nitric oxide synthase inhibition may decrease the toxic effects of 6-OHDA on dopaminergic terminals and on dopamine cell bodies in sub-regions of the SN and on neuronal nitric oxide synthase cell density in the rat brain. 2008 Elsevier B.V. All rights reserved.

Substantia nigra Globus pallidus Cingulate cortex Parkinson's disease Neuronal nitric oxide synthase

1.

Introduction

Parkinson's disease (PD) is a major neurological disorder that affects movement, balance and fine motor control. These impairments are related to the progressive degeneration of DA

neurons in the substantia nigra pars compacta (SN), with a concomitant reduction of striatal dopamine levels (Agid, 1991). In addition to the striatum, the DA neurons from the SN are known to also innervate various extrastriatal structures, including the globus pallidus (GP) and the cortex, that are also

Corresponding author. Fax: +55 16 36330999. E-mail address: eadelbel@forp.usp.br (E.A. Del Bel). Abbreviations: 6-OHDA, 6-hydroxydopamine; L-NOARG, NG-nitro-L-arginine; DA, dopaminergic; NOS, nitric oxide synthase; nNOS, neuronal nitric oxide synthase; TH, tyrosine-hydroxylase; PD, Parkinson's disease; TH+, tyrosine-hydroxylase positive; nNOS+, neuronal nitric oxide synthase positive; L-NAME, N(G)-nitro-L-arginine methyl ester; 7-NI, 7-nitroindazole; MPTP, 1-methyl-4-phenyl-1,2,3,6tetrehydropyridine; MPP+, 1-methyl-4-phenylpiridinium iodide 0006-8993/$ see front matter 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2008.01.088

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affected in PD and in animal models of the disease (Solis et al., 2007; Anaya-Martinez et al., 2006; Debeir et al., 2005; Freeman et al., 2001; Gauthier et al., 1999; Prensa and Parent, 2001). Although the etiology of PD is unknown, several lines of evidence suggest that oxidative stress may contribute to the mechanism of nerve cell death by the production of reactive oxygen species such as nitric oxide (Jenner, 2003). Nitric oxide (NO), a free radical gas, is a highly reactive molecule (Dawson et al., 1994). It is produced from L-arginine and it acts as a neurotransmitter or neuromodulator when synthesized by the neuronal form of the enzyme NOS (Forstermann et al., 1991; Bredt and Snyder, 1990). nNOS is a constitutive, cytosolic, Ca2+/calmodulin-dependent enzyme (Bredt and Snyder, 1990; Klatt et al., 1992) that occurs in neuronal cell bodies, dendrites, and axons (Bredt et al., 1990). Because NO has a short action time the inhibitors of NOS became a very effective way to study NO system function. Systemic application of L-arginine analogues such as L-NOARG (Dwyer et al., 1991) and N(G)-nitro-L-arginine methyl ester (L-NAME) has been shown to produce an in vivo (Carreau et al.,

1994) and in vitro (Traystman et al., 1995) time-dependent irreversible inhibition of nNOS. Systemic and intrastriatal microinjection of selective (7-nitroindazole) and non-selective (L-NOARG and L-NAME) nNOS inhibitors in rodents interfere with motor behavior (Del Bel et al., 2002; De Oliveira et al., 1997; for review see Del Bel et al., 2005). In experimental models of PD (reviewed by Kavya et al., 2006), immunoreactivity for nNOS in the SN is consistently increased 5 h after 1-methyl-4-phenyl-1,2,3,6-tetrehydropyridine (MPTP) treatment (Muramatsu et al., 2002), but decreased 1 7 days after injection. MPTP treatment also augmented the expression and activity of NO-activated guanylate cyclase (Chalimoniuk et al., 2006; Chalimoniuk et al., 2004). We have previously found an increase in the nNOS cell density 35 days after 6-OHDA medial forebrain bundle-induced lesion (Gomes and Del Bel, 2003). Moreover, nNOS inhibitors, 7-NI and Smethyl thiocitrulline, exhibited protection in mice and baboons from MPTP-induced nigral cell loss (Schulz et al., 1995; Hantraye et al., 1996; Watanabe et al., 2007). Treatment with 7-NI was also reported to be protective against DA neuronal damage

Fig. 1 Effect of intrastriatal 6-OHDA and of the L-NOARG treatment on striatal and pallidal TH+ fibers. Photomicrographs show the sections at the anterior (1.3 mm), medial (0.2 mm) and caudal ( 1.3 mm) striatum from Bregma of the sham (AD), 6-OHDA (A'D') and 6-OHDA treated with L-NOARG (A"D"). The 6-OHDA lesion caused a reduction in the TH+ fibers primarily in the middle and caudal part of the dorsal striatum (B and C) The treatment with L-NOARG attenuated the loss of TH+ fibers (B" and C"). Similar profiles of effects were observed in the GP (DD). Scale bar AC": 0.4 mm; DD": 0.06 mm.

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induced by in vivo infusion of 1-methyl-4-phenylpiridinium iodide (MPP(+)) in freely moving rats (Di Matteo et al., 2006) and non-selective NOS inhibitors reduce amphetamine-induced rotations in a 6-OHDA model (Barthwal et al., 2001). Also Singh et al. (2005) found animals pretreated with a NOS inhibitor, L-NAME, exhibited complete protection against amphetamineinduced rotations in a 6-OHDA unilateral intrastriatal model. The authors concluded that augmented NO availability subsequent to inducible isoform of NOS (iNOS) induction play an important role in the initial phase of neurodegeneration. Furthermore, in nNOS knockout mice, MPP+ or MPTP-induced neuronal damage was less marked than in wild type mice (Przedborski et al., 1996). However there are no studies concerning the influence of in vivo nNOS inhibition neither on the DA loss after intrastriatal 6-OHDA-induced lesion or in the subsequent modifications of nNOS. This neurotoxin, when injected into striatum, induces progressive and partial lesions of the nigrostriatal DA system in rat (Blandini et al., 2007; Winkler et al., 2002; Kirik et al., 1998; Sauer and Oertel, 1994) and mice (Pavon et al., 2006). Thus, the aim of this work was to assess the effects of the NOS inhibition by L-NOARG given systemically on both the DA neuronal loss and the neuronal NOS cell density in rats after intrastriatal injections of 6-OHDA.

2.

Results

First, the group of rats microinjected with saline (sham) and treated with L-NOARG did not show significant differences from sham rats treated with saline in this study.

2.1. Effect of intrastriatal 6-OHDA injections on the density of TH+ fibers and cells and the L-NOARG treatment
In the striatum: Representative cases from three different anatomical levels of striatum of the sham (AC), 6-OHDA (AC) and 6-OHDA treated with L-NOARG (AC) rats are illustrated in the Fig. 1. Densitometry revealed a significant reduction of TH+ in the caudal part of striatum of 6-OHDA rats in the lesioned side of the brain. This rats presented 22.7% of the optical density when compared to sham rats (F[214] = 25.354; P < 0.0001). More moderate, but still significant reduction was observed in the middle part of this structure (F[214] = 14.227; P = 0.001, Fig. 2), which optical density was 51.8% remaining. Anterior portion did not differ significantly from sham rats (F[214] = 0.752; P = 0.492 Fig. 2), presenting 94.2% optical density. We did not find significant differences in the contralateral striatum. Qualitative analysis indicated that the nucleus acumbens was not affected (results not shown). Densitometry revealed a significant loss in the caudal striatum of the 6-OHDA treated with L-NOARG group, when compared with same side sham rats (70% optical density, F[214] = 25.354; P < 0.0001; Fig. 2). Reductions in the anterior and middle portions were not significantly different from sham (respectively 97.1% and 82.8% optical density). Regardless the 6-OHDA group, the reductions in the medial and caudal level were significantly smaller (respectively F[214] = 14.227; P = 0.001 and F[214] = 25.354; P < 0.0001, Fig. 2).

Fig. 2 Upper panel: Quantification of striatal TH+ fibers by optical density measurements at three levels along the anteroposterior axis of the striatum: effect of intrastriatal 6-OHDA injection and of the L-NOARG treatment. Values represent the percentage (mean SEM) of the optical density from the same striatal side of sham rat sections. Numbers at the base of each bar indicate the distance (in mm) from the Bregma. The greatest lesion-induced reduction of TH+ was observed in the caudal portion of the striatum ( 1.3 mm) and in a lesser intensity at the mid striatum section (0.2 mm). Smaller reductions were observed with the L-NOARG treatment, significantly at the medial (0.2 mm) and caudal levels (1.3 mm). Lower panel: Quantification of nNOS+ cell density at three levels along the anteroposterior axis of the striatum: effect of intrastriatal 6-OHDA injection and of the L-NOARG treatment. Values represent the percentage (mean SEM) of the cell density from the same striatal side of sham rat sections. Numbers at the base of each bar indicate the anteroposterior distance (in mm) from the Bregma. The greatest lesion-induced increase of nNOS+ was observed in the anterior and middle striatum. L-NOARG treatment promoted reduction of nNOS+ cell density in both levels. Number of rats per group = 5. * indicates significant difference from sham rats (effect of 6-OHDA) and indicates significant difference from 6-OHDA group (effect of L-NOARG), one-way ANOVA followed by Duncan's test, P < 0.001. In the SN: Representative sections of SN from sham, 6OHDA and 6-OHDA treated with L-NOARG rats are illustrated in the Fig. 3A, A' and A" respectively. The number of TH+ cell counts was significantly reduced in the ipsilateral SN of 6-OHDA (F[213] = 9.134; P = 0.005), with 50.7% remaining cells. The lesion induced significant loss of TH+ cells in the SNv (F[211] = 7.812; P = 0.011), in the SNl (F[213] = 7.832; P = 0.008) and in the SN dorsal (F[212] = 7.175; P = 0.012). The loss of TH+ cells was greater in the SNv (46% present cells) and SNl (42%) than in the SN dorsal (67.4%, Fig. 3). Contralateral SN did not show significant modifications (results not shown). The TH+ cell counts were significantly reduced in the ipsilateral SN of 6-OHDA treated with L-NOARG rats (Fig. 3B; F[213] = 9.134; P = 0.005), with 73.3% remaining cells. TH+ cell count

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Fig. 3 (A) Effect of intrastriatal 6-OHDA injections on the TH+ neurons in the caudal substantia nigra (SN; 5.8 mm from Bregma). Photomicrographs show the SN in sham (A), 6-OHDA (A') and 6-OHDA treated with L-NOARG rats (A") (ipsilateral side). Scale bar: 0.3 mm. (B) Quantification of the TH+ neurons in the SN. Values represent the mean numbers ( SEM) of TH+ neurons within the ipsilateral SN and its dorsal (SNd), ventral (SNv) and lateral (SNl) portions. White columns represents sham group, black represents the 6-OHDA group and gray represents 6-OHDA treated with L-NOARG. Cell loss in the ventral and lateral SN was greater than in dorsal SN. L-NOARG treatment significantly reduced the TH+ cell loss in the ventral and lateral SN but not in the dorsal SN. * indicates significant difference from sham (effect of 6-OHDA). indicates significant difference from 6-OHDA group (effect of L-NOARG). Number of rats per group = 5 (P < 0.05, one-way ANOVA followed by Duncan's test).

reduction was also observed in the dorsal part of SN (F[212] =7.175; P =0.012, 68.7% present cells). However, the treatment resulted in a significantly smaller loss of TH+ cells in the SNv (79.6% cells) and SNl (75% cells) in comparison to 6-OHDA (F[211] =7.812; P =0.011 and F[213] =7.832; P = 0.008, respectively, Fig. 3B). In the extrastriatal structures: In sham rats, TH immunostaining in the GP was much less dense compared to the striatum (Fig. 1D). Qualitative analyses of TH+ fibers in the ipsilateral GP at the level 1.3 mm from Bregma revealed a loss in 6-OHDA rats when compared to sham animals, as described previously (Debeir et al., 2005); loss of labeling was mostly limited to the dorsal-lateral part of the structure (Fig. 1D'). Examination at high magnification indicates that both the large and the thin TH+ fiber types were lost (Fig. 1D'). The loss of TH in the GP+ was less marked in the animals treated with L-NOARG (Fig. 1D). The pattern of TH+ fibers in the cortex of sham rats resembled that described by other investigators (e.g., Debeir et al., 2005). In the 6-OHDA rats, a substantial loss of fibers occurred in the anterior cingulate cortex (Fig. 4B' and B"). The same pattern of loss could be observed in the 6-OHDA treated with L-NOARG rats (Fig. 4A" and B").

2.2. Effect of intrastriatal 6-OHDA injections on nNOS+ neurons and the L-NOARG treatment
In the striatum: Representative cases of ipsilateral striatum of the sham rats treated with saline (A) or L-NOARG (C) and of the 6-OHDA rats treated with saline or (B) with L-NOARG (D) are illustrated in Fig. 5. The mean density of nNOS+ neurons in the

ipsilateral striatum of 6-OHDA rats was significantly increased when compared with sham group and this effect was modified by L-NOARG treatment (F[214] = 7.876; P = 0.007). The mean density increase of nNOS+ neurons in the ipsilateral striatum of 6-OHDA rats was significant at the anterior (+31%, F[213] = 6.768; P N 0,05) and medial (+17.5%, F[214] = 7.876; P = 0,006) levels when compared to sham. At the caudal portion, the mean density of nNOS+ cell did not differ significantly from sham rats. Fig. 2 shows the 6-OHDA-induced increase in the mean density of nNOS+ neurons and the L-NOARG modification of this effect along the striatum anteroposterior axis in percentage from sham group. The mean density of nNOS+ cells was not significantly increased in the anterior striatum of 6-OHDA rats treated with L-NOARG when compared to sham group. But, treatment with L-NOARG resulted in a reduction of nNOS+ cells, from +31% in the 6-OHDA rats treated with saline compared to + 17.3% 6-OHDA rats treated with L-NOARG. In the medial ( 7.4%) and caudal levels ( 14.1%), also, a significant decrease was found in the mean density of nNOS+ cells in the group 6-OHDA treated with L-NOARG when compared with 6-OHDA (respectively: F[214] = 5.453; P < 0.05 and F[214] = 6.356; P < 0.05). We did not find significant differences in the striatum contralateral to microinjection (results not shown). Mean density of striatal nNOS+ neurons: sham = 30 1.7, 6-OHDA = 35 2.0 and 6-OHDA treated with L-NOARG = 27 1.5). In the SN: As the nNOS+ neurons could be found only in the dorsal tier of SN, we counted the number of nNOS positive neurons in this sub-region.

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Fig. 4 TH+ fibers in the anterior cingulate cortex of sham (A), 6-OHDA (A') and 6-OHDA treated with L-NOARG rats (A"). In the 6-OHDA group, a substantial loss of fibers was observed in the anterior cingulate cortex and its effect was not modified by L-NOARG treatment. In B, B' and B", three representative cases of ipsilateral sham (B), 6-OHDA (B') and 6-OHDA treated with L-NOARG (B"), respectively, at higher magnification. Scale bar AA": 0.05 mm; BB": 0.02 mm. The mean density of nNOS+ cells was significantly reduced (47% remaining) in the ipsilateral SN of 6-OHDA (F[212] = 3.787; P < 0.06) from the same side in sham rats. Contralateral SN did not present significant modifications (results not shown) after 6-OHDA. The nNOS+ cell density was not significantly reduced in the ipsilateral SN of 6-OHDA treated with L-NOARG rats from sham group. Thus, as the 6-OHDA induced a significant decrease in the nNOS+ density of cells, treatment with L-NOARG resulted in an appreciable smaller loss of the density of nNOS+ cells in the SN (77.6% presenting in the 6-OHDA treated with L-NOARG X 53% in the 6-OHDA). Mean density of nNOS+ neurons total cell counts: 14.5 (0.8) in the sham group, 7.7 (0.8) in the 6-OHDA group and (11.2 2.0) in the 6-OHDA treated with L-NOARG group.

3.

Discussion

Fig. 5 Representative photomicrographies of nNOS positive neurons in the ipsilateral dorsal-lateral portions of the striatum in sham (A), sham treated with L-NOARG (C), 6-OHDA (B) and 6-OHDA treated with L-NOARG (D) rats. Scale bar: 0.4 mm.

Unilateral intrastriatal injections of 6-OHDA in rats resulted in a loss of TH+ fibers in the striatum and a retrograde loss of TH+ neurons in the SN, particularly in the ventral and lateral part. A concomitant decrease of TH+ fibers occurred in extrastriatal structures including the GP and the cingulate cortex. The pattern of TH+ fiber and cell loss in the protocol we used, which is considered a good model of progressive loss of DAcontaining structures in PD (Kirik et al., 1998; Sauer and Oertel, 1994), was consistent with that reported by others (Przedborski et al., 1995; Winkler et al., 2002; Anaya-Martinez et al., 2006, Debeir et al., 2005). nNOS immunohistochemistry revealed an increase of nNOS cell density in the striatum and a decrease in the substantia nigra compacta after 6-OHDA. Our results suggest a protective effect of NOS inhibition by L-NOARG. This effect can be due to activity of NO on dopamine transporter (DAT) and/or its biochemical interaction with the 6-OHDA. It has been proposed that NO donors diminish the capacity of the DAT in a dose-, time- and temperature dependent manner (Pogun et al., 1994; Lonart and Johnson, 1994; Kiss et al., 2004). However, Volz and Schenk (2004) have reported that NO produced from L-arginine, a NOS substrate, affects DAT in a different manner to NO derived from NO donors. In that study, L-arginine increased DAT activity in the rat striatum by a NOSdependent mechanism. Since 6-OHDA is a substrate for DAT (Blum et al., 2001), it is reasonable to propose that NOS inhibition would lead to less NO production, less DAT activity and hence could protect DA terminal from 6-OHDA access. On the other hand, NO reacts with dopamine (Antunes et al., 2005) and 6-OHDA (Riobo et al., 2002; Palumbo et al., 2001) producing semiquinones, peroxinitrite and 6-hydroxydopamine quinone. These compounds are related to oxidative stress, which is recognized as an important mediator of neuronal degeneration in the 6-OHDA model (Smith and Cass, 2006; Henze et al., 2005) and in PD (Jenner, 2003; Fahn and Cohen, 1992). In

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agreement, Hanrott et al. (2006) recently published that extracellular auto-oxidation of 6-OHDA induces apoptosis in PC12 cells. Thus, an inhibition of NO production could offer some protection from these events. Our results are in agreement with previous studies showing that NOS inhibition can protect against MPTP-induced neurotoxicity in experimental animals (Schulz et al., 1995; Hantraye et al., 1996; Watanabe et al., 2007). The intrastriatal 6-OHDA lesion induced a preferential loss of TH+ neurons in the ventral SN as previously observed (Van den Munckhof et al., 2006; Debeir et al., 2005). This ventral cluster of cells provides a massive projection to the dorsal striatum and lateral GP (Prensa and Parent, 2001; Debeir et al., 2005). In addition, a lateral to medial gradient of TH+ loss in the SN was described in PD (Hu et al., 2006; Hutchinson and Raff, 1999; Hutchinson et al., 2003) and in rat 6-OHDA model (Olds et al., 2006; Fallon and Moore, 1978). Like the striatum and GP, the pattern of DA degeneration observed in the cingulate cortex can be associated with the pattern of neuronal loss in the dorsal SN (Deutch et al., 1988; Haber and Fudge, 1997). Thus, our results are consistent with previous anatomical reports since the effects of the L-NOARG in some SN sub-regions were reflected in their projection targets and collaterals. One of the main findings of the present study is the L-NOARG protective effect on the 6-OHDA induced modifications of nNOS cell density, which was increased in the striatum and decreased in the SN. The mechanisms by which deafferentation affects the density of nNOS+ cells in the denervated circuitries is not fully understood. The interaction between nNOS and TH+ neurons and fibers in the striatum is complex. NO has been proposed to regulate the DA release and uptake (West et al., 2002; West and Galloway, 1998) and the increased nNOS cell density could result from a compensatory augmentation in the activity of striatal nNOS+ interneurons, which are excited by an intact nigrostriatal pathway through a D1-like receptor mechanism (Centonze et al., 2002). Also, there are indications in the literature of an increase in the nNOS cell density or NADPH-diaphorase activity in the striatum after chronic deafferentation (Sancesario et al., 2004; Morton et al., 1993; Gomes and Del Bel, 2003). Neurodegeneration probably leads to rearrangements of the remaining neurocircuitries, preserving and/or further impairing the affected functions. Similar rearrangements may perhaps provide a background for functional alterations. NOS is required for striatal plasticity events like long-term depression (LTD) of cortico-striatal transmission, as well as metabotropic glutamate group I receptors, and both events seem to be linked by release of endocannabinoids (Sergeeva et al., 2007). LTD can be protective as glutamate release is enhanced at these synapses in PD, so we can speculate that nNOS in the striatum will contribute to the depression of cortical inputs and that the increase of nNOS cell density could represent a compensatory mechanism. Direct synaptic contact was demonstrated along the mesocortical and mesostriatal dopamine pathway either between DA and NADPH-diaphorase (Fujiyama and Masuko, 1996) or NOScontaining neurons (de Vente et al., 2000; Benavides-Piccione and DeFelipe, 2003) in the adult. In sections of the mesencephalon from two-day-old pups, NOSTH double-labeled neurons could be seen in the SN, VTA, cingulate and frontoparietal cortex (Gomez-Urquijo et al., 1999). However, such neurons became

infrequent in sections from one-month-old rats. Occasionally double labeling for NOS and TH could be observed of nNOS in some TH+ neurons in rat primary mesencephalic cultures (Salum et al., in press). Thus, the reduction in nNOS+ neurons in the substantia nigra compacta following nigrostriatal deafferentation was probably due to a loss of mesencephalic neurons. In conclusion, our results suggest that L-NOARG treatment has a protective effect on nigrostriatal 6-OHDA-induced lesion. It was consistent in terms of projection pathways of the DA system and because it was able to reduce the lesion effects on NO system. However, we can only speculate the events carried out by NOS inhibition and more studies are necessary to elucidate the mechanism by which NOS inhibitors modify the effects of 6-OHDA.

4.
4.1.

Experimental procedures
Animals

Adult male Wistar rats weighting 200250 g were housed in groups of five per cage in a temperature-controlled room (23 C), under 12 h lightdark cycle, with free access to food and water. All experiments were performed in accordance with the guidelines described in the European Communities Council Directive of 24 November 1986 (86/609/EEC), and the experimental protocol was carried out in compliance with the guidelines of the Brazilian Society of Neuroscience and Behavior for the care and use of laboratory animals. Every effort was made to keep the number of animals to a minimum and to minimize suffering.

4.2.

Drugs

Ascorbic acid (vitamin C, Sigma U.S.A.) and L-NOARG (Sigma) were dissolved in saline. All drugs with exception to 6-OHDA were administered intraperitoneally (i.p.), in a volume of 4 ml/ kg. Doses were chosen based on previous studies (6-OHDA: Gomes and Del Bel, 2003; L-NOARG: Marras et al., 1995; Del Bel and Guimares, 2000).

4.3.

Surgical procedure

The animals were anesthetized with 2.5% 2,2,2-tribromoethanol (10 ml/kg i.p., Aldrich) and fixed on a stereotaxic frame (DavidKopf) with the incisor bar 5 mm above the interaural line. A striatal lesion was induced by four microinjections of 2 l of the 6-OHDA (4 7 g/2 l of saline containing 0.02% ascorbic acid) into the right striatum. The microinjections were made at four locations within the striatum: 1) A: +1.3 L: 2.6 D: 5.0; 2) A +0.4 L: 3.2 D: 5.0; 3) A: 0.4 L: 4.2 D: 5.0; 4) A: 1.3 L: 4.5 D: 5.0 from Bregma, according to the atlas of Paxinos and Watson (1998). The microinjections were performed in a rate of 1 l/min with an infusion pump (Scientific, USA), and the needle was left in place for an additional 180 s to prevent reflux. The movement of an air bubble inside the PE-10 polyethylene tubing connecting the micro-syringe with the needle confirmed drug flow. Shamoperated animals were submitted to the same procedure but received vehicle (saline containing 0.02% ascorbic acid) instead of the neurotoxin.

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4.4. Experimental groups

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4.6.

Immunohistochemistry of TH and nNOS

On day one animals received the first dose of either L-NOARG 40 mg/kg (n = 5, i.p. dissolved in saline 4 ml/kg) or saline (n = 5, i.p). On day two they were subjected to the stereotaxic surgical procedure to inject the 6-OHDA or saline into the striatum. Both L-NOARG and saline i.p. administrations were continued for three further days (total of 5 injections), once a day at 14:00 o'clock. The experimental groups were: saline/saline (sham, n = 6); saline treated with L-NOARG (n = 4); 6-OHDA/saline (named 6-OHDA, n = 5); 6-OHDA treated with L-NOARG (n = 5). Animals were sacrificed on day 36 after surgical procedure.

4.5.

Histological analysis

Rats were sacrificed by terminal anesthesia (1.5 g/kg; 0.4 ml/kg urethane, i.p.) and perfused intracardially with 200 ml of 10 mM phosphate buffered saline, pH 7.4, followed immediately by 200 ml of freshly prepared 4% ice-cold paraformaldehyde in PBS. The brains were removed and placed in 4% paraformaldehyde in PBS for 2 h, cryoprotected in 30% sucrose in PBS, and then snapfrozen in isopentane ( 40 C cooled in dry ice) and stored at 70 C until the immunohistochemical procedures were carried out. Thirty five micrometer serial sections were cut within a cryostat (Leica). Neuroanatomical sites were identified using the Paxinos and Watson (1998) atlas. The anteroposterior (AP) localizations from Bregma of the analyzed areas were AP: 1.3, anterior striatum, AP: 0.2, medial striatum, AP: 1.3 mm, caudal striatum and AP: 5.8 mm, caudal SN (Fig. 6). To ensure that neurons were not counted twice analysis was performed in every sixth section (i.e. separated by 210 m). Adjacent brain sections were stained for TH, a marker for dopaminergic neurons, and for nNOS immunoreactivity.

TH immunohistochemistry was performed as previously described (Douhou et al., 2002). The same procedure was carried out for NOS. Briefly, tissue sections were washed and then incubated 1824 h with the primary antibody (TH: 1/1000, Peel Freez or nNOS: 1/1000, Emson see Herbison et al., 1996). Sections were processed by the avidin-biotin immunoperoxidase method (Vectastain ABC kit, Vector Lab) and immunopositive cells and terminals were visualized by addition of the chromogen 3,3-diaminobenzidine (DAB; Sigma, 1 mg/ml) and hydrogen peroxide (0.2%). Sections were mounted onto gelatin-coated glass slides, dehydrated in ethanol, cleared in xylene, and cover-slipped for microscopic observations. Immunopositive cells were revealed by a brown reaction product. Tissue from sham and lesion groups was always processed at the same time. In all experiments tissues from every group were always processed in the same assay.

4.7.

Quantitative morphology

Fig. 6 Representation of the chosen distance from Bregma comprising the substantia nigra sub-regions for analysis: lateral (SNl), dorsal (SNd) and ventral (SNv) tiers. Extracted from Paxinos and Watson, 1998. The optical dissector method was used to count TH+ and nNOS+ neurons in the SN at the caudal level of this structure ( 5.8 mm from Bregma). We counted TH+ cells in the dorsal, ventral and lateral sub-regions of the SN. The boundaries of the SN subdivisions in the coronal plane were determined microscopically (4 objective) and marked on line drawings using a computer. As the nNOS+ neurons can be found only in the dorsal tier of SN, we counted the number of nNOS positive neurons in this sub-region.

All the histological quantification was performed blindly. A preliminary qualitative analysis identified the regions with high neuronal labeling. Neurons which were positively stained exhibited labeled soma, dendrites and axons. The number of TH positive neurons in the SN and the number of nNOS positive neurons in the SN (Fig. 6) and striatum were counted, bilaterally, using a microscope (Nikon) equipped with a 60 objective (numerical aperture 1.4) and connected to an image analysis system (Mercator, Explora Nova, LaRochelle, France) equipped for stereological application. Results are expressed as the mean density of cells (number of positive neurons/0.5 mm2 of the structure), calculated from results obtained in each brain side. Labeling of TH positive fibers in the striatum was assessed by optical density measuring average pixel optical density over cell body and network. The optical density of a tissue area devoid of staining (corpus callosum) was measured (Mercator Explora Nova computerized image analysis system), being the light intensity adjusted to give a background density value. The background was subtracted from all subsequent measurements. A mean value for staining intensity was calculated and expressed in arbitrary gray scale (relative optical density) units (ROD units; Morris et al., 1997). Results are expressed as a percentage of the same side in sham brains. The counts and measurements were performed in 34 sections and then we calculate the mean value per rat.

4.8.

Statistical analysis

All values are expressed as the mean SEM. TH positive (TH+) and nNOS positive (nNOS+) cell counts as well as TH+ optical densities measurements from sham, lesioned and lesioned treated with L-NOARG rats were compared using one-way analysis of variance (ANOVA) followed by Duncan's test, factor being treatment. A significance level of P < 0.05 was considered to be statistically significant. The degrees of freedom were designed as F. All statistical analyses were performed using SPSS for Windows (version 8.0).

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Acknowledgments
The authors acknowledge Celia Aparecida da Silva and Laure Ginestet for their helpful technical support provided. We also thank Prof. Paul Bolam, Prof. Francisco Silveira Guimares and Prof. Vitor Tumas for the manuscript discussion and suggestions. Research supported by grants from FAPESP (projects number 01/14612-0 and number 02/13197-2), CAPES-COFECUB (project number 491/05/07) and CPNQ.

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