Behavioural Brain Research 266 (2014) 29–36

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Neuropeptide S counteracts 6-OHDA-induced motor deficits in mice Julia J. Didonet a , Judney C. Cavalcante b , Lisiane de S. Souza a , Miriam S.M.O. Costa b , Eunice André c , Vanessa de P. Soares-Rachetti a , Remo Guerrini d , Girolamo Calo’ e , Elaine C. Gavioli a,∗ a

Behavioral Pharmacology Laboratory, Department of Biophysics and Pharmacology, Federal University of Rio Grande do Norte, Natal, RN, Brazil Laboratory of Neuroanatomy, Department of Morphology, Biosciences Center, Federal University of Rio Grande do Norte, Natal, RN, Brazil Department of Pharmacology, Federal University of Parana, Curitiba, PR, Brazil d Department of Chemistry and Pharmaceutical Sciences, University of Ferrara, Ferrara, Italy e Department of Medical Sciences, Section of Pharmacology, and National Institute of Neuroscience, University of Ferrara, Ferrara, Italy b c

h i g h l i g h t s • Central 6-OHDA produced a notorious motor impairment in mice. • Central administration of NPS attenuated 6-OHDA-induced motor impairments. • This study candidates selective NPSR agonists as an innovative treatment for Parkinson disease.

a r t i c l e

i n f o

Article history: Received 25 January 2014 Received in revised form 28 February 2014 Accepted 3 March 2014 Available online 11 March 2014 Keywords: Dopamine Parkinson disease Mouse Neuropeptide S Motor activity 6-OHDA

a b s t r a c t Neuropeptide S (NPS) is a 20-aminoacid peptide that selectively activates a G-protein coupled receptor named NPSR. Preclinical studies have shown that NPSR activation promotes anxiolysis, hyperlocomotion, arousal and weakfullness. Previous findings suggest that dopamine neurotransmission plays a role in the actions of NPS. Based on the close relationship between dopamine and Parkinson disease (PD) and on the evidence that NPSR are expressed on brain dopaminergic nuclei, the present study investigated the effects of NPS in motor deficits induced by intracerebroventricular (icv) administration of the dopaminergic neurotoxin 6-OHDA in the mouse rotarod test. 6-OHDA injection evoked motor deficits and significantly reduced tyrosine hidroxylase (TH)-positive cells in the substantia nigra (SN) and ventral tegmental area. However, a positive correlation was found only between the motor performance of 6-OHDA-injected mice and the number of TH-positive cells in SN. The systemic administration of lDOPA + benserazide (25 + 6.25 mg/kg) counteracted 6-OHDA-induced motor deficits in mice. Similar to l-DOPA, the icv injection of NPS (0.1 and 1 nmol) reversed motor deficits evoked by 6-OHDA. In conclusion, NPS attenuated 6-OHDA-induced motor impairments in mice assessed in the rota-rod test. We discussed the beneficial actions of NPS based on a putative facilitation of dopaminergic neurotransmission in the brain. Finally, these findings candidate NPSR agonists as a potential innovative treatment for PD. © 2014 Elsevier B.V. All rights reserved.

1. Introduction

Abbreviations: 6-OHDA, 6-hydroxydopamine; icv, intracerebroventricular injection; NPS, neuropeptide S; SN, substantia nigra; TH, tyrosine hidroxylase. ∗ Corresponding author at: Behavioral Pharmacology Laboratory, Department of Biophysics and Pharmacology, Federal University of Rio Grande do Norte, Av. Senador Salgado Filho, s/n, Campus Universitário – Lagoa Nova, Natal 59072-970, RN, Brazil. Tel.: +55 84 3215 3419; fax: +55 84 3215 3419. E-mail address: [email protected] (E.C. Gavioli). http://dx.doi.org/10.1016/j.bbr.2014.03.002 0166-4328/© 2014 Elsevier B.V. All rights reserved.

Parkinson’ disease (PD) is a chronic and progressive neurodegenerative disease, characterized by resting tremor, rigidity, bradykinesia and akinesia; clinical features that are a consequence of dopamine neurons degeneration within the substantia nigra [1]. 6-OHDA is a widely used tool to induce parkinsonism in rodents. Once inside dopaminergic neurons, the toxin initiates degeneration through a combination of oxidative stress and mitochondrial respiratory dysfunction, thereby supporting a high degree of construct validity for the 6-OHDA model (for a review see [2]). According

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to Rodriguez-Diaz et al. [3], 6-OHDA intracerebroventricular (icv) injection produces a motor syndrome and an anatomical pattern of cell loss that resembles those of Parkinson disease. The pharmacological therapy of PD only provided symptomatic control, since clinically effective neuroprotector agents, able to slowing the degeneration of nigral dopaminergic neurons, have not been identified yet. The use of l-DOPA, the gold standard treatment for PD, is associated with motor complications including wearing off, dyskinesia and on-off phenomenon [4]. In this scenario the development of new therapeutic approach is important for future management of PD. NPS is a 20-aminoacid peptide, which is the endogenous ligand for a G-protein-coupled receptor named NPSR [5]. In cells expressing the recombinant NPSR, NPS increases Ca2+ mobilization, intracellular cAMP formation and phosphorylation of extracellular signal-regulated kinase, suggesting that NPS receptors may be coupled with Gq and Gs proteins [5,6]. High levels of NPSR mRNA are expressed in various brain regions such as cortex, olfactory nuclei, thalamus, hypothalamus, amygdala, and subiculum [7]. In the brain stem, NPSR is lowly expressed in the substantia nigra and the ventral tegmental area, brain regions that are important centers of dopamine synthesis [7,8]. The brain distribution of the NPS-NPSR receptor system suggests that this neuropeptidergic system may play a role in controlling multiple physiological functions. Indeed, in rodents, central administration of NPS increases arousal, suppresses anxiety-like behaviors and stimulates locomotor activity as repeatedly demonstrated across experimental conditions and animal species (for a review see [9]). NPS-induced hyperlocomotion behavior has been shown to be modulated by few neurotransmitters including dopamine [10–13]. Microdialysis studies support the view that NPS administration facilitates dopamine release in the brain [11,14]. Therefore, the expression of NPSR in important dopaminergic brain sites points to a modulatory effect exerted by the NPS-NPSR receptor system over dopamine neurotransmission in the brain [7]. Considering literature findings, which associated some biological effects of NPSR activation with dopaminergic neurotransmission, and the involvement of dopaminergic system in the pathophysiology of PD, the present study aimed to investigate the effects of NPS in mice displaying 6-OHDA-induced motor deficits.

6-OHDA administration was performed 3 days before behavioral and immunoistochemical assays. l-DOPA (Sigma–Aldrich Corporation, St. Louis, MO, EUA) was dissolved in carboxymethylcellulose 0.5% and was administrated orally (po) at 25 mg/kg, 60 min before behavioral assays. In order to prevent systemic metabolism of l-DOPA, an inhibitor of aromatic l-amino acid decarboxylase enzyme, benserazide (Sigma–Aldrich Corporation, St. Louis, MO, EUA) was solubilized in saline, and ip administered, at 6.25 mg/kg, 30 min before l-DOPA. The doses of l-DOPA and benserazide herein employed were adapted from a previous study performed in rats [16]. Human NPS, the endogenous ligand of the NPSR, was synthesized in house by Dr. Guerrini (Department of Chemistry and Pharmaceutical Sciences, University of Ferrara, Italy) and solubilized in saline. NPS was administrated icv, at 2 ␮l/min rate to a total volume of 2 ␮l, the doses used were 0.1 and 1 nmol. These doses were previously shown by different groups to be able to evoke hyperlocomotion (for a review see [9]). An 8 mm guide cannula (25 mm × 0.7 mm) was stereotaxically implanted for icv injections, it was placed in the left lateral ventricle, according to the following coordinates from bregma: AP: −0.6 mm, ML: +1.1 mm, DV: −1.0 mm [17]. Surgery happened under ketamine–xylazine anesthesia (ip, 100 and 10 mg/kg, respectively). The cannula was secured to the skull by acrylic dental cement. Four to five days after surgery, mice were subjected to behavioral and immunoistochemical assays. 2.3. Rotarod behavioral test The protocol used in this experiment was a modified version of the fixed-speed rotarod test described by Rozas et al. [18] for rats. Animals were pre-trained for 3 consecutive days on an automated rotarod unit at 16 and 35 rpm speed, during training sessions animals were kept on the rotating rod for 300 s for each speed. The rotarod apparatus (AVS Projetos, Riberão Preto, SP, Brazil) is composed by a 5 lane rod, which has 3 cm of diameter and is elevated 22 cm from the platform. During test session, animals were placed on the rod, and sequentially tested at 16, 28, 35, 50 and 58 rpm for a maximum of 300 s for each speed with a 5 min rest time between trials. The time of the first fall was measured for all speeds.

2. Materials and methods 2.4. Parkinson-induced by 6-OHDA administration 2.1. Animals Female Swiss mice (28–35 g) were used in this study. Animals were housed in a temperature-controlled room (23 ± 1 ◦ C) with a 12 h light/dark cycle and were given free access to food and water. All experiments were conducted in accordance with Brazilian Law No. 11.714/2008 for animal experimental use. All efforts were made to minimize animal suffering and to reduce the number of animals used. Protocol was approved by Ethic Committee for Animal Use of Federal University of Rio Grande do Norte (Protocol No. 020/2011).

2.2. Drugs and treatments Mice received a 2 ␮l solution of 6-OHDA (50 ␮g/mouse) dissolved in 0.01% ascorbic acid (Sigma–Aldrich Corporation, St. Louis, MO, EUA) through an intracerebroventricular (icv) injection. The dose of 6-OHDA used in this study was based on previous findings of Naudin et al. [15], which showed reductions on dopamine striatal levels to 50% of controls in mice. Nortriptyline (Pamelor® , NOVARTIS Biociências S.A., São Paulo, Brazil) was administrated intraperitoneally (ip, 30 mg/kg), 30 min before 6-OHDA injection, in order to prevent the uptake of 6-OHDA by noradrenergic neurons.

After the 3 training sessions, animals were submitted to stereotaxic surgery. The day after, animals received a 2 ␮l 6-OHDA solution, being previously anesthetized with thiopental 60 mg/kg to lower the risk of convulsions. All mice were treated with nortriptyline 30 mg/kg ip 30 min before 6-OHDA icv injection. Behavioral and immunoistochemical experiments were performed 3 days after 6-OHDA injection. The present experimental design considered that the same animals were subjected to all 3 test sessions. In that manner, on the day after surgery, before receiving 6-OHDA injection, mouse performance was assessed in the rotarod apparatus (baseline). On the same day, mice received the toxin injection and 3 days later, a new measure of their motor coordination on rotarod was taken; any eventual motor impairment evoked by 6-OHDA was detected at this point. Those animals treated with 6-OHDA and able to stay on the rotating rod of the apparatus at the lowest speed (16 rpm) until the cut off time (300 s) were excluded from subsequent experiments; these animals (approximately 50%) were considered non responders. No deaths were detected after treatment with the toxin under our experimental conditions. Animals detected with motor incoordination on the rod were treated with l-DOPA or NPS and submitted once more to test session on rotarod.

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To pharmacologically validate the 6-OHDA-induced motor deficits in mice, animals received l-DOPA (25 mg/kg, po) plus benserazide (6.25 mg/kg, ip), and behavioral evaluation occurred 60 min after l-DOPA injection. In a separate series of experiments, NPS (0.1 and 1 nmol) was given icv and behavioral evaluation occurred 15 min after NPS injection. 2.5. Immunoistochemical assays To quantify the amount of TH-positive cells in substantia nigra, 30 min after the rotarod assay, mice were anesthetized with thiopental and transcardially perfused with saline and 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were removed and stored for 2 h and then moved to a sucrose 30% solution at 4 ◦ C for at least 48 h. Coronal sections (30 ␮m) were obtained with a microtome. Sections were processed for TH immunohistochemistry as following. Floating sections were immersed for 30 min in 0.3% H2 O2 in phosphate buffer and 0.4% Triton X-100 to inactivate endogenous peroxidase. After several rinses they were incubated overnight in a solution containing a mouse anti-TH monoclonal antibody (Sigma–Aldrich, 1:5000) and 2% normal donkey serum in Triton X-100 0.4%. After several rinses, sections were incubated for 120 min in donkey antiserum (Jackson, 1:1000) containing antimouse antibody in phosphate buffer and Triton X-100 0.4%. After several rinses, sections were put in an avidine-biotine HRP solution (Protocol ABC, Vector Kit Elite, 1:333) in phosphate buffer for 120 min in room temperature. After several rinses, immunoreactions were visible after exposure to DAB and H2 O2 0.003% for 6 min. In order to stop the DAB reaction, sections were rinsed. The sections were mounted onto gelatin-coated slides, dehydrated sequentially until absolute alcohol, cleared in xylene and coverslipped with DPX. The photomicrographs were captured with a Nikon DXM1200 camera connected to an Olympus BX41 microscope. The images were digitalized using Nikon ACT-1 software and the TH-positive

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cells were counted using the Image J program (NIH) at three levels of the nucleus of substantia nigra bilaterally (between figures 56 and 57, between figures 58 and 59, and between figures 62 and 63 of Paxinos and Franklin [17]). In the ventral tegmental area, the TH-positive cells were counted bilaterally at one level (between figures 58 and 59 of Paxinos and Franklin [17]). The photomicrographs plate was mounted using Photoshop CS6 image-editing software. Only sharpness, brightness and contrast were adjusted.

2.6. Statistical analysis Data are presented as mean ± SEM. Raw data from rotarod experiments were converted to the area under the curve (AUC), by plotting the time on the rod (s) against rod speed (rpm); AUC data were used for statistical analysis. Significant differences between motor performances of different treatment groups were evaluated through ANOVA for repeated measures followed by Newman–Keuls post hoc test. Differences between control and 6OHDA-treated mice in the number of TH-positive neurons were evaluated with the Student’s t-test. Aiming to estimate the motor improvement evoked by l-DOPA and NPS on the motor deficits induced by 6-OHDA, a percentage of motor performance was calculated according to the following ratio: AUC (6-OHDA-injected mice treated with L-DOPA or NPS) AUC (6-OHDA-injected mice) Correlation coefficient was obtained by Pearson correlation test. For all cases p < 0.05 was considered statistical significant. All statistical analyses were performed using the software GraphPad Prism version 5.0.

Fig. 1. Effect of 6-OHDA on the TH immunoreactivity in substantia nigra. Brightfield photomicrographs showing TH-positive neurons in the midbrain of a mouse 3 days after icv injection of saline (A) or 6-OHDA (B). Panels C and D show high magnifications of the boxes in A and B, respectively. Note the differences on number of neurons and intensity of labeling between saline (C) and 6-OHDA (D) treated animals. Abbreviations: cp, cerebral peduncle; IP, interpeduncular nucleus; ml, medial lemniscus; SNC, substantia nigra, compact part; SNR, substantia nigra, reticular part; VTA, ventral tegmental area. Bar: 300 ␮m A and B; 50 ␮m C and D.

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B)

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Fig. 2. Number of TH-positive cells in the mouse substantia nigra (A) and ventral tegmental area (B) 3 days after icv injection of 6-OHDA 50 ␮g or saline. The 6-OHDA group is representing only the number of TH-positive cells found in those mice which displayed motor deficits in the rotarod test. Data are shown as mean ± S.E.M. of 5 animals. *p < 0.05 vs. control (Student’s t test).

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Fig. 3. Correlation between mice motor performance in the rotarod test (calculated by the area under the curve data – see Section 2) and the number of TH-positive cells in substantia nigra (Pearson correlation; r = 0.574, p = 0.032, n = 14) (A) and ventral tegmental area (Pearson correlation; r = 0.062, p = 0.39, n = 14) (B) on the third day after icv injection of 6-OHDA 50 ␮g.

3. Results 3.1. TH-positive cells in substantia nigra in 6-OHDA- treated mice Fig. 1 shows a photomicrography of substantia nigra slice (at 300 and 50 ␮m) from saline and 6-OHDA-treated animals. A notable reduction in the number of neurons and intensity of labeling in 6-OHDA-treated animals is depicted. A quantitative analysis of TH-positive cells is represented in Fig. 2. Student’s t test indicated a significant reduction in the number of TH-positive cells in substantia nigra (Fig. 2A, p = 0.016), and ventral tegmental area (Fig. 2B, p = 0.018) of 6-OHDA-treated mice on the third day after the injection compared to controls. Considering that 6-OHDA was injected in one of the lateral ventricles, no differences in the number of TH-positive cells in the substantia nigra were detected in 6-OHDA-treated mice between left and right hemispheres (Fig. S1). Fig. 3 shows that there is a positive correlation between mouse performance in rotarod test (calculated by the AUC; see Section 2) and number of TH-positive neurons in the substantia nigra (Fig. 3A, r = 0.574, p = 0.032, n = 14). However, no correlation was found between the motor performance of mice after 6-OHDA injection and the number of TH-positive cells in the ventral tegmental area (Fig. 3B, r = 0.062, p = 0.39, n = 14). 3.2. Effects of 6-OHDA and l-DOPA on mouse motor performance To evaluate the baseline performance in the rotarod test, mice (before 6-OHDA treatment) were subjected to the apparatus. For 16 rpm, all animals reached the cut-off (i.e., 300 s), while at higher

Fig. 4. Effects of l-DOPA (25 mg/kg) oral administration in mice motor incoordination induced by 6-OHDA assessed by rotarod test. The same group of mice went through all three conditions: (i) before receiving 6-OHDA treatment (baseline); (ii) 3 days after the injection of 6-OHDA, and iii) 60 min after l-DOPA administration. Data are shown as mean ± S.E.M. *p < 0.05 vs. baseline, and # vs. 6-OHDA treatment (ANOVA repeated measures, Newman–Keuls test were performed on area under the curve data – Section 2; n = 5 for l-DOPA).

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speeds, mice fell from the rotating bar at lower times. Three days after treatment with 6-OHDA, mice were again subjected to the rotarod test and a dramatic impairment in motor performance was detected (Fig. 4). It should be noted that only those mice that displayed a considerable motor impairment in the rotarod test were subsequently subjected to the treatment with l-DOPA or NPS (see details in Section 2). Fig. 4 illustrates the effects of l-DOPA 25 mg/kg po on motor performance of mice previously treated with 6-OHDA. Repeated measures ANOVA followed by Newman–Keuls post hoc test showed that l-DOPA administration was able to alleviate motor deficits of 6-OHDA-treated mice, as calculated by the AUC (Fig. 4; p = 0.007; F(2,8) = 10.04). 3.3. Effect of NPS in 6-OHDA-treated mice As illustrated in Fig. 5, the icv injection of NPS (0.1 nmol panel A, and 1 nmol panel B) significantly attenuated the motor deficits induced by 6-OHDA. Repeated measures ANOVA followed by the Newman–Keuls post hoc test showed a significant motor improvement promoted by the NPS injection (0.1 nmol, p < 0.001,

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F(2,16) = 40.35; 1 nmol, p < 0.001, F(2,18) = 57.03) compared to the performance of 6-OHDA. Interestingly, motor improvements promoted by NPS injection (at both doses tested) in 6-OHDA-treated mice were not significantly different from those effects evoked by administration of the reference drug, l-DOPA (Fig. 5C; p > 0.05, F(2,21) = 0.59).

4. Discussion In this study the effects of NPS on motor behavior were assessed in mice icv injected with 6-OHDA. This toxin produced motor deficits in the rotarod performance that were sensitive to the standard anti-PD drug l-DOPA. Similar beneficial effects were measured in response to supraspinal NPS. These findings suggest that the selective activation of the NPSR might represent an innovative strategy for the treatment of PD. The 6-OHDA icv administration significantly impaired motor coordination in the rotarod test. Paralleling the motor deficit induced by 6-OHDA, a significant bilateral loss of TH-positive neurons in the substantia nigra of animals injected with the toxin was

Fig. 5. Effect of NPS 0.1 nmol (A) and NPS 1 nmol (B) on mice motor incoordination induced by 6-OHDA assessed by rotarod test. The same group of animals went through all three conditions: (i) before receiving 6-OHDA treatment (baseline); (ii) 3 days after 6-OHDA treatment, and (iii) 15 min after NPS administration. Comparison of the beneficial effects of l-DOPA and NPS 0.1 and 1 nmol on motor impairment evoked by 6-OHDA (C). Data are shown as mean ± S.E.M. *p < 0.05 vs. baseline, and # vs. 6-OHDA treatment (ANOVA repeated measures, Newman–Keuls test were performed on area under the curve data – see Section 2; n = 9 for NPS 0.1 nmol, and n = 10 for NPS 1 nmol).

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observed. A significant reduction of TH-positive cells in the ventral tegmental area was found in 6-OHDA-treated mice; however, no correlation was observed between motor performance of 6OHDA-injected mice and the number of TH-positive cells in ventral tegmental area. Acute oral administration of l-DOPA attenuated 6OHDA-induced motor impairments in mice. Thus, the icv injection of 6-OHDA in mice is a useful method for behaviorally screening innovative antiparkinsonian drugs. Few literature information is available about the motor effects of 6-OHDA after icv injection. This is possibly due to the fact that the lesion extension induced by the ventricular injection of the toxin affects more dopaminergic nuclei than the microinjection of 6-OHDA into the substantia nigra or striatum (for a review see [19,20]). In fact, we observed that seven days after icv injection of 6-OHDA, animals lost significant weight, become immobile on the cage, displayed piloerection and hypothermia. To minimize the effects of 6-OHDA on animal gross behavior, behavioral tests and immunohistochemical analysis were performed on the third day after toxin administration. Despite the absence of correlation between motor performance and number of TH-positive cells in ventral tegmental area, we cannot exclude that behavioral alterations in the rotarod test could be due to a partial lesion of ventral tegmental area, a dopaminergic nucleus which innervates the limbic system and cortical areas and is involved in emotional, rewarding and motivational regulation [21–23]. As showed in Fig. 2, animals displaying motor impairments had a significant loss of TH-positive neurons in substantia nigra after 6OHDA icv administration compared to controls. Additionally, the motor performance of our 6-OHDA-treated mice in the rotarod test was positively correlated to the number of TH-positive cells in substantia nigra. Besides the positive correlation between motor performance and number of TH-positive cells in the substantia nigra, our behavioral findings showed that only about 50% of 6OHDA-treated mice displayed significant motor deficits. In order to counteracting the motor deficits generated by dopaminergic neuronal deaths, compensatory mechanisms take place when this loss is lower than the threshold [24]. In this scenario, half of our 6OHDA-treated mice achieved this critical threshold. Importantly, the effects of l-DOPA and NPS evaluated in this study were obtained in the subset of mice displaying significant motor impairment in the rotarod (approximately 50% of animals tested). It should be mentioned that high levels of animals’ exclusion were found. The gender could be one of the aspects that is affecting the lesion extension of 6-OHDA. A behavioral study showed that male animals were more susceptible to 6-OHDA than females; female rats had a significantly less dopaminergic cell loss and responded to 6-OHDA with a significantly higher degree of behavioral recovery after the injury [25]. Estrogen levels could also be an important neuroprotective factor to the effects of 6-OHDA in the brain (for a review see [26]). In this regard, the estrous cycle could also influence the sensitive to 6-OHDA-induced dopaminergic damage. Rationally, the dose of 6-OHDA can contribute to the high levels of animals’ exclusion. However, in this study, higher dose of 6-OHDA were tried, but a significant mouse mortality was found. Based on these observations, the animal model herein used for testing the behavioral effects of NPS need to be improved. Possibly the use of male mice could reduce significantly the high levels of animals’ exclusion. l-DOPA is still the gold standard drug for the treatment of PD patients. This treatment is highly effective, however it is associated with a number of disadvantages; for example, it does not arrest the progress of the disease. During chronic l-DOPA treatment, the drug loses its effectiveness and several serious side-effects such as dyskinesia, psychotic symptoms, and on-off phenomena appear [27]. Thus, the search for novel, more effective and/or better tolerated pharmacological treatments for Parkinson disease is mandatory.

NPS is a neuropeptide discovered in the brain of mammalians and other vertebrate species almost 10 years ago [5]. The first biological activities reported for NPS were anxiolysis associated with hyperlocomotion and wakefulness [5]. Literature findings suggest that the locomotor stimulatory effects of NPS could involve dopaminergic and adenosinergic neurotransmission; interestingly both systems are closely related to the pathophysiology and pharmacotherapy of Parkinson disease. Mochizuki et al. [11] observed that microinjection of NPS into the ventral tegmental area significantly and dose-dependently increased locomotor activity in rats. This effect of NPS was dose-dependently inhibited by sulpiride, a selective D2 receptor antagonist, into the shell part of the nucleus accumbens. Regarding the adenosinergic system, a selective A2A receptor antagonist was able to counteract the hyperlocomotion evoked by NPS in mice [10]. Moreover, Pacheco et al. [12] showed that the hyperlocomotor effect of NPS depends on extracellular adenosine synthesized by ecto-5 -nucleotidase. Although these biological findings suggest a close relationship between NPS and dopamine and adenosine systems, no literature information is available about a putative effect of NPS on Parkinson disease. The present findings demonstrated that the icv administration of NPS at 0.1 and 1 nmol, significantly improved motor performance of mice treated with 6-OHDA. These doses of NPS have been previously showed to induce hyperlocomotion in rodents under distinct experimental conditions [5,28–32]. In our Swiss mice, the administration of NPS 1 nmol, but not 0.1 nmol, increased locomotor activity in the open field test (Fig. S2), thus suggesting that NPS can restore motor deficits in mice injected with 6-OHDA at doses that do not affect locomotor activity in normal animals. Regarding the mechanisms by which NPS improves motor deficits evoked by 6-OHDA, it could be suggested that NPS stimulates dopamine release via selective activation of NPSR receptors expressed in dopaminergic nuclei. Literature findings support this proposal. Using in vivo microdialysis in rats, Mochizuki et al. [11] found elevated extracellular dopamine metabolites in the nucleus accumbens shell after injection of NPS into the ventral tegmental area. Another microdialysis study found enhanced extracellular levels of dopamine and its metabolite in the rat medial prefrontal cortex after NPS supraspinal administration [14]. Despite this growing body of evidence which suggests an increase in dopamine release evoked by NPS, Raiteri et al. [33] using in vitro synaptosomes showed that NPS at high concentrations weakly reduced the overflow of [3 H]dopamine in mouse frontal cortex. Addictive behaviors are classically associated to the activation of the dopaminergic system. It is known that NPSR are expressed in reward-associated regions throughout the brain [7]. Nevertheless, literature findings suggest a paradoxical involvement of the NPS-NPSR receptor system in the modulation of addictive behaviors. Central injections of NPS displayed reward-like effects, weakly facilitated seeking and induced positive reinforcement [34]. ICV NPS increased cocaine seeking behavior [13,35,36]; similar results were also observed in ethanol abstinent rats [37]. By contrast, some findings provide an indirect evidence suggesting lack of effects of NPS on the reward system. Cannella et al. [38] has been shown that ICV NPS does not give conditioned place preference, which strongly support lack of rewarding properties of this peptide. Besides the ˜ et al. [39] have been shown studies by Kallupi et al. [35] and Paneda that NPS does affect cocaine self-administration, no effects of NPS antagonists on cocaine self-administration were observed [36]. A possible role of other structures downstream of substantia nigra, such as globus pallidus and subthalamic nucleus, and ventral tegmental area in mediating the motor improvements of NPS in 6-OHDA lesioned mice could also be suggested. In fact, a very low expression of NPSR is found in some of these brain areas in rats [7] and mice [8]. Further studies aimed to investigate the expression of NPSR in the basal ganglia and in structures downstream of ventral

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tegmental area are required for giving support to the mechanisms by which NPS improves 6-OHDA-induced motor deficits. Taken together, the motor improvements of NPS in lesioned mice could be due to the activation of NPSR expressed in substantia nigra, ventral tegmental area, and other structures downstream, such as globus pallidus and subthalamic nucleus. Additionally, a study aimed to investigate the effects of NPS on dosage of striatal dopamine levels by HPLC is mandatory for supporting the therapeutic potential of selective NPSR agonists in PD. Considering the main clinical challenges of Parkinson’s disease treatment (i.e., neuroprotection, disease-modifying and antidyskinetic effects), our research group has previously reported neuroprotective effects of NPS against oxidative stress evoked by the icv administration procedure [28,29], and chemically induced by pentylenetetrazole [40]. Studies in humans and animal models of PD reveal that mitochondrial dysfunction might be a defect that occurs in PD which can lead to a decline in energy production, generation of reactive oxygen species and induction of stress-induced apoptosis [41]. Antioxidant compounds seem to be interesting candidates for the treatment of PD [42], and in this regard the NPS has potential antioxidant effects. The main limitations of investigating the behavioral, neurochemical and also other aspects involved in the clinical challenges of PD is the availability of NPSR agonists able to cross the blood brain barrier. Therefore, brain penetrant non peptidic NPSR agonists are mandatory for further investigating the potential therapeutic effects in PD. 5. Conclusions In summary, we showed that the icv administration of NPS attenuated motor deficits evoked by 6-OHDA in the mouse rotarod test. 6-OHDA icv injected caused significant lost of TH-positive cells in the substantia nigra and ventral tegmental area. However, a positive correlation was only found between the number of TH-positive cells in the substantia nigra and the performance in the rotarod test. Based on the close relationship between dopamine and PD, and on the fact that NPSR receptors are expressed on dopaminergic nuclei in the brain, we proposed that the beneficial effects of NPS in 6-OHDA-treated mice may derive from the stimulation of dopaminergic neurotransmission. Further studies aimed to investigate the role of NPS on dopamine release in the nigrastriatal pathway are required to understand the mechanisms by which NPS restores 6-OHDA-induced motor deficits. These studies may provide important information to determine the potential of selective NPSR agonists as innovative drugs for the treatment of PD. Acknowledgments This work was supported by funds from Brazilian National Council Research (CNPq Grant Nos. 476832/2009-8, 475188/2011-0 and 305742/2009-4 to ECG), CAPES (PNPD 2783/2011), and from the University of Ferrara (FAR grants to GC and RG). JJD held a MSc fellowship founded by CAPES/Brazil. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bbr.2014.03.002. References [1] Hughes AJ, Daniel SE, Kilford L, Lees AJ. Accuracy of clinical diagnosis of idiopathic Parkinson’ disease: a clinic-pathologic study of 100 cases. J Neurol Neurosurg Psychiatry 1992;55:181–4. [2] Duty S, Jenner P. Animal models of Parkinson’s disease: a source of novel treatments and clues to the cause of the disease. Br J Pharmacol 2011;8:1–35.

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Neuropeptide S counteracts 6-OHDA-induced motor deficits in mice.

Neuropeptide S (NPS) is a 20-aminoacid peptide that selectively activates a G-protein coupled receptor named NPSR. Preclinical studies have shown that...
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