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Neurotox Res (2012) 21:291–301 DOI 10.1007/s12640-011-9278-3

Behavioral, Neurochemical and Histological Alterations Promoted by Bilateral Intranigral Rotenone Administration: A New Approach for an Old Neurotoxin Camila G. Moreira • Janaı´na K. Barbiero • Deborah Ariza • Patrı´cia A. Dombrowski • Pamela Sabioni • Mariza Bortolanza • Claudio Da Cunha • Maria A. B. F. Vital • Marcelo M. S. Lima

Received: 26 July 2011 / Revised: 6 September 2011 / Accepted: 17 September 2011 / Published online: 28 September 2011 Ó Springer Science+Business Media, LLC 2011

Abstract Rotenone exposure in rodents provides an interesting model for studying mechanisms of toxininduced dopaminergic neuronal injury. However, several aspects remain unclear regarding the effects and the accuracy of rotenone as an animal model of Parkinson’s disease (PD). In order to counteract these limitations, this study characterized a precise neurotoxin-delivery strategy employing the bilateral intranigral administration protocol of rotenone as a reliable model of PD. We performed bilateral intranigral injections of rotenone (12 lg) and subsequent general activity (1, 10, 20, and 30 days after rotenone) and cognitive (7, 8, 15, and 30 days after rotenone) evaluations followed by neurochemical and immunohistochemical tests. We have observed that rotenone was able to produce a remarkable reduction on the percentage of tyrosine hydroxylase immunoreactive neurons (about 60%) within the substantia nigra pars compacta. Dopamine (DA) was severely depleted at 30 days after rotenone administration, similarly to its metabolites. In addition, an increase in DA turnover was detected at the same timepoint. In parallel, striatal serotonin and its metabolite were found to be increased 30 days after the neurotoxic insult, without apparent modification in the serotonin turnover. C. G. Moreira J. K. Barbiero D. Ariza P. A. Dombrowski P. Sabioni M. Bortolanza C. D. Cunha M. A. B. F. Vital Laborato´rio de Fisiologia e Farmacologia do Sistema Nervoso Central, Departamento de Farmacologia, Universidade Federal do Parana´, Curitiba, PR, Brasil M. M. S. Lima (&) Laborato´rio de Neurofisiologia, Departamento de Fisiologia, Setor de Cieˆncias Biolo´gicas, Universidade Federal do Parana´, Av. Francisco H. dos Santos s/n, Caixa Postal: 19031, Curitiba, PR 81531-990, Brasil e-mail: [email protected]; [email protected]

Besides, motor behavior was impaired, mainly 1 day after rotenone. Furthermore, learning and memory processes were severely disrupted in different time-points, particularly at the training and test session (30 days). We now provide further evidence of a time-dependent neurodegeneration associated to cognitive impairment after the single bilateral intranigral administration of rotenone. Thus, it is proposed that the current rotenone protocol provides an improvement regarding the existing rotenone models of PD. Keywords Rotenone Intranigral Dopamine Substantia nigra pars compacta Parkinson’s disease

Introduction Neurodegenerative diseases are complex to model mainly due to the occurrence of several behavioral, cognitive, neurochemical and neuroanatomical abnormalities. Parkinson’s disease (PD) is a prototypical example of disorder that presents such body of characteristics that are not fully recapitulated by the existing animal models. While many models successfully reproduce certain features of the disease, their predictive value for neuroprotective interventions is uncertain, and the number of relevant outcome variables that can be assessed is limited (Cannon et al. 2009). The literature widely explores the key neurotoxic models of PD, for example induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 6-hydroxydopamine (6-OHDA) and more recently by rotenone (see Lima et al. 2009). Ideally, an animal model should have similar etiology and function to the human equivalent, and it is necessary that as many aspects of the disease are replicated as possible to avoid confounding and misleading results, which in turn may contribute to the

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development of new therapeutic approaches (Lane and Dunnett 2008). Rotenone is gaining increasing attention because it is widely used as herbicide in private gardens and in several powders for delousing humans or animals and thus a real threat that an environmental substance can cause (Nehru et al. 2008). Therefore, rotenone exposure in rodents provides an interesting model for studying mechanisms of toxin-induced dopaminergic neuronal injury (Betarbet et al. 2000; Greenamyre et al. 2003; Segura Aguilar and Kostrzewa 2004; Drolet et al. 2009). In this model, a massive inhibition of mitochondrial complex I produces selective degeneration of the dopaminergic nigrostriatal system and reproduces key pathological features of clinical PD (Sherer et al. 2003; Alam and Schmidt 2004). Indeed, rotenone administration affects many of the pathogenic pathways including: oxidative stress, alpha-synuclein phosphorylation and aggregation and Lewy pathology, DJ-1 acidification and translocation, proteasomal dysfunction and nigral iron accumulation (Betarbet et al. 2006). Previous studies have shown that systemic administration of rotenone is able to produce a clear pattern of neurodegeneration, mainly affecting the substantia nigra pars compacta (SNpc) (Thiffault et al. 2000), but also affecting the striatum and globus pallidus (Ferrante et al. 1997). More specific delivery strategies of the toxin—e.g., directly to the medial forebrain bundle (Alam and Schmidt 2004), or to the SNpc (Saravanan et al. 2005; Santiago et al. 2010)—could cause selective damage to the dopaminergic nigrostriatal system resulting in a more accurate model. However, several aspects remain unclear regarding the effects and the accuracy of rotenone as an animal model of PD (Lapointe et al. 2004). Accordingly, the main limitations of the rotenone model have been variability (i) in the percentage of animals that develop general activity and cognitive alterations, (ii) lesion magnitude, and (iii) characterization of neurochemical deficits (Schmidt and Alam 2006; Cannon et al. 2009). In order to counteract the limitations stated, the objective of this study was to characterize a precise neurotoxin-delivery strategy employing the bilateral intranigral administration protocol of rotenone as a reliable model of PD. To test this rationale, we performed intranigral injections of rotenone 12 lg (Saravanan et al. 2005; Santiago et al. 2010), partially destroying dopaminergic neurons located at the SNpc. Different open-field and cognitive parameters that are intensely modulated by dopamine (DA) were tested. In addition, neurochemical quantifications of neurotransmitters—DA, serotonin (5-HT) and their respective metabolites—within the striatum, as well as determination of the tyrosine hydroxylase immunoreactive neurons (TH-ir) neurons in the SNpc were performed.

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Materials and Methods Animals Male Wistar rats from our breeding colony weighing 280–320 g at the beginning of the experiments were used. The animals were randomly housed in groups of five in polypropylene cages with wood shavings as bedding and maintained in a temperature-controlled room (22 ± 2°C) on a 12-h light–dark cycle (lights on at 7:00 a.m.). The animals had free access to water and food throughout the experiment. Ethics Statement The studies were carried out in accordance with the guidelines of the Committee on the Care and Use of Laboratory Animals, United States National Institutes of Health. In addition, the protocol complies with the recommendations of Federal University of Parana´ and was approved by the Institutional Ethics Committee (approval ID #0144). Stereotaxic Surgery All the rats received atropine sulfate (0.4 mg/kg, intraperitoneal) and penicillin G-procaine (20.000 U in 0.1 ml, intramuscular) and were anesthetized with equitesin (chlornembutal, 0.3 ml/kg, intraperitoneal). Bilateral intranigral infusions of rotenone (12 lg in 1 ll of polyethylene glycol 400; Sigma, St. Louis, MO, USA) were performed according to previous studies (Saravanan et al. 2005; Santiago et al. 2010). In order to deliver the neurotoxin specifically into the SNpc, the following stereotaxic coordinates were adopted: anteroposterior (AP): -5.0 mm from the bregma; mediolateral (ML): ±2.1 mm from the midline; dorsoventral (DV): 8.0 mm from the skull (Paxinos and Watson 2005). The control of the flow of the injections were made by using an electronic pump (Harvard Apparatus, USA) at a rate of 0.33 ll/min, for 3 min, followed by 2 min with the needle in the injection site to avoid reflux. Sham operations followed the same procedure but 1 ll of polyethylene glycol was injected instead. Experimental Design Before the stereotaxic surgeries the animals were distributed randomly in three groups: control (n = 10), sham (n = 10) and rotenone (n = 10). Behavioral motor evaluation was performed through the open-field test in several time-points: 1, 10, 20, and 30 days after the rotenone infusion. After the last session of open-field, the rat brains were collected for TH-ir neurons quantification. A different

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set of animals—control (n = 10), sham (n = 10) and rotenone (n = 10)—underwent similar randomization and surgical lesion however; they were tested only in the twoway active avoidance task 7 (training session), 8, 15 and 30 days after rotenone. The same histological quantification was made after the last behavioral task. Finally, an additional set of animals was used exclusively for the neurochemical study performed 1 and 30 days after the rotenone. For this experiment, 30 rats were employed for each time-point and the groups followed the same randomization: control (n = 10), sham (n = 10), and rotenone (n = 10). Open-Field Test The apparatus consists of a rectangular box (40 9 50 9 63 cm) with a floor divided into 20 (10 9 10 cm) rectangular units. A 100-W ceiling light was situated 48 cm above the arena floor, providing illumination around 300 lx. The groups were firstly exposed to the apparatus at 1 day after rotenone. Throughout the subsequent timepoints the same rats were re-exposed to the open-field up to 30 days after the neurotoxin injection. The animals were gently placed in the right corner of the arena and were allowed to freely explore the area for 5 min. Four parameters were quantified during this test: latency time (time taken to initiate the first movement), locomotor activity (number of crossings from one rectangle to the other), rearing activity (number of times the animals stood on their hind paws) and immobility time (number of seconds of lack of movement during testing). The open-field was washed with a 5% water–ethanol solution before behavioral testing to eliminate possible bias due to odors left by previous rats. Two-Way Active Avoidance Task The two-way avoidance task apparatus consisted of an automated 23 9 50 9 23 cm shuttle-box (GEMINI Avoidance System, San Diego Instruments, CA, USA) with a dark front glass and a floor made of parallel 5 mm calibre stainless steel bars spaced 15 mm apart. The box is divided into two compartments of the same size by a guillotine door that remained opened during the test. The walls of each compartment were either black or white. The white compartment was illuminated while the black remained dark. In the training session the animals were individually placed in the light chamber facing the wall. The door was opened and the time to enter the dark compartment was recorded automatically by the apparatus. After 3 min of habituation, 50 sound cues (conditioned stimulus, 1.5 kHz, 60 dB, duration of 5 s) were paired with subsequent 0.4 mA foot shock (unconditioned stimulus,

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duration of 5 s) until the animal crossed to the other compartment. The animal could avoid the shock by crossing to the other side during the presentation of the conditioned stimulus (active avoidance). The time between each conditioned stimulus presentation varied randomly, ranging from 10 to 50 s. The number of active avoidances, the latency to cross to the other side of the box after the beginning of each conditioned stimulus, and the number of inter-trial crossing between the two box compartments were recorded automatically by the apparatus. The test session conducted 8, 15, and 30 days after rotenone were identical to the training one except for the absence of the foot shock. After 30 s of habituation, the door was opened, and the time taken by the rat to enter the dark compartment was recorded, with a ceiling of 300 s before removal of the animals that did not enter the dark compartment. The apparatus was cleaned with a 5% water– ethanol solution before behavioral testing to eliminate possible bias due to odors left by previous rats. Quantification of Striatal Neurotransmitters, Metabolites and Turnovers The striatum of the rats were rapidly dissected and stored at -80°C until the neurochemical quantification. The endogenous concentrations of DA, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) were assayed by reverse-phase high performance liquid chromatography (HPLC) with electrochemical detection. Briefly, the system consisted of a Synergi Fusion-RP C-18 reverse-phase column (150 9 4.6 mm i.d., 4 lm particle size) fitted with a 4 9 3.0 mm pre-column (Security Guard Cartridges Fusion-RP); an electrochemical detector (ESA Coulochem III Electrochemical Detector) equipped with a guard cell (ESA 5020) with the electrode set at 350 mV and a dual electrode analytical cell (ESA 5011A); a LC-20AT pump (Shimadzu) equipped with a manual Rheodyne 7725 injector with a 20 ll loop. The column was maintained inside in a temperature-controlled oven (25°C). The cell contained two chambers in series: each chamber including a porous graphite coulometric electrode, a double counter electrode and a double reference electrode. Oxidizing potentials were set at 100 mV for the first electrode and at 450 mV for the second electrode. The tissue samples were homogenized with an ultrasonic cell disrupter (Sonics) in 0.1 M perchloric acid-containing sodium metabisulfite 0.02% and internal standard. After centrifugation at 10,0009g for 30 min at 4°C, 20 ll of the supernatant was injected into the chromatograph. The mobile phase, used at a flow rate of 1 ml/min, had the following composition: 20 g citric acid monohydrated (Merck), 200 mg octane-1sulfonic acid sodium salt (Merck), 40 mg ethylenediaminetetraacetic acid (EDTA) (Sigma), 900 ml HPLC-grade

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water. The pH of the buffer running solution was adjusted to 4.0 then filtered through a 0.45 lm filter. Methanol (Merck) was added to give a final composition of 10% methanol (v/v). The neurotransmitters and metabolites concentrations were calculated using standard curves that were generated by determining in triplicate the ratios between three different known amounts of the internal standard. The unit was expressed as ng/g of wet weight. Striatal DA and 5-HT turnovers were calculated according to the equations: (DOPAC ? HVA)/DA and 5-HIAA/5HT, respectively. The values are expressed without units.

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Statistical Analysis Homogeneity of variance was assessed by the Bartlett test and normal distribution of the data by the Kolmogorov– Smirnov test. Differences between groups in the behavioral and neurochemical tests were analyzed by two-way analysis of variance (ANOVA)—with group as the between-subjects factor and repeated measures as the within-subjects factor—followed by the Newman–Keuls post hoc test. Immunohistochemistry findings were analyzed be one-way ANOVA followed by the Newman–Keuls post hoc test. Values are expresses as mean ± standard error of mean (SEM). The level of significance was set at P B 0.05.

TH Immunostaining For the immunohistochemical study, the rats were deeply anesthetized with ketamine immediately after the behavioral test, and were intracardially perfused with saline first, then with 4% of the fixative solution formaldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were removed from the skulls and were immersed during 1 week in that fixative solution at 4°C. Subsequently, the brains were placed in 30% sucrose solution for 48 h before sectioning. For each rat, 12 sections of 30 lm thick were cut on a cryostat in the coronal plane covering about 360 lm (-4.92 to -5.28 from bregma) of the midbrain (Paxinos and Watson 2005). These coordinates correspond to the maximal extent of the dopaminergic neurons within the SNpc (Reksidler et al. 2008; Santiago et al. 2010). Tissue sections were incubated with primary antibody anti-TH, raised in rabbits, diluted in PBS containing 0.3% Triton X-100 (1:500; Chemicon, CA, USA) overnight at 4°C. Biotin conjugated secondary antibody incubation (1:200; Vector Laboratories, USA), was made for 2 h at room temperature. After several washes in PBS, antibody complex was localized using a variation of the ABC system (Vectastain ABC Elite kit, Vector Laboratories, USA) followed by 3,30 -diaminobenzidine reaction with nickel enhancement. Slides were then dehydrated in ascending ethanol concentrations, cleared in xylene and cover slipped. Cell counts were conducted making use of the software Image-Pro Express 6 (Media Cybernetics, USA). Each slice was digitized with a digital camera connected to a microscope BX51 (Olympus Optical Co, Japan). A digital area was created in order to delimitate the boundaries of the SNpc. For each analysis, the same area was adopted. A ‘‘manual tag’’ tool was used to count the neurons inside the area. All the counts were performed in images obtained in 9400 magnification. Stereological parameters (counting frame, sampling grid) were calculated according to recommendations of straightforward counting neurons based on a computer approach (Schmitz and Hof 2000). The results were represented as % of the control group.

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Results Open-Field Test As can be seen in Fig. 1, latency time was significantly increased for the rotenone group at the time-point 1 day, in comparison to the control and sham groups, as revealed by the group factor [F(2.72) = 6.27; P = 0.0062], time factor [F(3.72) = 19.38; P \ 0.0001], and its interaction [F(6.72) = 6.52; P \ 0.0001] (Fig. 1a). Locomotion frequencies obtained after rotenone administration were significantly reduced 1 (P \ 0.001) and 10 (P \ 0.05) days, respectively, in comparison to the control and sham group, as indicated by the group factor [F(2.72) = 10.65; P = 0.0005], time factor [F(3.72) = 7.67; P = 0.0002], and interaction [F(6.72) = 2.70; P = 0.024] (Fig. 1b). Moreover, rearing frequency demonstrated to be reduced, for the rotenone group, only at the time-point 1 day, in comparison to the control (P \ 0.001) and sham (P \ 0.01) groups as specified by the group [F(2.72) = 14.33; P \ 0.0001] and time [F(3.72) = 19.31; P \ 0.0001] factors, but not by its interaction [F(6.72) = 1.89; P = 0.0942] (Fig. 1c). In contrast, concerning the immobility parameter, a significant increase was detected for the rotenone group only at the time-point 1 day, when compared to the control group, as demonstrated by the group [F(2.72) = 7.10; P = 0.0038], time [F(3.72) = 6.86; P = 0.0004], and interaction [F(6.72) = 5.31; P = 0.0001] factors (Fig. 1d). Two-Way Active Avoidance Task The results presented in Fig. 2 demonstrate that intranigral rotenone was able to promote a remarkable impairment in the learning and memory scores in this task. A significant reduction in the number of avoidances were found for the rotenone group in comparison to the control (P \ 0.05) and sham (P \ 0.05) groups, during the training session, as demonstrated by the group [F(6.72) = 8.89; P = 0.011],

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number of avoidances during the test sessions peaking at the time-point 30 days, with significant differences (P \ 0.05, for both groups) when compared to its own performances during the training session. On the contrary, the rotenone group exhibited a significant reduction in the number of avoidances, in comparison to the control (P \ 0.01) and sham (P \ 0.01) groups, at the time-point 30 days. Quantification of Striatal Neurotransmitters, Metabolites and Turnovers

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Fig. 1 Temporal motor behavior alterations quantified after bilateral intranigral rotenone administration. a Latency time (time taken to initiate the first movement); b locomotor activity (number of crossings from one rectangle to the other); c rearing activity (number of times the animals stood on their hind paws); d immobility time (number of seconds of lack of movement during testing). Values are expressed as mean ± SEM (n = 10/group). *P \ 0.05, ***P \ 0.001 compared to the sham group at the same time-point. Twoway ANOVA followed by the Newman–Keuls test

time [F(3.72) = 14.15; P = 0.0001], and interaction [F(6.72) = 5.30; P = 0.025] factors. Conversely, Control and sham groups revealed expected increases in the

Figure 3 shows the neurochemical comparison of two timepoints (1 and 30 days) that circumscribed the most noteworthy behavioral alterations inflicted by the intranigral administration of rotenone. Accordingly, DA revealed to be reduced only at 30 days, in comparison to the control (P \ 0.001) and sham (P \ 0.001) groups, as indicated by the group [F(2.72) = 18.49; P \ 0.0001], time [F(1.72) = 53.03; P \ 0.0001], and interaction [F(2.72) = 46.98; P \ 0.0001] factors (Fig. 3a). Similarly, striatal DOPAC levels were significantly reduced in the rotenone group at 30 days, when compared to the control (P \ 0.001) and sham (P \ 0.001) groups, as demonstrated by the group [F(2.72) = 5.54; P = 0.0012], time [F(1.72) = 0.69; P = 0.42] and interaction [F(2.72) = 6.12; P = 0.008] factors (Fig. 3b). Conversely, a significant increase in the HVA content was found for the rotenone group 1 day after its intranigral infusion, compared to the control (P \ 0.05) and sham (P \ 0.05) groups. Interestingly, a remarkable reduction of HVA content was detected for the rotenone group at the time-point 30 days, when compared to the control (P \ 0.01) and sham (P \ 0.01) groups as revealed by the group [F(2.72) = 0.10; P = 0.90], time [F(1.72) = 15.29; P = 0.0009], and interaction [F(2.72) = 12.20; P = 0.0003] factors (Fig. 3c). In contrast, the rotenone group

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TH Immunostaining Representative photomicrographs of TH immunohistochemistry in the SNpc obtained for the time-point 30 days are shown in Fig. 5a. The TH-ir neurons were prominently detectable in the SNpc of control and sham rats. The bodies and fibers of dopaminergic neurons showed intense staining with evident immunopositive processes. In opposite, the rotenone group exhibited a remarkable reduction of TH staining with an apparent neuroanatomical profile of nigral lesion including the presence of noticeable injection tracks (not present in the sham group). The unbiased quantification of the neuronal loss unveiled a significant reduction of about 60% in the percentage of TH-ir neurons within the bilateral SNpc for the rotenone group compared to the control (P \ 0.001) and sham (P \ 0.001) groups [F(2.69) = 14.83; P \ 0.0001] (Fig. 5b).

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Days Fig. 3 Neurochemical examination of the striatal content of a DA, b DOPAC, c HVA and d DA turnover after bilateral intranigral rotenone administration. Values are expressed as mean ± SEM (n = 10/group). *P \ 0.05, **P \ 0.01, ***P \ 0.001 compared to the sham group at the same time-point. Two-way ANOVA followed by the Newman–Keuls test

exhibited a significant increase in the striatal DA turnover when compared to the control (P \ 0.01) and sham (P \ 0!.001) groups only at 30 days, as demonstrated by the group

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[F(2.72) = 5.89; P = 0.01], time [F(1.72) = 13.15; P = 0.001] and interaction [F(2.72) = 4.00; P = 0.035] factors (Fig. 3d). Figure 4 depicts the alterations promoted by intranigral rotenone in the striatal levels of 5-HT, 5-HIAA, and 5-HT turnover occurred at 1 and 30 days after neurotoxin exposure. Rotenone elicited a significant increase in the 5-HT content only at the time-point 30 days, in comparison to the control (P \ 0.001) and sham (P \ 0.001) groups, as pointed out by the group [F(2.72) = 10.12; P = 0.001], time [F(1.72) = 3.71; P = 0.07], and interaction [F(2.72) = 9.98; P = 0.0012] factors (Fig. 4a). Similarly, striatal 5-HIAA content was found to be increased for the rotenone group, compared to the control (P \ 0.01) and sham (P \ 0.01) groups, as indicated by the group [F(2.72) = 10.90; P = 0.0007], time [F(1.72) = 1.70; P = 0.208], and interaction [F(2.72) = 1.04; P = 0.37] factors (Fig. 4b). In addition, the analysis of the 5-HT turnover did not reveal significant differences among the groups as expressed by the group [F(2.72) = 0.15; P = 0.86], time [F(1.72) = 0.35; P = 0.55] and interaction [F(2.72) = 1.74; P = 0.20] factors (Fig. 4c).

Discussion This study demonstrated the suitability of the bilateral intranigral infusion of rotenone as a reliable animal model of the presymptomatic state of PD. This proposal is validated due to several behavioral, neurochemical and histological alterations promoted by intranigral rotenone that resembles PD pathophysiology. We have observed that bilateral intranigral administration of rotenone was able to produce a remarkable reduction on the percentage of TH-ir neurons (about 60%) within the SNpc, with noticeable

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repercussions in the dopaminergic and serotoninergic neurotransmission. That is, DA was severely depleted at 30 days after rotenone administration, similarly to its metabolites. In addition, an increase in DA turnover was detected at the same time-point. In parallel, striatal 5-HT and its metabolite 5-HIAA were found to be increased 30 days after the neurotoxic insult, without apparent modification in the 5-HT turnover. Besides, general activity behavior was impaired, mainly 1 day after rotenone. Furthermore, learning and memory processes were severely disrupted in different time-points, particularly at the training and test session (30 days). Hence, the current findings describe a model of PD that apparently presents a confined neurodegeneration attributable to the specific

neurotoxin delivery and subsequent occurrence of a late dopaminergic and serotoninergic deficits, associated to a sustained learning and memory impairment. Several studies using different protocols of rotenone administration reported DA depletion and locomotor abnormalities (Alam and Schmidt 2002; Santiago et al. 2010), occasionally in a progressive fashion (Cannon et al. 2009), however, usually with high mortality (Biehlmaier et al. 2007) or repeated exposure paradigm (Ferrante et al. 1997; Betarbet et al. 2000). Such circumstances imply in reproducibility restrictions and increased contamination risk for the laboratory staff, which must manipulate the toxin in a long-term protocol. In addition, variability in neurochemical and behavioral parameters are frequent, mainly as a result of the low percentage of animals that express the neuronal damage in the dopaminergic nigrostriatal system. With the current protocol we describe that a single intranigral infusion of a small volume of the neurotoxin suspension is capable to mimic several desired features of PD. A multitude of studies previously showed nigrostriatal toxicity after rotenone exposure, although only a few demonstrated such events after intracerebral injections (Antkiewicz-Michaluk et al. 2004; Saravanan et al. 2005; Ravenstijn et al. 2008; Santiago et al. 2010). In this sense, the present results denoted an initial general activity behavior impairment similar to that induced by MPTP (Lima et al. 2006; Reksidler et al. 2007, 2009) or 6-OHDA (Lima et al. 2006; Santiago et al. 2010) that is compensated over time. However, rotenone elicited a significant reduction in the locomotion frequency at 10 days after surgery, suggesting that this parameter is possibly more affected by rotenone than the others collected during the open-field test (Reksidler et al. 2007). Considering that the main alterations in the open-field test were concentrated only at 1 day after rotenone, we decided to initiate the two-way active avoidance task 7 days after the neurotoxin infusion. In addition, repeated sessions were performed, in order to exclude any possible locomotor bias. Accordingly, a clear learning and memory deficit was achieved by the use of intranigral rotenone, corroborating the role of basal ganglia on select the actions based on environmental stimuli and store adaptative associations as nondeclarative memories such as motor skills, habits, and memories formed by different instrumental conditioning (Da Cunha et al. 2009). In view of that, we propose that the bilateral intranigral rotenone administration is a more adequate model to promote cognitive deficits than locomotor ones. We believe that this apparent limitation of the model (absence of a sustained locomotor disruption) indicates that rotenone mimics the presymptomatic state of PD. The reduced levels of DA, HVA and DOPAC, only detected 30 days after the insult, suggest that lesion exerts

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Fig. 5 Immunohistochemical analysis of the SNpc (left and right) at the end of the experiments (30 days after bilateral intranigral rotenone administration). Representative photomicrographs of the TH-ir neurons in the ventral midbrain of the groups are depicted in the a (magnification: 9100). b Shows the stereological quantification of the TH-ir neurons. Legend: SNpc substantia nigra pars compacta (indicated by the arrow), SNr substantia nigra reticular. Values are expressed as mean ± SEM (n = 10/group). ***P \ 0.001 compared to the sham group at the same timepoint. One-way ANOVA followed by the Newman–Keuls test

a time-dependent neurodegeneration of the striatal dopaminergic terminals. Moreover, the turnover calculation revealed an attempt to compensate this DA deficit by the dopaminergic neurons, which is a classical mechanism of plasticity, also observed with other neurotoxins (Hsieh

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et al. 2002; Ariza et al. 2010; Barbiero et al. 2010). Intriguingly, striatal content of 5-HT and 5-HIAA were both increased 30 days after rotenone infusion. However, the augment of 5-HIAA suggests an increase in the 5-HT degradation, that probably will result in a subsequent

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decrease in the content of 5-HT. This notion is substantiated by the absence of increase in the 5-HT turnover that, if detected, would suggest an engagement of plasticity mechanisms. Future studies adopting longer time-points are necessary to further investigate this hypothesis. Tryptophan-dietary depletion impairs object-recognition memory and is accompanied by a reduction in 5-HT in several brain areas, such as the hippocampus, frontal cortex, and striatum (Jenkins et al. 2010). Also, it is reported that intra-striatal hypoxanthine administration provoke impairment of spatial learning and memory in rats without affecting striatal dopaminergic system, although serotoninergic pathways seem to have been affected (Bavaresco et al. 2007). According to the current results it would be expected some level of compensatory effect on learning and memory (Mitchell et al. 2009). Previous studies in primates and rodents had also shown that MPTP-induced destruction of the nigrostriatal dopaminergic pathway leads to hyperinnervation of caudate nucleus and the putamen by serotoninergic fibres (Zhou et al. 1991; Gaspar et al. 1993; Maeda et al. 2003; Zeng et al. 2010). Thus, the present data is consistent with a rotenone induced compensatory sprouting of striatal serotoninergic fibres and consequent increase in the striatal 5-HT release. Besides, the magnitude of increase in DA release may depend, to some extent, on the density of 5-HT innervation which compels a new balance in DA neurotransmission within the striatum (Navailles et al. 2010). In other words, it is reasonable to suggest that the increase in the striatal 5-HT content would be a compensatory mechanism due to the massive decrease in the DA levels. We now provide further evidence of a possible timedependent neurodegeneration associated to cognitive impairment after the single bilateral intranigral administration of rotenone. Considering the current results, it is proposed that the intranigral rotenone is an accurate model to induce neurochemical, histological, and cognitive-like alterations similar to PD. In addition, the histological evidence denoted a remarkable reduction of about 60% in the number of TH-ir within the SNpc, a slightly increased neuronal loss compared to MPTP (Lima et al. 2007; Reksidler et al. 2008). Moreover, we did not detect significant alterations in the number of TH-ir neurons in the ventral tegmental area (data not shown), indicating that the neurotoxin delivery occurred in a site-specific fashion, as expected. A recent comparison between the rotenone infusion into the medial forebrain bundle and the intranigral infusion revealed that the former causes relatively minimal necrotic damage and the later provokes dopaminergic neuronal and associated striatal innervation loss as a result of an acute inflammatory response, and not exclusively due to rotenone exposure per se (Norazit et al. 2010). In fact this notion is rather attractive because one of

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the most recent features of PD is the participation of the neuroinflammatory process during (and/or before) the development of the disease. Thus, it is encouraged to find animal models that promote neuroinflammatory alterations similar to PD (Teismann et al. 2003; Lima et al. 2006; Reksidler et al. 2007; Choi et al. 2009; Ariza et al. 2010). In conclusion, the present data provide evidence that bilateral intranigral rotenone administration promotes cognitive, neurochemical and histological deficits similar to PD. Since other neurotoxin-induced animal models of PD—such as the MPTP (Prediger et al. 2006; Reksidler et al. 2008) or 6-OHDA (Deumens et al. 2002; Tadaiesky et al. 2008)—still undergo refinement in an attempt to more closely replicate the disease, we propose that the current rotenone protocol provides an improvement regarding the existing rotenone models of PD. Acknowledgments This study was supported by grants from CNPq, CAPES, and REUNI that had no further role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication. Conflict of interests interest exists.

The authors have declared that no conflict of

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