The pesticide fipronil injected into the substantia nigra of male rats decreases striatal dopamine content: A neurochemical, immunohistochemical and behavioral study

Behavioural Brain Research 384 (2020) 112562

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The pesticide fipronil injected into the substantia nigra of male rats decreases striatal dopamine content: A neurochemical, immunohistochemical and behavioral study

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Rahul Bharatiyaa, Jessica Bratzua, Carla Lobinac, Giulia Cordad, Cristina Coccod, Philippe De Deurwaerderee, Antonio Argiolasa,b,c, Maria Rosaria Melisa,b,*, Fabrizio Sannaa a

Department of Biomedical Sciences, Section of Neuroscience and Clinical Pharmacology, University of Cagliari, Cagliari, Italy Centre of Excellence for the Neurobiology of Addictions, University of Cagliari, Cagliari, Italy Institute of Neuroscience, National Research Council, Cagliari Section, University of Cagliari, Cagliari, Italy d Department of Biomedical Sciences, Neuro-Endocrine-Fluorescence (NEF) Laboratory, Cagliari, Italy e Institute of Cognitive and Integrative Neurosciences of Aquitaine, CNRS UMR 5287, University of Bordeaux, Bordeaux, France b c

ARTICLE INFO

ABSTRACT

Keywords: Fipronil Dopamine Motor activity Novel object recognition Social interaction Nociception

Experimental evidence shows that the phenylpyrazole pesticide fipronil exerts neurotoxic effects at central level in rodents, and in particular on nigrostriatal dopaminergic neurons, whose degeneration is well known to cause motor and non-motor deficits in animals and in humans. In order to characterize better the central neurotoxic effect of fipronil, we injected fipronil (15 and 25 μg) dissolved in dimethyl sulfoxide (DMSO) unilaterally into the substantia nigra of male rats. Male rats injected with DMSO unilaterally into the substantia nigra were used as controls. Control and fipronil-treated rats were then tested in different motor (i.e., open field arena, rotarod, tail flick) and non motor tests (novel object recognition, social interaction) 15 days after injection. A systemic challenge dose of the dopamine-agonist apomorphine was also used to study the presence of a rotational behavior. Sixteen days after fipronil or DMSO injection into the substantia nigra, rats were sacrificed, and either striatal dopamine content or substantia nigra tyrosine hydroxylase (TH) immunoreactivity were measured. The results confirm that the unilateral injection of fipronil into the substantia nigra caused the degeneration of nigrostriatal dopaminergic neurons, which leads to a decrease around 50 % in striatal dopamine content and substantia nigra TH imunoreactivity. This occurred together with changes in motor activity and coordination, and in nociception but not in recognition memory and in social interaction, as revealed by the results of the behavioral experiments performed in fipronil-treated rats compared to vehicle-treated rats 15 days after treatment, as found with other compounds that destroy nigrostriatal dopaminergic neurons.

1. Introduction Fipronil (FPN) belongs to the phenylpyrazole class of pesticides, which have a highly effective broad-spectrum activity as insecticides and are widely used for agricultural and non agricultural purposes, from soil injection, use on fruits, vegetables, coffee, rice and other crops and treatment of seeds to the use in poultry farms and in topical pet care products (see [1,2]). FPN acts as a GABAergic insecticide, by binding to the γ-gamma-aminobutyric acid (GABA) receptors and consequently by blocking chloride ion cellular uptake in invertebrates, leading to uncontrolled hyper-excitation of the central nervous system, convulsions, and cell death (see [3]). Due to its high lipophilic nature,

FPN can become sequestered in highly lipidic tissues, including the brain, for an extended period of time [4]. Exposure to micromolar concentrations of FPN has been also reported to be able to induce cell death in vitro, either in Caco-2 cells, which are used as a model to mimic the cellular barrier of the intestinal epithelium [5,6], or in human dopaminergic neuroblastoma SH-SY5Y cells, via an apoptotic pathway mediated by reactive oxygen species (ROS) and inflammatory response [7–9]. The latter finding suggests that dopaminergic neurons may be a possible target of FPN neurotoxicity at central level. In agreement with this hypothesis, a progressive loss of nigrostriatal dopaminergic neurons induced by inflammatory responses to FPN has been recently reported after injection

⁎ Corresponding author at: University of Cagliari, Department of Biomedical Sciences, Section of Neuroscience and Clinical Pharmacology, Cittadella Universitaria, SS 554, km 4,500, 09042, Monserrato, Cagliari, Italy. E-mail address: [email protected] (M.R. Melis).

https://doi.org/10.1016/j.bbr.2020.112562 Received 14 November 2019; Received in revised form 29 January 2020; Accepted 14 February 2020 Available online 15 February 2020 0166-4328/ © 2020 Elsevier B.V. All rights reserved.

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of FPN into the substantia nigra (SN) of male rats [9,10]. The inflammatory responses caused by FPN injected into the SN were revealed either by increased levels of pro-inflammatory factors such as inducible nitric oxide synthase (iNOS), ciclooxygenase 2 (COX-2), and tumor necrosis factor alpha (TNF-α) in the SN and in the striatum of FPNtreated rats, or by the up-regulation of glial fibrillary acidic protein expression and by the activation of microglia [9,10]. The increase of the above pro-inflammatory factors in FPN-treated rats was inversely correlated to the loss of nigrostriatal dopaminergic neurons, shown by the decrease in tyrosine hydroxylase (TH) immunoreactive neurons in the striatum and SN, decrease also confirmed by the decrease of TH expression by western blot analysis [9,10]. These results are in line with those obtained with other pesticides, such as rotenone and paraquat, in inducing a loss of dopaminergic neurons in the SN [11–17], as well as with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxy-dopamine, which are extensively used to realize animal models of Parkinson’s disease [18–23]. Many studies show also that degeneration of nigrostriatal dopaminergic neurons induced by the above compounds is often correlated to deficits in the performance of the treated animals in spontaneous motor activity in the open field [24–29] as well as in motor coordination in the rotarod test [30–34], in memory recognition (novel object recognition), social interaction and nociception [35–39]. In order to better characterize the central neurotoxic effects of FPN, the effect of two doses of FPN (15 μg and 25 μg) microinjected unilaterally into the SN of male rats was studied on spontaneous locomotor activity, motor coordination, the induction of rotational behavior after challenge with the dopamine agonist apomorphine, nociception, object recognition memory and social interaction, measured between 7 and 15 days after treatment. The results of these experiments show that FPN injected into the SN induces changes in spontaneous locomotor activity, motor coordination, apomorphine-induced rotational behavior, nociception, but not in novel object recognition and social interaction. These changes are related, at least in part, to the decrease of the striatal concentration of dopamine and its main metabolite 3, 4-dihydroxyphenylacetic acid (DOPAC) and of nigral TH immunoreactivity caused by the pesticide injected in the SN.

2.3. Microinjections into the substantia nigra (SN) After a preliminary examination of the performance of the animals on the Rotarod test, on the locomotor activity apparatus, and the control of the body weight, rats were divided into three groups: group-1 (DMSO-controls), group-2 (FPN 15 μg) and group-3 (FPN 25 μg). FPN doses were chosen taking into account those used in earlier studies (10). For microinjections into the SN, rats were positioned in the Stoelting stereotaxic apparatus under isoflurane anesthesia (1.5–2.0 %), the skin over the skull was cut with a lancet, the two edges of skin separated with pincers and a small hole was made in the skull with a dentist’s drill at the SN coordinates (AP: -5.3 mm; ML: -2.0 mm; DV: -8.0 mm) [40]. FPN dissolved in DMSO and diluted to the used concentrations (15 μg/ μL and 25 μg/μL, respectively), or DMSO alone (1 μL), were infused unilaterally into the SN in a volume of 1 μL/site/min by means of a 10 μL Hamilton microsyringe mounted on the holder of the Stoelting stereotaxic apparatus and driven by hand. The needle of the microsyringe was kept in the injection site for additional 3 min to allow a better diffusion of the injected solution before being slowly retracted. All rats were given 7 days to recover after the microinjections and behavioral tests were performed thereafter as described below. The body weight of all animals was recorded after the recovery period daily up to 15 days post-intranigral microinjection. The Rotarod test was performed after 7 days from surgery once daily until day 15 and other behavioral tests such as locomotor activity, apomorphine-induced rotation test, tail flick test, novel object recognition test and social interaction test were performed between the14th-16th day after FPN microinjection. 2.4. Locomotor activity Locomotor activity was measured as already described [41]. Rats were individually tested for motor activity under standardized environmental conditions (in a soundproof room with a light level of 30 lx) with a Digiscan Animal Activity Analyzer (Omnitech Electronics, Columbus, Ohio) after a two hr-habituation session in order to prevent the influence of novelty factors linked to the experimental procedure and motility apparatus, 15 days after intranigral microinjections. Each cage (42 cm x 42 cm x 63 cm) had two sets of 16 photocells located at right angles to each other, projecting horizontal infrared beams 2.5 cm apart and 2 cm above the cage floor and a further set of 16 horizontal beams whose height was adapted to the size of the animals (20 cm). Horizontal and vertical activities were measured as total number of sequential infrared beam breaks (counts) in the horizontal or vertical sensors, recorded every 5 min, beginning immediately after placing the animals into the cage, over a test period of 60 min.

2. Material and methods 2.1. Animals Adult three months old male Sprague Dawley rats, weighing 250−300 g (at the beginning of the experiments), were used in the study. Animals were housed in groups of 4 per cage, and maintained under standard conditions with 12-h light/dark cycles at room temperature (22 ± 2 °C, 60 ± 5% humidity). They were fed with standard pellet diet and tap water ad libitum along the study. The rats were handled once daily for 10 days before the injections into the SN in order to avoid the stress induced by the experimental procedures during the experimental session and also to familiarize them with the experimental operators. At the end of this period, each rat underwent a habituation session in order to prevent the influence of novelty factors linked to the kind of experimental procedure in which the animals were to be tested. The behavioral experiments were performed between 09:00–16:00 h, according to the guidelines of the European Communities Directive of September 22, 2010 (2010/63/EU) and the Italian Legislation (D.L. March 4, 2014, n. 26), and approved by the Ethical Committee for Animal Experimentation of the University of Cagliari.

2.5. Rotarod test Motor performance was checked using the Rotarod apparatus according to a procedure already described [42]. All animals were trained for 7 days before microinjection on the Rotarod apparatus in order to reach a stable performance. During the training phase and experimental sessions the rats were placed perpendicular to the rotating axis and the head against the direction of the rotation; the animal must therefore move forwards in order to stay on the rod. The rats were tested under the following protocol: beginning at the constant rotation speed of 2 rpm for the first 5 min, then progressively accelerated from 2 to 20 rpm for the next 5 min (acceleration phase) and finally under the constant speed of 20 rpm for other 5 min. The latency to fall was recorded at the beginning of the acceleration phase in a total 10 min (600 s) test period. Only the rats which successfully completed the training sessions (600 s) were selected. [43,44].

2.2. Drugs and reagents FPN (fipronil), apomorphine−HCl, dimethyl sulfoxide (DMSO) were purchased from Sigma Aldrich, (Dusseldorf, Germany), normal saline (0.9 % NaCl) was prepared before experiments. All other reagents were from available commercial sources.

2.6. Apomorphine-induced rotation test FPN-treated rats were compared to control rats for rotational 2

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index expressed by the ratio TN/(TF + TN) [TN = time spent exploring the novel object N; TF = time spent exploring the familiar object F] was first calculated for each animal [48,49]. As this measure can be biased by differences in overall levels of exploration, the discrimination index (DI) expressed by the ratio (TN - TF)/(TN + TF) was also calculated [50]. Between trials, the objects were carefully washed with 70 % ethanol solution to avoid olfactory cues.

behavior with a challenge dose of apomorphine−HCl (0.5 mg/kg). This test should be able to reveal the extent of the FPN-induced lesion at the level of the nigrostriatal dopaminergic neurons [45]. The rats were placed in a circular Plexiglas apparatus located in a dimly-lit, quiet room before test, partially filled with sawdust, to acclimatize with it. Apomorphine hydrochloride was dissolved in bidistilled water and injected subcutaneously (s.c.) at a dose of 0.5 mg/kg [45]. Two minutes after apomorphine injection, the rats were placed in the rotameter chamber and full 360-degree rotations (either ipsilateral or contralateral) were recorded for a 30 min test period by means of a digital video-camera and video-files stored in a backup device for following analyses.

2.9. Social interaction test The test rats (always one animal per time) were first habituated individually as described in the novel object recognition test. On the test day, to minimize transfer effects and avoid possible visual or olfactory influences, test rat and pairing rat, were transferred to testing room and allowed to acclimate for 10 min with the surrounding environment. For this, the rat pairs were weight-matched (a difference of no more than ± 5 g) and made up of rats that were unknown to each other (i.e. rats that did not share the same home cage from birth to single housing). Prior to the test, each test rat was marked by a vertical line on the back for differentiation between the two. Each pair of rats was placed in the open field arena (45 × 45 × 30 cm) and the behavior (such as sniffing, genital investigation, crawling under and climbing over) was recorded for 10 min test period by means of a digital videocamera and video-files stored in a backup device for following analyses [44,51].

2.7. Tail flick test The animal’s response to phasic pain was tested by measuring the latency of tail flick to a high intensity light beam. The test was performed with a Tail Flick instrument for the recording of the tail flick latency to radiant heat (TSE Systems, Bad Homburg, Germany). The animal was placed on the recording platform of the apparatus with its tail placed on the radiant heat window. A beam of light at 56 °C was projected and the latency to removal of the tail in response to the noxious stimulus was recorded. For each animal, the thermal stimulus was applied on three different parts of the tail and the latency to removal of the tail was considered as the mean of the three measurements. A cut-off exposure time of 20 s was set to prevent tissue damage [46].

2.10. Brain tissue dissections Once the behavioral studies were completed for each animal, for dopamine and DOPAC measurement 8 DMSO-, 8 FPN-15 μg and 5 FPN25 μg -treated rats were deeply anesthetized and sacrificed by decapitation. The brain was quickly removed and washed with ice-cold saline. After cooling, the right and left striatum were micro-punched [52,53] and transferred to 2 mL centrifuge tubes. The tissues were weighed and immediately stored at −80 °C until processing. Briefly, the striatal tissues were weighed, homogenized by sonication in 0.1 M perchloric acid (HClO4) (1 mg of wet tissue in 20 μL of 0.1 M HClO4) [54] and centrifuged at 23,000 rpm for 30 min. Then the supernatant obtained was filtered using microspin centrifuge tubes (0.22-μm nylon filter) at 10,000 rpm for 10 min. The filtered supernatant was stored at −80 °C until dopamine and DOPAC measurement.

2.8. Novel object-recognition test The novel object-recognition test was used to assess cognitive alterations associated with FPN treatment. Training in the object recognition task took place in an open field arena (45 × 45 × 30 cm), with the floor covered with sawdust. Rats (always one animal per time) were first habituated individually in the open field arena, in a dimly-lit quiet room, for 10 min to acclimatize with the arena and then put back in their home cages. Twenty four hr after habituation the rats were placed again in the open field arena and two identical objects (a plastic bottle with a heavy magnet at its bottom devoid of any natural significance) were positioned in two adjacent corners of the open field arena 9 cm apart from the walls and the rats were placed in remaining corner, facing towards the wall of the apparatus and familiarized with the object for 3 min [47] (Trial 1). At least 1 h after familiarization, rats explored the open field arena for other 3 min in presence of the familiar object (F) (a plastic bottle identical to that used in Trial 1) and a novel object (N) (a cube-shaped plastic box with a heavy magnet at its bottom, made with the same plastic of the bottle used in Trial 1) positioned in similar way (Trial 2). The exploration of the objects (defined as sniffing or touching the object with the nose and/or forepaws but not by turning around or sitting on the object) was recorded by means of a digital video-camera, and video-files stored in a backup device for the following analyses. The time spent exploring the test objects (e.g., the duration of exploration) of each rat was measured and the total exploration times in trial 1 and trial 2 calculated (Table 1). A recognition

2.11. Brain histology In order to verify the injection site in the SN, the remaining portion of the brain was immediately put in 4% paraformaldehyde solution until processing. The brain portion was then transferred in 30 % sucrose solution stored at 4 °C for at least 48 h before the section procedure. Brain sections of 40 μm were made by using a cryostat maintained at −20 °C, placed on a glass slide, stained with Neutral Red solution, then allowed to dry overnight under dark and on next day observed with a contrast phase microscope. Only the rats which showed the track of the microinjection needle positioned correctly in the SN (Fig. 1) were considered for the statistical analysis of the results.

Table 1 Novel object recognition test: total 3 min exploration times.

Total exploration times (s) in Trial 1 Total exploration times (s) in Trial 2 Novel object exploration times (s) in Trial 2 Familiar object exploration times (s) in Trial 2

DMSO-treated rats Mean ± SEM

FPN 15 μg-treated rats Mean ± SEM

FPN 25 μg-treated rats Mean ± SEM

49.93 ± 4.05 79.87 ± 6.17** 50.67 ± 4.07# 29.20 ± 3.53

42.60 ± 5.52 79.47 ± 6.58**** 49.40 ± 6.80# 30.07 ± 3.55

48.27 ± 3.97 81.09 ± 6.18** 51.73 ± 6.17# 29.36 ± 3.98

Total exploration time in Trial 2 is given by the sum of the novel object exploration time and of the familiar object exploration time in Trial 2. Values are means ± SEM of 11–15 rats per group; **: P < 0.01, ****: P < 0.0001 Total exploration time in Trial 2 vs. Trial 1; #: P < 0.05 Novel vs. Familiar object in Trial 2 (two-way ANOVA followed by Tukey’s multicomparison test). 3

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with PBS, coverslipped with PBS-glycerol and visualized using an Olympus BX41/BX51 fluorescence microscope (Milan, Italy), equipped with a Fuji FinePix S2 and S3 Pro digital camera (Fujifilm, Milan, Italy). Controls included negative controls (replacement of the primary antibody by antibody diluent alone). In order to obtain a semi-quantitative determination of the immunofluorescent signal, the FIJI image processing package, based on ImageJ (NIH) was used. The values of three sections (encompassing the initial, medial and final portion of the SN) were acquired in order to cover the entire SN of each animal. The means ± SEM of density values for TH were then calculated for each group of experimental animals (control DMSO, FPN 15 μg/μL and FPN 25 μg/μL), and changes in the injected SN among groups reported as percent of the values of the intact SN (intact SN IR = 100 %) [41]. 2.14. Statistical analysis Data are presented as mean values ± SEM. Total motility scores, rotational behavior, tail flick, novel object recognition, social interaction, neurochemical and immunohistochemical data were analyzed by means of one- or two-way factorial ANOVAs. Body weight changes, time-fractioned motor activity and rotarod data were analyzed by means of two-way ANOVAs for repeated measures with the treatment as between subjects factor and the time (i.e., day test or test fraction) as within subjects factor. When ANOVAs revealed statistically significant main effects and/or interactions, pair wise comparisons were performed by using the Tukey’s multicomparison test. Statistical analyses were all carried out with PRISM, Graph Pad 8 Software (San Diego, USA) with the significance level set at P < 0.05.

Fig. 1. Schematic representation of a coronal section of the rat brain showing the tip of the microinjection needle in the SN [40]. The portion of the Neutral Red-stained section showing the tip of microinjection needle into the SN (marked by the black arrow) is magnified in the insert. Abbreviations: SN = substantia nigra; VTA = ventral tegmental area.

2.12. Measurement of striatal dopamine and DOPAC concentration Dopamine and DOPAC were measured by injecting a 20 μL aliquot of the supernatant obtained from striatal homogenates (as described above, brain tissue dissection) by using high pressure liquid chromatography (HPLC) coupled to electrochemical detection using a 4011dual cell (Coulochem II, ESA, Cambridge, MA, USA) as already described [55,56]. Detection was performed in reduction mode at +350 and −180 mV. The HPLC was equipped with a Supelcosil C18 column (7.5 cm ×3.0 mm i.d., 3 μm particle size; Supelco, Supelchem, Milan, Italy), eluted with 0.06 M citrate/acetate pH 4.2, containing methanol 20 % v/v, 0.1 mM EDTA (ethylenediaminetetraacetic acid), 1 μM triethylamine, and 0.03 mM sodium dodecyl sulfate as a mobile phase, at a flow rate of 0.6 mL/min and room temperature. The sensitivity of the assay was 0.125 pg for dopamine and 0.1 pg for DOPAC.

3. Results 3.1. Body weight As shown in Fig. 2, there was no significant difference in the body weight pattern between rats unilaterally injected into the SN either with vehicle (DMSO 1 μl, control rats) or FPN at the dose of either 15 μg or 25 μg. A slight decrease in body weight was observed in the first week after surgery in the majority of rats. The weight gain pattern, from 8 to 12 days after the microinjection, was progressive and slightly higher in control DMSO-treated rats as compared to FPN-treated rats, but the differences were not statistically significant. Accordingly, two-way ANOVA detected a significant effect of Day [F(9,342) = 240.5, P < 0.0001], a significant Day x Treatment interaction [F(18,342) = 5.347, P < 0.0001], but not a significant effect of Treatment [F(2,38) = 0.224, P > 0.05]. Thereafter, the weight gain pattern seemed nearly similar until the 15th day of the test period in all the rats.

2.13. Immunohistochemistry For immunohistochemistry, 5 DMSO-, 5 FPN-15 μg and 4 FPN-25 μg-treated rats were deeply anaesthetized with chloral hydrate (400 mg/kg i.p.) and transcardially perfused-fixed with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2-7.4. Brains were rapidly removed; washed overnight in PBS containing 7% sucrose and 0.01 % NaN3, and orientated in aluminium foil moulds in cryo-embedding medium (in g/ L: polyvinyl alcohol, 80; polyethylene glycol, 42.6; Tween-20, 10; and NaN3, 0.5) [57], and frozen in melting freon (cooled with liquid nitrogen). Coronal cryosections (10 μm) comprehensive of the whole SN obtained from the midbrain (starting from section with AP≈−6.5 up to section with AP≈−4.5) [40], were collected onto poly-L-lysine-coated slides and stored in the vapor phase of a liquid nitrogen tank until used. For immunohistochemistry, sections prepared as reported above obtained from 4 to 5 rats for each group, were brought to room temperature and washed in Triton X-100 (1 mL/L, in PBS solution). Sections (10 μm) of the SN were selected and then labeled with polyclonal sheep anti-TH (Millipore, Darmstadt Germany, AB_373131, 1:600), followed by the incubation with the corresponding speciesspecific donkey secondary antibody conjugated with Cy3 (Jackson Immunoresearch Laboratories, West Grove, PA) to reveal the immunoreactivity of the primary antibody. Primary and secondary antibodies were routinely diluted in PBS containing 30 mL/L of normal donkey serum, 30 mL/L of normal rat serum and 0.02 g/L NaN3, in order to prevent the non specific binding. Sections were finally washed

Fig. 2. Body weight of fipronil (FPN, 15 μg and 25 μg)- and control DMSOtreated rats. Values are expressed as means ± SEM (n = 11-15 rats per group) (two-way ANOVA followed by Tukey’s multicomparison test). B.S. = before surgery. 4

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Fig. 4. Total counts of horizontal locomotor activity in the first 30 min (A) and in the whole 60 min test (B) of fipronil (FPN, 15 μg and 25 μg)-treated rats and control DMSO-treated rats. FPN and DMSO were injected unilaterally into the SN 15 days before the experiment. Values are expressed as means ± SEM (n = 11-15 rats per group). #P < 0.05, FPN 25 μg.-treated rats vs. control DMSOtreated rats (one-way ANOVA followed by Tukey’s multicomparison test).

Fig. 3. Horizontal (A) and vertical (B) locomotor activity of fipronil (FPN, 15 μg and 25 μg)- and control DMSO-treated rats. FPN and DMSO were injected unilaterally into the SN 15 days before the experiment. Rats were put individually inside the apparatus and locomotor activity was recorded for 60 min (12 consecutive periods of 5 min). Values are expressed as means ± SEM (n = 11-15 rats per group) (two-way ANOVA followed by Tukey’s multicomparison test).

3.2. Locomotor activity As shown in Fig. 3, FPN 15 μg and 25 μg-treated rats did not show any change in horizontal (Fig. 3A) or vertical activity (Fig. 3B) as compared to control DMSO-treated rats. Indeed, the horizontal and vertical activities decreased in the 60 min of the experiment in a similar way in all the three groups of rats. However, both FPN 15 μg- and 25 μg-treated rats expressed a tendency to show higher levels of horizontal activity (Fig. 3A) in the first 30 min of the test when compared to control DMSOtreated rats, although this increase did not reach a statistical significance. Accordingly, two-way ANOVA of horizontal activity detected a significant effect of Time fraction (F(11,418) = 93.78, P < 0.0001), a significant Time fraction x Treatment interaction (F(22,418) = 1.865, P < 0.05), but not a significant effect of Treatment (F(2,38) = 2.415, P > 0.05), while two-way ANOVA of vertical activity detected a significant effect of Time fraction (F(11,418) = 53.86, P < 0.0001), but not a significant effect of Treatment (F(2,38) = 0.05487, P > 0.05), nor a significant Time fraction x Treatment interaction (F(22,418) = 0.9865, P > 0.05). In line with the results shown in Fig. 3, additional analyses revealed that FPN 25 μg-treated rats show an increase in the total counts of horizontal activity, when compared to the control DMSO-treated counterparts (P < 0.05) in the first 30 min of the test (one-way ANOVA, F(2,38) = 4.70, P < 0.05) (Fig. 4A); however, this difference was not anymore detectable when considering the whole 60 min duration of the test (Fig. 4B) (one-way ANOVA, F(2,38) = 2.359, P > 0.05). In contrast, no increase was detected in the total counts of vertical activity of FPNtreated compared to DMSO-treated rats in the first 30 min or total 60 min of the test (one-way ANOVA, 30 min: F(2,38) = 0.142, P > 0.05; 60 min: F(2,38) = 0.045, P > 0.05) (data not shown).

Fig. 5. Percent impairment of motor performance of rats in Rotarod test after unilateral SN microinjections of fipronil (FPN) at 15 days after injection. Percent impairment of motor performance is defined as [(T1-T2)/T1] x 100, where T1 and T2 are the amount of time each rat remained on the rotating drum in the two trials conducted before and after microinjections, respectively. Values are expressed as means ± SEM (n = 11-12 rats per group). *P < 0.05, **P < 0.01, ***P < 0.001, FPN 15 μg-treated rats vs. control DMSO-treated rats; #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001, FPN 25 μg.treated rats vs. control DMSO-treated rats (two-way ANOVA followed by Tukey’s multicomparison test). B.S. = before surgery.

3.3. Rotarod test As shown in Fig. 5, FPN-treated rats (FPN 15 μg and FPN 25 μg) displayed a marked impairment of motor performance as compared to control DMSO-treated rats in the Rotarod task. The test clearly shows a significant impairment of motor performance in FPN-treated rats compared to control rats in all the days of test after intranigral microinjection (two way ANOVA, Treatment: F(2,38) = 8.44, P < 0.001; Day: F(9,342) = 28.91, P < 0.0001; Treatment x Day: F(18,342) = 3.56, P < 0.0001; pair wise comparisons: from P < 0.05 to P < 0.0001 5

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Fig. 6. Apomorphine (0.5 mg/kg, s.c.) induces rotations mainly ipsilateral to the injection site in fipronil (FPN, 15 μg and 25 μg)-treated rats as compared to control DMSO-treated rats. Fipronil (FPN) and DMSO were injected unilaterally into the SN 15 days before the experiment. Values are expressed as means ± SEM (n = 11-15 rats per group). ***P < 0.001, FPN-treated rats vs. control DMSO rats, ###P < 0.001, ####P < 0.0001 ipsilateral vs. contralateral turns (two-way ANOVA followed by Tukey’s multicomparison test).

Fig. 7. Pain sensitivity of fipronil (FPN)- and DMSO-treated rats in presence of acute thermal noxious stimuli, evaluated using tail flick test 15 days after injection. FPN (15 μg and 25 μg) and DMSO were injected unilaterally into the SN 15 days before the experiment. Values are expressed as means ± SEM (n = 1115 rats per group). ****P < 0.0001, FPN (15 μg or 25 μg)-treated rats vs. control DMSO rats (one-way ANOVA followed by Tukey’s multicomparison test).

FPN- vs. DMSO-treated rats). Some impairment (although not significant) in motor performance is also seen in control DMSO-treated rats, which may be attributed to the habituation on the Rotarod apparatus that may cause a gradual decrease in motor performance per se (Fig. 5).

approach the objects (Fig. 8C) (two-way ANOVA, Treatment: F (2,76) = 0.070, Object: F (1,76) = 2.058, Treatment x Object: F (2,76) = 0.069, all P > 0.05) between the FPN (15 μg and 25 μg)- and control DMSO-treated rats. On the other hand, an increase in the number of approaches to the novel object was observed as compared to the familiar object (two-way ANOVA, Treatment: F (2,76) = 1.298, P > 0.05, Object: F (1,76) = 9.393, P < 0.01, Treatment x Object: F (2,76) = 0.386, P > 0.05) (Fig. 8D), which was significant (pair wise comparisons: P < 0.05) in the DMSO- but not FPN-treated groups.

3.4. Apomorphine-induced rotation test As shown in Fig. 6, apomorphine (0.5 mg/kg s.c.) induces a greater number of ipsilateral than contralateral rotations to the FPN injected side in FPN-treated rats when compared to DMSO-treated rats (two way ANOVA, Treatment: F(2,76) = 5.83, P < 0.01; Rotation side: F(1,76) = 50.48, P < 0.0001; Treatment x Rotation side: F(2,76) = 5.52, P < 0.01; pairwise comparisons: P < 0.0001 and P < 0.001 for 15 μg and 25 μg FPN-treated rats, respectively). Moreover, pairwise comparisons showed that FPN (15 μg and 25 μg)-treated rats displayed a significant higher number of ipsilateral rotations to the FPN-injected side as compared to control DMSO-treated rats (both P < 0.001) while no differences were detected in the number of the contralateral rotations.

3.7. Social interaction test The social interaction test failed to show significant differences in the time of interaction (contact) (Fig. 9A) and number of contacts (Fig. 9B) and between FPN (15 μg and 25 μg) and control DMSO-treated rats (two-way ANOVA, interaction time, F(2,38) = 0.339, number of contacts, F(2,38) = 0.524, both P > 0.05).

3.5. Tail flick test

3.8. Striatal dopamine and DOPAC concentrations

The tail flick test was used to evaluate pain sensitivity of FPN (15 μg and 25 μg) and control DMSO-treated rats in presence of acute noxious stimuli. FPN (15 μg and 25 μg)-treated rats show significant less time to flick the tail (one-way ANOVA, F(2,38) = 22.69, P < 0.0001) when exposed to the acute noxious thermal stimuli on tail as compared to control DMSO-treated rats (DMSO- vs. FPN-treated rats both P < 0.0001) (Fig. 7). This suggests that FPN-treated rats have a lower (thermal) threshold to pain when compared to DMSO-treated rats.

As shown in Fig. 10, FPN 15 μg and 25 μg microinjected unilaterally into the SN induced both a marked decrease of the concentration of dopamine (40–50 %) and DOPAC (30–40 %) in the striatum ipsilateral to the injected SN when compared to the contralateral striatum (two way ANOVA, dopamine, Treatment: F(2,36) = 34.44, p < 0.0001; Injection side: F(1,36) = 118.3, p < 0.0001; Treatment x Injection side: F (2,36) = 34.44, p < 0.0001; DOPAC, Treatment: F(2,36) = 8.00, P < 0.01; Injection side: F(1,36) = 22.14, P < 0.0001; Treatment x Injection side: F(2,36) = 8.00, p < 0.01; pair wise comparisons: from P < 0.05 to P < 0.0001). The concentrations of dopamine and DOPAC in the striatum ipsilateral to the injected SN of both FPN-treated rats (15 μg and 25 μg) were also highly significantly lower than those found in the striatum ipsilateral to the injected SN of the DMSO-treated rats (from P < 0.01 to P < 0.0001 FPN-vs. DMSO-treated rats). Accordingly, absolute dopamine and DOPAC contents were 8912 ± 848 pg and 2083 ± 184 pg, 10437 ± 1374 pg and 2364 ± 271 pg and 8751 ± 1478 pg and 1986 ± 312 pg per mg of tissue, respectively, in the striatum contralateral and 4548 ± 355 pg and 1284 ± 119 pg, 6851 ± 1266 and 1858 ± 302 pg, and 8753 ± 1686 pg and 1966 ± 331 pg per mg of tissue, respectively, in the striatum ipsilateral to the injected SN of FPN 15 μg-, SN FPN 25 μg- and SN DMSO-treated rats, respectively. Moreover, the DOPAC/dopamine ratio in the striatum ipsilateral to the injected SN of FPN-15 μg and 25 μg-treated

3.6. Novel object-recognition test As shown in Table 1, total exploration time of Trial 1 was shorter than that of Trial 2 in all the three groups of rats as expected; however, no significant difference was found between the total exploration time for the familiar object in Trial 1 and the familiar object and the novel object in Trial 2 among the three groups (two-way ANOVA, Treatment: F (2,76) = 0.314, P > 0.05, Trial: F (1,76) = 51.13, P < 0.0001, Treatment x Trial: F (2,76) = 0.210, P > 0.05). No significant difference was also detected in the recognition index (calculated for each rat by the ratio TN/(TF + TN) [TF = time spent exploring the familiar object (F); TN = time spent exploring the novel object (N)] (Fig. 8A) (one way ANOVA, F(2,38) = 0.364, P > 0.05) or in the discrimination index calculated by the ratio (TN - TF)/(TF + TN) (Fig. 8B) (one way ANOVA, F(2,38) = 0.347, P > 0.05) or in the latency (seconds) to 6

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Fig. 8. Novel object recognition task performed on fipronil (FPN, 15 μg and 25 μg)- and control DMSO-treated rats: effect on A) the recognition index calculated for each rat by the ratio TN/(TF + TN) [TF = time spent exploring the familiar object (F); TN = time spent exploring the novel object (N)]; B) the discrimination index calculated for each rat by the ratio (TN - TF)/(TF + TN) [TN = time spent exploring the novel object (N) in trial 2; TF = time spent exploring the familiar object (F) in trial 2]; C) the latency to approach objects (s); and D) the number of approaches on objects. FPN and DMSO were injected unilaterally into the SN 15 days before the experiment. Values are expressed as means ± SEM (n = 11-15 rats per group). *P < 0.05 novel vs. familiar object (one-way or two-way ANOVA followed by Tukey’s multicomparison test).

4. Discussion

rats was ≈20 % higher than that of SN DMSO-treated rats (0.28 vs. 0.23) (two-way ANOVA, Treatment F(2,36) = 7.796, P < 0.01, Side F (1,36) = 21.06, P < 0.0001, Treatment x Side F(2,36) = 5.235, P < 0.05, pair wise comparisons: from P < 0.01 to P < 0.001), which showed a DOPAC/dopamine ratio similar to that of the contralateral striatum (both values: 0.23). These findings indicate a significant neurodegenerative/neurotoxic effect of FPN injected in the SN and suggest that SN FPN-treated rats have a striatal dopamine turnover higher than that of SN DMSO-treated rats.

This study shows for the first time that FPN injected unilaterally in the SN induces changes in spontaneous locomotor activity and in motor coordination in the motility and rotarod apparatus and in rotational circling when challenged with the dopaminergic drug apomorphine. In particular, only a modest but significant time-dependent increase was seen on horizontal but not vertical activity in the first 30 min of the motility test at the dose of 25 μg of the pesticide injected into the SN (this increase was not longer detectable when considering the whole 60 min of the test). It is unlikely that this increase is due to a reduced thigmotaxis (e.g., to higher levels of anxiety of FPN- vs. DMSO-treated rats at the beginning of the behavioral testing), since all rats were previously habituated for 2 h to the motility apparatus. However, this result resembles the increase in locomotor activity found 6 days after treatment in rats with lesions of the nigrostriatal dopaminergic neurons induced by MPTP given at doses that produce a 50–60 % decrease in striatal dopamine content [27], which is slightly higher than that found in FPN-treated rats in this study. However in the above study a decrease rather than an increase in locomotor activity was found 18 days after MPTP treatment, which is similar to the decrease in locomotor activity found in rotenone- and MPP+-treated rats, with a 50–60 % decrease in striatal dopamine content [28,29] as well as in 6-hydroxy-dopaminetreated rats, given at doses that produce up to 80 % dopamine decrease in the striatum [24–26]. This difference may be due to the fact that MPTP, MPP+, rotenone and 6-hydroxy-dopamine in the above studies were given bilaterally while FPN was given unilaterally. FPN injected into the SN at both doses tested also induced a marked impairment of motor performance in the rotarod task when compared to DMSO-treated rats. The impairment in the rotarod performance task induced by both doses of FPN was long lasting as it was always found

3.9. Immunohistochemistry Differences in TH immunostaining between the treated and untreated SN were found using the TH antibody. Accordingly, as shown in Fig. 11, dopaminergic neurons were reduced in number in the SN after FPN (15 μg and 25 μg) treatment when compared to the intact contralateral SN or to the DMSO-treated SN. In both cases, the decrease was found to be extended through the different sections examined. The differences in TH immunostaining between contralateral intact and FPN- and DMSO-injected SNs were found significant after ImageJ analysis (Fig. 12) (two way ANOVA, Treatment: F(2,39) = 13.58, P < 0.0001; Injection side: F(1,39) = 55.73, P < 0.0001; Treatment x Injection side: F(2,39) = 13.58, P < 0.0001). Accordingly, FPN, 15 μg and 25 μg microinjected unilaterally into the SN induced both a marked decrease (≈40–50 %) in the TH immunofluorescence in the injected SN when compared to the contralateral intact SN and in the FPN-treated SN when compared to the DMSO-treated SN (all P < 0.0001). Apparently FPN 25 μg induces a similar decrease in TH immunofluorescence when compared to FPN 15 μg (P > 0.05), showing a significant neurodegenerative/neurotoxic effect of FPN injected in the SN as compared to DMSO treatment. 7

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Fig. 9. Social interaction test in fipronil (FPN, 15 μg and 25 μg)- and control DMSO-treated rats. A) Time of interaction (sec), B) Number of contacts. FPN and DMSO were injected unilaterally into the SN 15 days before the experiment. Values are expressed as means ± SEM (n = 11-15 rats per group) (oneway ANOVA).

Fig. 10. A) Percent (%) dopamine (DA) and B) DOPAC in the ipsilateral and contralateral striatum of SN Fipronil (FPN, 15 μg and 25 μg)-treated rats compared to control DMSO-treated rats. FPN and DMSO were injected unilaterally into the SN 16 days before the sacrifice. Absolute dopamine and DOPAC contents per mg of striatal tissue are those reported in the Results section. Values are expressed as means ± SEM (n = 5-8 rats per group). **: P < 0.01, ****: P < 0.0001 FPN-treated rats vs. control DMSO, #: P < 0.05, ####: P < 0.0001 striatum ipsilateral to the injected SN vs. striatum ipsilateral to the intact SN, (two-way ANOVA followed by Tukey’s multicomparison test).

from day 7 to day 15 after FPN injection into the SN, when compared to the rotarod performance of DMSO-treated rats. The impairment of SN FPN-treated rats in the rotarod test is in line with earlier experiments reporting a marked decrease in the latency to fall from the rotarod in SN 6-hydroxy-dopamine- or MPP+- treated rats starting two weeks after the injection of these compounds either in the striatum or the SN [34,35]. However, in these studies the decline in motor performance in the rotarod occurred together with a marked decrease in locomotor activity in the open field test [34,35], as found also in rats systemically treated with rotenone [33] at variance from the FPN-treated rats of this study, which did not show a decrease in locomotor activity (see above). This study also shows that FPN-treated rats when challenged with the direct dopamine agonist apomorphine also showed a significantly higher number of rotations ipsilateral to the injected SN when compared to DMSO- treated rats. This resembles the results of earlier experiments showing that rats with a partial unilateral lesion of the nigrostriatal dopaminergic system (which causes a 50–60 % decrease in striatal dopamine when compared to that of the intact striatum, similar to that found in the FPN-treated rats) show often rotational behavior ipsilateral to the lesion side or even no rotational behavior at all, when challenged with systemic apomorphine [24,58–60]. This has been explained by assuming that when dopamine content is still around 50 % of the control values, compensatory mechanisms occur in the striatum making turning/circling after apomorphine challenge either ipsilateral or even not visible. These mechanisms, which may be secondary to changes in dopamine activity in lesioned areas (i.e. increased dopamine synthesis in or release from surviving neurons) or to changes in the activity of other neurotransmitter systems in the basal ganglia (i.e., GABA, glutamic acid, serotonin and others) (see [41]), may help mitigate the progressive loss of dopaminergic innervations and prevent the appearance of postsynaptic dopamine receptor hypersensitivity, explaining in part the failure of apomorphine to induce contralateral

turning in SN FPN-treated rats. In line with this hypothesis, and as discussed below, a higher DOPAC/dopamine ratio (suggestive of a higher dopamine turnover) was found in the striatum ipsilateral to the injected SN of FPN- compared to DMSO-treated rats. This study also shows that FPN injected unilaterally into the SN reduces significantly the latency of the animals to flick the tail when exposed to a thermal stimulus as compared to control DMSO-treated rats in a classical tail flick test used for testing drug-induced analgesia. This suggests that FPN-treated rats have a lower threshold to thermal stimuli when compared to DMSO-treated rats. In general, this finding is in line with experimental evidence implicating a role of the basal ganglia in general and of dopamine in particular, in the regulation of nociception. Accordingly, the destruction of dopaminergic neurons in the SN may impair natural analgesia by disrupting the dopaminemediated descending pathways that block neurotransmission of ascending nociceptive signals from the spinal cord [61]. In line with this hypothesis, in rats with unilateral injection of 6-hydroxy-dopamine into the medial forebrain bundle inducing a depletion of striatal dopamine around 80 %, 6-hydroxy-dopamine -lesioned animals were found to exhibit lower thermal thresholds than sham control rats although the response latency of the tail flick test was only slightly shorter in the 6hydroxy-dopamine-lesioned rats respect to sham control rats [62]. However, in contrast to the above results, a recent study shows that rats with unilateral injection of 6-hydroxy-dopamine given in the striatum showed no change in thermal nociceptive threshold in the tail flick test, while exhibiting a significant decrease in withdrawal threshold to non 8

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Fig. 11. Tyrosine hydroxylase (TH) staining in the substantia nigra (SN) of DMSO- and FPN- treated groups (untreated vs. treated sides). TH immunostaining is similarly revealed within the two sides of DMSO treated groups but it was decreased after intranigral injection of fipronil (FPN) at both concentrations (15 μg and 25 μg) compared to contralateral untreated sides. TH: Cy3, red labeling. Scale bar: 40 μm.

painful mechanical stimuli [38]. Differences in the experimental conditions (i.e. the type of drug injected, the site of injection, and others), may be responsible for these discrepancies. Another result of this study is that in the novel object recognition test, FPN injected unilaterally into the SN at both the doses tested failed to change both the recognition index and the discrimination index and the latency to approach both the familiar and the novel object when compared to the DMSO-treated rats. As the novel object recognition test allows the evaluation of recognition memory and the estimation of the cognition state of the animal by measuring the time spent/dedicated to a new object presented to the animal when compared to that dedicated to a familiar object [50,63], the failure of FPN injected into the SN to modify the above parameters, suggests that the pesticide did not cause significant sensorimotor, attentional or motivational deficits when injected unilaterally into the SN at the doses used in this study. Together,

these results are in contrast with the results of earlier studies showing that rats bilaterally injected with rotenone in the SN exhibited a significant decrease in the time spent in exploring novel objects when compared to familiar objects [29]. Rotenone-treated rats in the above studies, at variance from FPN-treated rats, also showed a significant decrease in locomotor activity in the open field test [29]. Nonetheless, this study also shows that while a significant increase in the number of approaches to the novel object compared to the familiar object occurred in SN DMSO treated rats, this did not occur in both SN FPN-treated rats. Although the meaning of this finding is unclear, it suggests that FPN induces some kind of impairment not necessarily related to the object recognition memory measured by this recognition test, when given at the dose used in this study. Further experiments are necessary to verify this possibility. No difference was also found between unilateral SN FPN- (both 9

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decrease in dopamine and DOPAC content in the ipsilateral striatum and TH immunoreactivity in the injected SN, respectively, of FPNtreated rats when compared to the control values of rats unilaterally injected with DMSO in the SN as well as to those of the intact contralateral SN and striatum. Interestingly, the decrease in striatal dopamine and TH immunoreactivity found in SN FPN-treated rats are also accompanied by changes in locomotor activity (measured in the motility apparatus), motor coordination (evaluated in the rotarod test), nociception (measured with the tail flick test), but not in the novel object recognition test and in the social interaction test. Surprisingly with the two doses of FPN used in this study, dose dependent effects were seen only in one case (i.e., in the 30 min horizontal locomotor activity). The reason of the absence of dose-dependent effects is unknown. One possibility is that a ceiling effect has been already reached with the doses of FPN used in this study; alternatively, FPN may be metabolized at the SN level to metabolites (i.e., FPN sulfone, FPN desulfinyl, FPN sulfide) [69] that might interfere and even counteract the actions of the parent pesticide. Further studies are required to verify these possibilities. Nevertheless, the neurochemical, immunoistochemical and behavioral changes found in SN FPN-treated rats are in part coincident with those found with other compounds (i.e., 6hydroxy-dopamine, MPTP, MPP+) including pesticides (i.e., rotenone) when injected into the SN, the striatum, medial forebrain bundle or VTA, which induce marked lesions of the nigrostriatal dopaminergic neurons (originating in the SN and projecting to the striatum, but also to the nucleus accumbens and medial prefrontal cortex) when available. This suggests that FPN acts in the SN to induce loss of dopamine neurons by mechanisms that may be only partially similar to those induced by the above compounds (see [8–10]). However, it cannot be ruled out that a main reason for these behavioral differences is that FPN was injected unilaterally into the SN of rats whereas bilateral injections or systemic treatments were often used in the studies reporting the behavioral responses of rotenone-, MPTP- and MPP+-treated rats. Although studies with bilateral injections of FPN are necessary to clarify the above discrepancies, and it is still unknown whether fipronil given systemically is able to cause damage of nigrostriatal dopaminergic neurons in rodents and other mammals, the results of this study suggest that unilaterally SN FPN-treated rats, which show a 40–50 % decrease in striatal dopamine and SN TH immunoreactivity, may be considered a preclinical model of the early stage of Parkinson’s disease, as already suggested not only for rotenone-, MPTP- and MPP+-treated rats, but also for 6-hydroxy-dopamine-treated rats, when given at small doses that induce a 50 % decrease of striatal dopamine [36]. Moreover, although FPN is generally considered safe for humans [70–72], the results of this study confirming the ability of FPN to damage the nigrostriatal dopaminergic neurons, make this compound a possible reason of concern for human health. Accordingly, its increasing use as insecticide may lead to an increased chronic exposure to FPN not only as it occurs in the case of occupational exposure but also in human population in general. This possibility may be confirmed by follow-up studies with a chemical exposure of FPN that better models its human exposure/absorption.

Fig. 12. Percent (%) of TH immunofluorescence in the intact and injected SN of Fipronil (FPN, 15 μg and 25 μg)-treated rats compared to control DMSO-treated rats. FPN and DMSO were injected unilaterally into the SN 16 days before the sacrifice. Values are expressed as means ± SEM (n = 12-15 samples, 3 sections for each of 4-5 rats per group). ****: P < 0.001 FPN-treated rats vs. control DMSO, ####: P < 0.0001 injected SN vs. intact SN (two-way ANOVA followed by Tukey’s multicomparison test).

doses) and control SN DMSO-treated rats in the social interaction test, as indicated by the absence of significant differences in the number of contacts and the time of interaction with an unknown rat between FPNand DMSO-treated rats. This finding is in contrast with those of earlier studies showing that treatment with compounds that decrease dopamine content in the striatum (e.g., rotenone, 6-hydroxy-dopamine) may exert a negative impact in the social interaction test, the forced swimming test, the open field test or the sucrose preference test [64,65]. This discrepancy may be due to differences in the experimental conditions (i.e., different doses, different injection sites and/or administration routes, different tests after pesticide treatment) between this and the above studies. Finally, this study confirms that FPN induces a decrease in striatal dopamine and DOPAC content when injected into the SN [7,9,10]. Accordingly, the pesticide injected unilaterally directly into the SN, at the doses of 15 μg and 25 μg., induced a significant decrease in the content of dopamine (40–50 %) and DOPAC (30–40 %), respectively, in the ipsilateral striatum when compared either to the ipsilateral striatum of SN DMSO-treated rats and to the contralateral striatum as well, when the animals were sacrificed 15 days after FPN microinjection. The decrease in dopamine and DOPAC content occurred concomitantly to an increase in DOPAC/dopamine ratio, thus suggesting that an increased dopamine turnover occurs in the striatum ipsilateral to the injected SN of FPN-treated rats compared to DMSO-treated rats (0.28 in FPN 15and FPN 25 μg.-treated rats vs. 0.23 in DMSO-treated rats). As discussed above, the increased dopamine turnover in the striatum ipsilateral to the FPN-injected SN may in part contribute to explain the failure of apomorphine to induce contralateral turning in unilateral SN FPNtreated rats. The decrease in striatal dopamine and DOPAC content of FPNtreated rats was parallel to a 40–50 % decrease of TH immunoreactivity in the SN treated with FPN, decrease that was also very evident when compared to TH immunoreactivity found in the DMSO-treated SN or in the contralateral intact SN. These neurochemical and immunohistochemical changes were seen with only minor effects on the body weight pattern of FPN-treated rats when compared to DMSOtreated rats during the 15 days of the experiment. These findings resemble those obtained in rats with lesions of the nigrostriatal dopaminergic system induced by other compounds, such as 6-hydroxy-dopamine [30,35,66], MPTP and its oxidation product 1-methyl-4phenylpyridinium (MPP+) (believed to be responsible for MPTP neurotoxicity, at least in rats) [27,28,67] or rotenone [30,67], injected in the SN or in the medial forebrain bundle, and which are often used as preclinical animal models of Parkinson’s Disease [25,30,68]. In conclusion, this study confirms that the pesticide FPN unilaterally injected in the SN at the dose of 15 μg and 25 μg exerts a neurotoxic effect on nigrostriatal dopaminergic neurons, as revealed by the

Author statement RB, JB, and FS performed the behavioral and neurochemical experiments; RB, CL and FS the rotarod and nociception experiments; RB, GC and CC immunohistochemistry experiments, FS, RB and AA analyzed the experimental data; FS, PDD, AA, MRM organized and supervised the experimental design; RB, FS, AA and MRM wrote the manuscript. Declaration of Competing Interest The authors have nothing to declare. 10

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Acknowledgments

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