CX3CR1 Disruption Differentially Influences

Neurotox Res DOI 10.1007/s12640-015-9557-5

ORIGINAL ARTICLE

CX3CR1 Disruption Differentially Influences Dopaminergic Neuron Degeneration in Parkinsonian Mice Depending on the Neurotoxin and Route of Administration Fabrine Sales Massafera Tristaõ1,7,8 • Ma´rcio Lazzarini2 • Sabine Martin2,3 • Majid Amar1 • Walter Stu¨hmer2,3 • Frank Kirchhoff4 • Lucas Arau´jo Caldi Gomes2 • Laurance Lanfumey5 • Rui D. Prediger6 • Julia E. Sepulveda1 • Elaine A. Del-Bel7,8 • Rita Raisman-Vozari1 Received: 26 May 2015 / Revised: 3 August 2015 / Accepted: 18 August 2015 Ó Springer Science+Business Media New York 2015

Abstract Parkinson’s disease (PD) is characterized by progressive degeneration of dopaminergic neurons accompanied by an inflammatory reaction. The neuron-derived chemokine fractalkine (CX3CL1) is an exclusive ligand for the receptor CX3CR1 expressed on microglia. The CX3CL1/ CX3CR1 signaling is important for sustaining microglial activity. Using a recently developed PD model, in which the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxin is delivered intranasally, we hypothesized that CX3CR1 could play a role in neurotoxicity and glial activation. For this, we used CX3CR1 knock-in mice and compared results with those obtained using the classical PD models through intraperitonal MPTP or intrastriatal 6-hydroxydopamine (6-OHDA). The striatum from all genotypes (CX3CR1?/?, CX3CR1?/GFP and CX3CR1-deficient mice) showed a significant dopaminergic depletion after intranasal MPTP inoculation. In contrast to that, we could not see differences in the number of

dopaminergic neurons in the substantia nigra of CX3CR1deficient animals. Similarly, after 6-OHDA infusion, the CX3CR1 deletion decreased the amphetamine-induced turning behavior observed in CX3CR1?/GFP mice. After the 6-OHDA inoculation, a minor dopaminergic neuronal loss was observed in the substantia nigra from CX3CR1-deficient mice. Distinctly, a more extensive neuronal cell loss was observed in the substantia nigra after the intraperitoneal MPTP injection in CX3CR1 disrupted animals, corroborating previous results. Intranasal and intraperitoneal MPTP inoculation induced a similar microgliosis in CX3CR1-deficient mice but a dissimilar change in the astrocyte proliferation in the substantia nigra. Nigral astrocyte proliferation was observed only after intraperitoneal MPTP inoculation. In conclusion, intranasal MPTP and 6-OHDA lesion in CX3CR1-deficient mice yield no nigral dopaminergic neuron loss, linked to the absence of astroglial proliferation.

Electronic Supplementary Material The online version of this article (doi:10.1007/s12640-015-9557-5) contains supplementary material, which is available to authorized users. & Elaine A. Del-Bel [email protected]

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Department of Molecular Physiology, University of Saarland, Homburg, Germany

& Rita Raisman-Vozari [email protected]

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INSERM UMR S894, Universite´ Pierre et Marie Curie, UPMC, Paris, France

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Departamento de Farmacologia, Centro de Cieˆncias Biolo´gicas, Universidade Federal de Santa Catarina, UFSC, Campus Trindade, Floriano´polis, SC, Brazil

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Department of Morphology, Physiology and Basic Pathology (MFPb-Fisiologia), School of Odontology, University of Sao Paulo (USP), Av Cafe´ s/n 14040-220, Ribeiraõ Preto, SP, Brazil

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Center for Interdisciplinary Research on Applied Neurosciences (NAPNA), USP, Ribeiraõ Preto, Brazil

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Sorbonne Universite´ UPMC UM75 INSERM U1127, CNRS UMR 7225, Institut du Cerveau et de la Moelle Epinie`re, The´rapeutique Expe´rimentale de la neurode´geńe´rescence, Hoˆpital de la Salpeˆtrie`re – Baˆtiment ICM, 47 boulevard de l’Hoˆpital, 75651 Paris, France Department of Molecular Biology of Neuronal Signals, Max Planck Institute of Experimental Medicine, Go¨ttingen, Germany Center Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), Go¨ttingen, Germany

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Keywords Parkinson’s disease MPTP 6-OHDA Fractalkine (CX3CL1) Fractalkine receptor (CX3CR1) Neurodegeneration Neuroinflammation

Introduction Parkinson’s disease (PD) represents one of the more common neurodegenerative disorders in aged humans, with prevalence around 0.15–1 % in people over 60 years (Duvoisin 1991; Mayeux 2003). However, due to the phenomenon of population aging, this number is expected to increase dramatically in the coming years. The hallmarks of PD are the progressive degeneration of dopaminergic (DAergic) neurons located in the substantia nigra, the consequent depletion of the DAergic nerve terminals in the striatum (Blandini et al. 2000; Jellinger 1988; Savitt et al. 2006), and the presence of fibrillar aggregates, called Lewy bodies, in the cytoplasm of neurons (Dickson 2001). The causes of PD are unknown, but there is evidence that exposure to environmental agents, including a number of viruses, toxins, agricultural chemicals, dietary nutrients and metals (Cordova et al. 2012; Prediger et al. 2012), or genetic factors (Goedert 2001; Olanow and Tatton 1999) are associated with its development. There is growing indication that neuroinflammatory mechanisms might contribute to the cascade of events leading to neuronal degeneration in PD (McGeer et al. 1988; Hirsch and Hunot 2009; Tieu et al. 2003; Wu et al. 2002). Post-mortem analyses in the brain of Parkinsonians revealed increased number of activated glial cells in the substantia nigra (McGeer et al. 1988) and high concentrations of tumor necrosis factor alpha (TNF-a), interleukin (IL)-1b, IL-2, IL-4, and IL-6 in the striatum (Mogi et al. 1994a, b; Nagatsu et al. 2000). Microglial activation and accumulation of inflammatory mediators in the substantia nigra and striatum have been described after 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA) injections in rodents (Cicchetti et al. 2002; Liberatore et al. 1999; Sriram et al. 2002; Wang et al. 2009, 2010). However, it remains unidentified the mechanisms that trigger microglial activation in DAergic neuronal cell bodies and terminals in PD. Since the initial cloning of fractalkine/CX3CL1, it was proposed as the only known member of chemotactic cytokines that could play significant role in the nervous system, due to its high expression on neurons (Limatola and Ransohoff 2014; Hatori et al. 2002; Asensio and Campbell 1999). Recent studies have pointed the fractalkine signaling, via the activation of its single receptor CX3CR1, as a component of the cascade of microglial regulation (Harrison et al. 1998; Pabon et al. 2011; Wynne et al. 2010). Neurons might communicate with glial cells through this fractalkine/CX3CR1 interaction,

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thereby controlling cell survival (Fuhrmann et al. 2010) or neuron-glia communication. In several neurological and neurodegenerative diseases, the CX3CL1/CX3CR1 pathway has been implicated to mediate inflammatory responses (Cotter et al. 2002; Sato et al. 2006; Sunnemark et al. 2005). However, the effects of modifying CX3CL1/CX3CR1 pathways are extremely context dependent (Limatola and Ransohoff 2014). The disruption of CX3CR1 has been shown to either increase (Meucci et al. 2000; Cardona et al. 2006), decrease (Fuhrmann et al. 2010), or have no participation (Davalos et al. 2005; Jung et al. 2000) over the effects of neurotoxins on the CNS. More specifically, in the neurotoxininduced PD model, previous data demonstrated that the disruption of CX3CL1/CX3CR1 signaling increased the DAergic neuronal loss after the intraperitoneal MPTP inoculation (Cardona et al. 2006). Moreover, the microinjection of the soluble isoform of CX3CL1 in the substantia nigra was sufficient for neuroprotection after exposure to intraperitoneal MPTP in mice (Morganti et al. 2012). Although previous studies (Cardona et al. 2006; Shan et al. 2011; Cook et al. 2001; Jung et al. 2000) have evaluated the involvement of fractalkine or CX3CR1 in the brain, none has directly investigated the role of CX3CL1/ CX3CR1 signaling in the striatum, or even assessed its participation in the loss of DAergic system and neuroinflammation following the administration of different neurotoxins in distinct mice models of PD. The intranasal pathway, a direct route of communication between the environment and the brain, facilitates the entry of environmental neurotoxins (Prediger et al. 2012). Also, several clinical and epidemiological studies find an association between inflammatory factors affecting the intranasal pathway and neurological disorders such as PD (Elbaz and Tranchant 2007; Prediger et al. 2012). Recent studies of our research group (Aguiar et al. 2013, 2014; Prediger et al. 2006, 2010, 2012; Tristao et al. 2014; Kadar et al. 2014) and others (Rojo et al. 2006) have shown that the intranasal MPTP administration achieves direct and prompts CNS delivery, increasing its levels mainly in the striatum. Therefore, analogous to PD patients (Miyachi et al. 2006), intranasally MPTP-inoculated mice may show DAergic degeneration firstly in the striatum than in the substantia nigra. In this study, we analyzed the influence of CX3CR1 disruption in the degeneration of DAergic neurons and glial response in mice models of PD, through the MPTP administration by intranasal and intraperitoneal pathways, and intrastriatal microinjection of the neurotoxin 6-OHDA. We took advantage of knock-in mice that harbor a targeted replacement of the CX3CR1 gene by a green fluorescent protein (GFP)reporter (CX3CR1GFP). Heterozygous CX3CR1?/GFP mice retain the receptor function, in contrast to the CX3CR1GFP/GFP mice, in which the CX3CL1/CX3CR1 pathway is totally inactivated (Davalos et al. 2005; Jung et al. 2000; Nimmerjahn

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et al. 2005). Our results suggest that CX3CR1 disruption revealed a glial cell component related to dopaminergic neuron protection.

Materials and Methods Mice CX3CR1?/?, CX3CR1?/GFP, and CX3CR1GFP/GFP mice were generated at animal facilities of the University of Saarland, Germany, from heterozygous breeding pairs backcrossed for several generations to C57BL/6 strain. The generation of transgenic CX3CR1 knock-in mice that carry a targeted replacement of the CX3CR1 gene by a GFP reporter, was described previously (Jung et al. 2000). Male mice weighting *30 g were kept with free access to water and food in groups of six animals per cage and maintained in a 12 h light cycle room under controlled temperature (22 ± 1 °C). Each mice group received only one treatment (saline or neurotoxin), as showed in Fig. 1. All experiments were conducted according to European, French, and German guidelines for the welfare of experimental animals. Mice breeding and experiments were approved by local national ethic committees (Saarland state’s ‘‘Landesamt fu¨r Gesundheit und Verbraucherschutz’’, animal license number: 71/2010 and 72/2010; and French Ethical Committee, degree n° 2001-464). High-performance Liquid Chromatography (HPLC) of Brain Monoamines and Their Metabolites The CX3CR1?/? and CX3CR1GFP/GFP mice were decapitated, and after brain removal, the striatum and brainstem were dissected, immediately frozen in liquid nitrogen and stored at -80 °C until use. Next, the tissue was weighed and sonicated for 5 s in 10 volumes (v/wt) of 0.1 N perchloric acid/0.05 % disodium EDTA/0.05 % sodium metabisulfite. The levels of dopamine (DA) and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA); serotonin (5-HT) and its metabolite, 5-hydroxy-indole-acetic acid (5-HIAA), were measured by HPLC as previously described (Aguiar et al. 2013; Prediger et al. 2010, 2011). Briefly, the sonicated samples were centrifuged, and 10 lL of the resulting supernatant was injected onto a Beckman Ultrasphere 5 lm IP column (Beckman) (Hamon et al. 1988; Prediger et al. 2010). The monoamine levels were quantified electrochemically at 0.65 V, and concentrations were expressed in nanograms per gram of tissue. MPTP Intoxication MPTP-HCl (Sigma-Aldrich) was dissolved in 0.9 % NaCl (saline) at a concentration of 40 mg/mL for intranasal

Fig. 1 Experimental protocol. The mice were divided into three experimental groups based on neurotoxin and route of intoxication. a CX3CR1?/?, CX3CR1?/GFP, and CX3CR1GFP/GFP mice were intranasally infused with either saline or MPTP (three times a day, 2 h apart, 65 mg/kg total). Mice were perfused, and their brains were collected at 3 and 7 days after intoxication. b CX3CR1?/?, CX3CR1?/GFP, and CX3CR1GFP/GFP mice were intraperitoneally (i.p.) inoculated with saline or MPTP (three times a day, 3.5 h apart, 65 mg/kg). Mice were perfused, and their brains were collected after 7 days. c CX3CR1?/GFP and CX3CR1GFP/GFP mice were microinjected in the striatum with either saline or 6-OHDA (2 lL, 3 lg/lL, deposits at two points). The rotational behavior induced by amphetamine (0.5 mg/kg, subcutaneous) was evaluated at day 26 after the dopaminergic neuron lesion. Mice were perfused after 30 days, and their brains were collected

administration, or 6.7 mg/mL, for intraperitoneal intoxication. Briefly, CX3CR1?/?, CX3CR1?/GFP, and CX3CR1GFP/ GFP mice received 65 mg/kg of MPTP, three times in a single day, every other 2 h (intranasal) or 3.5 h (intraperitoneal). The experimental intranasal and intraperitoneal MPTP-intoxication protocols are shown in Fig. 1a and b, respectively. For intranasal intoxication, a PE-10 micropipette was used, and animals were given a 3-min interval to regain normal respiratory function before this procedure was repeated through the contralateral nostril (Prediger et al. 2010). Animals were held by the neck and were laid upside down to limit liquid flow down the trachea during intranasal intoxication. Control mice were similarly administrated with saline. Six mice per group were used in this study.

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Striatal 6-OHDA Lesion

Colorimetric Immunohistochemistry

CX3CR1?/GFP and CX3CR1GFP/GFP mice were anesthetized via an intraperitoneal ketamine 10 % (0.1 mL/100 g, CPPharma, Burgdorf, Germany) and xylazine 2 % (0.03 mL/ 100 g, Bayer, Leverkusen, Germany) injection and placed in a stereotaxic frame with a mouse adapter (Heidenhain stereotaxic alignment system, INC-USA). Saline was used to protect the eyes from drying during the surgery. Using a 50 lL Hamilton syringe (Hamilton, Bonaduz, Switzerland), 2 lL of 6-OHDA (3 lg/lL, Sigma–Aldrich) solution in 0.02 % ascorbic acid (Sigma–Aldrich) in saline was injected in the right striatum at the following stereotaxic coordinates, in two deposits (Lundblad et al. 2005; Paxinos and Franklin 2001), the medial (anterior: ?1.0; lateral: -2.1; vertical: -2.9) and lateral (anterior: ?0.3; lateral: -2.3; vertical: -2.9) portions of the striatum, from bregma. After injection, the syringe was left in place for an additional 3-min interval. Next, the skin was sutured, and the animals were removed from the stereotaxic frame and placed under a heating lamp for 1 h, as previously described (Lazzarini et al. 2013). The experimental 6-OHDA-inoculation protocol is shown in Fig. 1c.

Free-floating midbrain sections were firstly permeabilized with washing buffer (0.15 % Triton X-100 (Sigma-Aldrich) in 0.1 M PBS). Endogenous peroxidases and non-specific binding sites were respectively blocked for 30 min with 0.3 % H2O2, and after with 5 % normal serum, both diluted in washing buffer. Next, the sections were incubated with primary antibodies (diluted in washing buffer containing 0.02 % thimerosal) for 18 h at 4 °C (anti-tyrosine hydroxylase (TH), 1:500, US Biological; anti-DA transporter (DAT), 1:10000, kindly provided by B. Giros, Douglas Institute, Montreal, Canada; anti-glial fibrillary acidic protein (GFAP), 1:4000, Dako; anti-CD11b, 1:250, AbD Serotec). Bound antibodies were visualized using species-specific biotinylated secondary antibodies (1:250; Vector), standard avidin– biotin-peroxidase techniques (1:125; Vectastain Elite ABC kit or Vector), and 3,3-diaminobenzidine (DAB, SigmaAldrich), resulting in a brown color. After every step, slices were washed three times (5 min each) in washing buffer. All tissues were analyzed by bright-field microscopy.

Rotational Behavior CX3CR1?/GFP and CX3CR1GFP/GFP mice were tested for rotational behavior 26 days after administration of saline or 6-OHDA. The unilateral rotatory test is one of the most frequently used paradigms to investigate the DA loss induced by 6-OHDA microinjection (Blesa et al. 2012). Each mouse received a subcutaneous injection of D-amphetamine (0.5 mg/kg, RBI-Sigma-Aldrich), as previously described (Lazzarini et al. 2013), and the rotations were counted during 10 min.

Free-floating midbrain sections were washed three times (5 min each step) with washing buffer, and the non-specific binding sites were blocked for 30 min with 4 % BSA diluted in washing buffer. After first washing step, the sections were incubated with primary antibodies diluted in washing buffer containing 0.02 % thimerosal for 18 h at 4 °C (mouse antiTH, 1:250). Sections were then stained with donkey antimouse Alexa Fluor 647 (1:1000; Invitrogen) for 2 h. All images were acquired with an automated whole slide scanner (Hamamatsu Nanozoomer, Japan), and quantitative measurements were performed using ImageJ software.

Tissue Preparation for Immunostaining

Image Analysis

At days 3 and 7 following the end of the intranasal MPTP infusion, 7 days after the intraperitoneal MPTP inoculation, and 30 days after 6-OHDA lesion, mice were perfused transcardially with 25 mL of 1 unit/mL heparin in saline, followed by 50 mL of ice-cold 4 % paraformaldehyde in 0.1 M phosphate buffer (NaH2PO4, Na2HPO4; PFA/PB, pH 7.0). Animals were then decapitated and brains quickly removed, postfixed in 4 % PFA/PB for 2 h and cryoprotected for further 24 h at 4 °C in 0.1 M phosphate-buffered saline (NaH2PO4, Na2HPO4, NaCl; PBS) containing 30 % sucrose. Brains were then snap frozen in dry-ice-cooled isopentane at -30 °C and stored at -80 °C. Next, tissues were cut into series of 20-lmthick sections on a freezing microtome (HM450, Thermo Scientific), placed in anti-fungal solution (0.5 % NaN3 in 0.1 M PBS) and stored at 4 °C until use.

After performing the immunostaining reaction, the neuroanatomical sites were identified according to The Mouse Brain in Stereotaxic Coordinates (Paxinos and Franklin 2001). The stained TH and DAT neurons from MPTPintoxicated mice were counted in the substantia nigra using a 209 objective (total magnification, 2009) and the Mercator software (Explora Nova, France), as previously described (Tristao et al. 2014). To estimate the total amount of immunolabeled cells in the whole substantia nigra, a number of TH-positive neurons and GFAP-positive astrocytes were evaluated in a single hemisphere of a systematic subset (e.g., every tenth) of brain sections representative of this whole brain area (Cavalcanti-Kwiatkoski et al. 2010). The total number of neurons was estimated applying the following formula: number of TH-positive

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Immunofluorescence

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neurons x 2 hemispheres x 10 subsets. The optical density (O.D.) of striatal TH, DAT, and GFAP staining, and nigrostriatal CD11b-labeling were analyzed by automatic evaluation using appropriate softwares (MCID, Image Analysis Software Solutions for Life Sciences, Interfocus Imaging Ltd., Linton, UK; ImageJ, National Institutes of Health; http://rsbweb.nih.gov/ij/). For 6-OHDA-inoculated mice, images were captured using a 20x objective on an Axiovert 200 M Zeiss microscope connected to an image analysis system (Axiocam-Axio Vision 4.6.3 SP1). The TH-, CD11b-, and GFAP-positive labeling in the dorsolateral striatum and substantia nigra were captured from rostral to caudal and quantified as previous described (Lazzarini et al. 2013). Briefly, by using Image J software, the image analyzer reads O.D. as gray levels. Background was removed by subtracting values obtained from regions where no staining was observed. These were deep mesencephalic nucleus for substantia nigra, and corpus callosum for striatum for TH and CD11b immunoreactivity, and contralateral side of cortex for GFAP labeling. Statistical Analysis Data are expressed as the mean ± standard error of the means (SEM). Statistical analyses were performed using independent t test or 2-way analysis of variance (ANOVA) followed by the parametric Tukey’s Multiple Comparison Test. All analyses were performed with IBM SPSS Statistic software (version 20.0). The accepted level of significance was p B 0.05.

Results Monoamine Levels in the Striatum and Brainstem from CX3CR11/1 and CX3CR1GFP/GFP Mice We investigated by HPLC measurements whether the disruption of CX3CR1 leads to a change of monoamines in the striatum and brainstem. Our data showed a reduction of the DA concentration (*35 %) in the striatum from CX3CR1GFP/GFP (p B 0.05) when compared to CX3CR1?/? mice. The DOPAC, HVA, 5-HT, and 5-HIAA levels were similar in all analyzed groups (Table 1). In the brainstem, the concentration of every monoamine was not significantly altered by the genetic disruption of CX3CR1 (Table 1). In both striatum and brainstem, the ratios of DOPAC/DA, HVA/DA, and 5-HIAA/5-HT were similar in CX3CR1?/? and CX3CR1GFP/GFP mice. In accordance, previous data indicated no changes in the DA turnover in the whole brain of CX3CR1GFP/GFP mice (Corona et al. 2010).

Effect of CX3CR1 Disruption on MPTP-Induced DAergic Degeneration After intranasal MPTP infusion, the TH immunoreactivity was measured in the striatum and substantia nigra from CX3CR1?/?, CX3CR1?/GFP, and CX3CR1GFP/GFP mice at two different time points (Fig. 1a). The first time point (3 days) was chosen because previous literature described that the DAergic degeneration begins at 12 h post-injection and continues up to 4 days (Jackson-Lewis et al. 1995). At 7 days after MPTP inoculation (second time point), the more extensive MPTPinduced effects in the striatum are observed, with intense TH-immunoreactivity reduction. Our results revealed similar decrease in the striatal density of TH labeling in all CX3CR1?/? (-35 %), CX3CR1?/ GFP (-46 %), and CX3CR1GFP/GFP genotypes (-54 %) at day 3 after MPTP intoxication (Fig. 2a). At day 7, the TH labeling decreased in all groups (CX3CR1?/?, -64 %; CX3CR1?/GFP, -63 %; and CX3CR1GFP/GFP, -51 %), but there was no difference between genotypes (Fig. 2a, c). In the substantia nigra, at day 7 after the intranasal MPTP intoxication, we observed decreased number of THlabeled neurons in both the CX3CR1?/? (-47 %) and CX3CR1?/GFP mice (-38 %) (Fig. 2b, d). In contrast, the substantia nigra from CX3CR1GFP/GFP mice showed no change in the number of TH-positive neurons after intranasal MPTP inoculation either at 3 or 7 days after inoculation (Fig. 2b, d). Moreover, our results indicate that the effect of intranasal MPTP instillation was similar in both CX3CR1?/? and CX3CR1?/GFP mice. In the striatum, the reduction of TH immunoreactivity was more pronounced than in the substantia nigra from CX3CR1GFP/GFP mice. This increased striatal susceptibility after MPTP intraperitoneal inoculation was showed before (Gupta et al. 1986; Song and Haber 2000). Next, we investigated at day 7 after intranasal MPTP inoculation the effect of CX3CR1 disruption on DAT, a specific marker of DAergic neurons (Bannon 2005). When compared to saline-infused matched genotype, the striatal DAT density was similarly reduced in both MPTPinoculated CX3CR1?/? (*81 %) and CX3CR1GFP/GFP (*70 %) mice (p B 0.05, Fig. 3a, c). In the substantia nigra, there was a decrease in the number of DAT-positive neurons from CX3CR1?/? mice (*45 %, p B 0.05), in contrast to no significant reduction in CX3CR1GFP/GFP mice (Fig. 3b, d). Therefore, the TH and DAT immunostaining confirmed that CX3CR1 deletion was associated with reduced or absence of toxic effects over the DAergic neurons in the substantia nigra from intranasally MPTP-inoculated mice. The intraperitoneal MPTP administration induced loss of TH immunoreactivity in the striatum, regardless of the

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Neurotox Res Table 1 Monoamines and metabolite level in the striatum and the brainstem from CX3CR1?/? and CX3CR1GFP/ GFP mice

Striatum CX3CR1 DA

Brainstem ?/?

GFP/GFP

CX3CR1

CX3CR1?/?

CX3CR1GFP/GFP 186 ± 24

7013 ± 604

4549 ± 641*

160 ± 20

484 ± 58

350 ± 33

110 ± 14

94 ± 16

HVA

1676 ± 245

1187 ± 73

289 ± 30

340 ± 48

5-HT

295 ± 13

259 ± 12

530 ± 69

461 ± 42

5-HIAA

254 ± 31

214 ± 23

465 ± 66

448 ± 35

DOPAC

Results are expressed in ng/g of tissue. Values are the mean ± SEM (n = 4–5 per group). Statistical analyses were carried out using independent t test * p B 0.05, versus CX3CR1?/? mice

Fig. 2 TH immunoreactivity in the striatum and substantia nigra after intranasal MPTP intoxication. The CX3CR1?/?, CX3CR1?/GFP, and CX3CR1GFP/GFP mice were intranasally infused with either saline or MPTP (three times a day, 2 h apart, 65 mg/kg total). Their brains were collected at 3 and 7 days after intoxication. a Optical density (O.D.) of TH immunoreactivity in the striatum. b Number of TH-positive neurons in the substantia nigra. c–d Representative photomicrographs of TH

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immunoreactivity in the (c) striatum (Scale bar 0.05 mm) and d substantia nigra (Scale bar 500 lm). Data are expressed as mean ± S.E.M (n = 4–5 mice per group). The statistical analyses were performed using 2-way ANOVA, followed by the Tukey’s Multiple Comparison Test. *p B 0.05, versus saline-inoculated matched genotype. §p B 0.05, versus MPTP-inoculated matched genotype at 3 days. #p B 0.05, versus MPTP-inoculated CX3CR1?/? and CX3CR1?/GFP mice

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Fig. 3 Evaluation of DAT staining in the striatum and substantia nigra from CX3CR1?/? and CX3CR1GFP/GFP mice after the intranasal MPTP administration. Mice were intranasally infused with saline or MPTP (three times a day, 2 h apart, 65 mg/kg), and their brains were collected after 7 days. a Optical density (O.D.) of DAT immunoreactivity in the striatum. b Number of DAT-positive neurons in the substantia nigra. c–

d Representative photomicrographs of DAT immunoreactivity in the (c) striatum (Scale bar 500 lm) and d substantia nigra (Scale bar 300 lm). Data are expressed as mean ± S.E.M (n = 4–5 mice per group). The statistical analyses were performed using 2-way ANOVA, followed by the Tukey’s Multiple Comparison Test. *p B 0.05, versus saline-inoculated matched genotype

mice genotype (p [ 0.05; Fig. 4a, b). However, the substantia nigra from CX3CR1?/GFP mice showed less neurotoxic effect on DAergic neurons (*22 %) compared with CX3CR1GFP/GFP group (*56 %; Fig. 4c, d). Our results are in agreement with Cardona et al. (2006).

The CX3CR1?/GFP and CX3CR1GFP/GFP MPTP-intoxicated mice showed increased striatal GFAPstaining at both evaluated time points (p B 0.05; Fig. 5a, c). In the substantia nigra from CX3CR1?/GFP mice, there was a significant increase in the astrocyte density at 3 and 7 days after intoxication, 50 and 82 %, respectively (p B 0.05, Fig. 5b, d). In contrast, in the substantia nigra from CX3CR1GFP/GFP mice, the number of GFAP-labeled cells did not change significantly following the MPTP administration (Fig. 5b, d). Saline-inoculated CX3CR1?/GFP and CX3CR1 GFP/GFP mice showed no differences in the GFAP labeling (Fig. 5) in the striatum and substantia nigra.

Effect of CX3CR1 Disruption on Nigrostriatal Astrogliosis After MPTP Intoxication At days 3 and 7 after the intranasal MPTP intoxication, we evaluated the astroglial reaction in the striatum and substantia nigra using the specific astrocyte marker, GFAP.

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Fig. 4 Evaluation of neurodegenerative and neuroinflammatory processes in the striatum and substantia nigra from CX3CR1?/GFP and CX3CR1GFP/GFP mice after the intraperitoneal MPTP administration. The CX3CR1?/GFP and CX3CR1GFP/GFP mice were intraperitoneally (i.p.) inoculated with saline or MPTP (three times a day, 3.5 h apart, 65 mg/kg), and their brains were collected after 7 days. a Optical density (O.D.) of TH immunoreactivity in the striatum. b Representative photomicrographs of striatal TH immunoreactivity (Scale bar 500 lm). c Number of TH-positive neurons in the substantia nigra. d Representative photomicrographs of nigral TH immunoreactivity

(Scale bar 50 lm; Inset: Scale bar 500 lm). e O.D. of GFAP immunoreactivity in the striatum. f Number of GFAP-positive cells per 0.03 mm2 was counted in the substantia nigra. g Representative photomicrographs of nigral GFAP immunoreactivity (Magnification, 940). Number of GFP-fluorescent cells in the striatum (h) and substantia nigra (i). Data are expressed as mean ± S.E.M (n = 4–5 mice per group). The statistical analyses were performed using 2-way ANOVA, followed by the Tukey’s Multiple Comparison Test. *p B 0.05, versus saline-inoculated matched genotype. #p B 0.05, versus MPTP-inoculated CX3CR1?/GFP mice

The GFAP analysis, 7 days after the intraperitoneal MPTP delivery, revealed increased immunoreactivity in the striatum (p B 0.05, Fig. 4e) and increased number of GFAP-positive cells in the substantia nigra (Fig. 4f, g), which was similar in both CX3CR1?/GFP and CX3CR1GFP/ GFP genotypes (Fig. 4e–g).

Effect of CX3CR1 Deletion on Nigrostriatal GFPFluorescent Microglial Cells After the MPTP Inoculation

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Because GFP gene reporter identifies microglial cells (Davalos et al. 2005; Jung et al. 2000; Nimmerjahn et al.

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Fig. 5 Evaluation of GFAP immunoreactivity in the striatum and substantia nigra after the intranasal MPTP administration. The CX3CR1?/GFP and CX3CR1GFP/GFP mice were intranasally infused with saline or MPTP (three times a day, 2 h apart, 65 mg/kg). Their brains were collected at days 3 and 7 after intoxication. a Optical density (O.D.) of GFAP immunoreactivity in the striatum. b Number of GFAP-positive cells per 0.03 mm2 of substantia nigra. c–

d Representative photomicrographs of GFAP immunoreactivity in the (c) striatum (Scale bar 500 lm) and d substantia nigra (Scale bar 300 lm). Data are expressed as mean ± S.E.M (n = 4–5 mice per group). The statistical analyses were performed using 2-way ANOVA, followed by the Tukey’s Multiple Comparison Test. *p B 0.05, versus saline-inoculated matched genotype. #p B 0.05, versus MPTP-inoculated CX3CR1?/GFP mice

2005), we evaluated the nigrostriatal GFP fluorescence as an indicator of microglial reactivity in CX3CR1GFP mice. At day 3 after intranasal MPTP administration, there was a significant increase in the GFP fluorescence in the striatum from both CX3CR1?/GFP and CX3CR1GFP/GFP mice (p B 0.05, Fig. 6a, c). However, the CX3CR1-pathway disruption increased the optical density of striatal GFP-labeled cells (two times more) when compared to the CX3CR1?/GFP mice (p B 0.05, Fig. 6a). At day 7 after intranasal MPTP inoculation, we observed that the GFP fluorescence in the striatum decreased to control values in both genotypes (Fig. 6a, c). In the substantia nigra, only the CX3CR1GFP/GFP mice exhibited a remarkable increase in the number of microglial cells at day 3 after intranasal MPTP administration

when compared to saline-inoculated animals (p B 0.05; Fig. 6b). There was a decrease of GFP fluorescence at day 7 after the intranasal MPTP delivery, but it was still 45 % increased in comparison with CX3CR1?/GFP mice (p B 0.05, Fig. 6b). In saline-inoculated CX3CR1?/GFP and CX3CR1 GFP/ GFP mice, we found no differences in the density of GFP fluorescence in both striatum and substantia nigra (Fig. 6). At day 7 after the intraperitoneal MPTP intoxication, we originally showed increased number of GFP-fluorescent cells only in the striatum and substantia nigra from CX3CR1GFP/GFP mice, with no change in the CX3CR1?/ GFP (p B 0.05, Fig. 4h–j). This is in agreement with previous results from Cardona et al. (2006).

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Fig. 6 Evaluation of CX3CR1 GFP-fluorescent cells in the striatum and substantia nigra after the intranasal MPTP administration. The CX3CR1?/GFP and CX3CR1GFP/GFP mice were intranasally infused with saline or MPTP (three times a day, 2 h apart, 65 mg/kg). Their brains were collected at days 3 and 7 after intoxication. a Optical density (O.D.) of CX3CR1GFP fluorescence in the striatum. b Number of CX3CR1GFP-positive cells in the substantia nigra. c Representative photomicrographs of striatal CX3CR1-positive cells (green, GFP

fluorescent) and dopaminergic fibers (red, TH-positive cells). Scale bar 50 lm. Data are expressed as mean ± S.E.M (n = 4–5 mice per group). The statistical analyses were performed using 2-way ANOVA, followed by the Tukey’s Multiple Comparison Test. *p B 0.05, versus saline-inoculated matched genotype. §p B 0.05, versus MPTP-inoculated matched genotype at 3 days. #p B 0.05, versus MPTP-inoculated CX3CR1?/GFP mice at same time point

Effect of CX3CR1 Deletion on DAergic Cell Loss and Neuroinflammation in 6-OHDA-Lesioned Mice

The loss of TH labeling in the 6-OHDA-injured side (ipsilateral) was analyzed in the striatum 30 days after lesion (Table 2 and Online Resource 1). Our data showed that 6-OHDA induced a TH-density loss of 41.87 % (p B 0.05) in the ipsilateral striatum from CX3CR1?/GFP mice. However, no striatal TH immunoreactivity loss (2.09 %, p [ 0.05) was observed in CX3CR1GFP/GFP mice (Table 2 and Online Resource 1). In the substantia nigra from 6-OHDA-lesioned CX3CR1?/ GFP mice, our data showed a reduction of 37.07 % in the TH labeling in comparison with saline-inoculated matched genotype (p B 0.05, Table 2). In contrast, the 6-OHDA did

We investigated the role of CX3CR1 deletion in the 6-OHDA-induced mouse model of Parkinsonism. Amphetamine-induced ipsilateral rotation in unilaterally 6-OHDA-lesioned rodents is a widely accepted behavioral test to identify the DAergic cell loss (Blesa et al. 2012). After amphetamine injection, 6-OHDA-inoculated CX3CR1GFP/ GFP mice showed only *3.5 rotations in comparison to CX3CR1?/GFP mice showing 37 rotations in a 10-min interval (p B 0.05, Table 2).

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Neurotox Res Table 2 Evaluation of neurodegenerative and neuroinflammatory processes induced by 6-OHDA microinjection in the striatum

Amphetamine-induced rotations in 6-OHDA-inoculated mice Striatum

GFAP CD11b

CX3CR1GFP/GFP

37.000 ± 6.000

3.500 ± 3.175#

Substantia nigra

CX3CR1?/GFP TH

CX3CR1?/GFP

CX3CR1GFP/GFP

CX3CR1?/GFP

CX3CR1GFP/GFP

Saline

100 ± 11.817

100 ± 10.323

100 ± 8.419

100 ± 6.892

6-OHDA

58.135 ± 8.575*

97.916 ± 2.361#

62.926 ± 8.626*

93.354 ± 2.934#

Saline

11.254 ± 1.624

11.159 ± 1.176

13.971 ± 2.322

14.405 ± 1.958

6-OHDA

31.206 ± 1.994*

26.122 ± 1.710*

21.865 ± 2.694

20.027 ± 2.697

Saline

12.071 ± 2.444

3.910 ± 1.009

10.206 ± 1.071

10.530 ± 0.658

6-OHDA

8.651 ± 1.109

5.870 ± 0.942

11.705 ± 0.985

11.270 ± 1.029

The CX3CR1?/GFP and CX3CR1GFP/GFP mice were microinjected in the striatum with either saline or 6-OHDA (2 lL, 3 lg/lL, deposits at two points). The rotational behavior induced by amphetamine (0.5 mg/kg, subcutaneous) was evaluated at day 26 after the dopaminergic neuron lesion. The CX3CR1GFP/ GFP mice exhibited a significant decrease in the number of rotations when compared with lesioned CX3CR1?/GFP mice. After 30 days, the brains from saline and 6-OHDA-inoculated mice were collected and immunolabeled with specific antibodies. The optical density of TH, GFAP, and CD11b staining was evaluated in the striatum and in the substantia nigra. Data are expressed as mean ± S.E.M. The statistical analyses were performed using 2-way ANOVA, followed by the Tukey’s Multiple Comparison Test. * p B 0.05, versus saline-inoculated matched genotype. # p B 0.05, versus 6-OHDA-inoculated CX3CR1?/GFP mice

not lead to TH degeneration in the substantia nigra from CX3CR1GFP/GFP mice (6.64 %, p [ 0.05, Table 2). The population of astrocytes and microglial cells were analyzed in the striatum and substantia nigra from CX3CR1?/GFP and CX3CR1GFP/GFP mice through the density of GFAP and CD11b staining, respectively (Table 2 and Online Resource 1). In the striatum, both CX3CR1?/GFP and CX3CR1GFP/ GFP mice had a similar increase of GFAP staining (Table 2 and Online Resource 1). However, there was no significant change on the density of GFAP in the substantia nigra from both genotypes in comparison with the matched control (p [ 0.05; Table 2). Analysis of CD11b-reactivity in the striatum and in the substantia nigra showed no changes in the microglial cell population and absence of genotype-related differences after 6-OHDA striatal microinjection (p [ 0.05; Table 2).

Discussion In this study, we analyzed the influence of CX3CR1 disruption in the degeneration of DAergic neurons induced by two neurotoxins, MPTP and 6-OHDA. Both compounds are well known to lead to destruction of mesencephalic DAergic neurons and fibers. The MPTP was inoculated either via intranasal or intraperitoneal, and the 6-OHDA was microinjected in the striatum. Our findings concerning TH, GFAP, and CD11b immunoreactivity, and GFP fluorescence are summarized in Table 3: (i) after intranasal MPTP infusion, at days 3 and 7,

the absence of CX3CR1 in mice did not influence the striatal DAergic loss, the astrogliosis, or GFP fluorescence. In the substantia nigra, the CX3CR1 disruption did not modulate the TH immunoreactivity at day 3 but induced neuroprotection at day 7. However, in this last region at both 3 and 7 days, the absence of CX3CR1 was associated with no effect in the GFAP population but increased the GFP fluorescence, in contrast to controls, which showed increased GFAP and no effect in the GFP detection; (ii) after intraperitoneal MPTP injection, in the striatum and in the substantia nigra, the lack of CX3CR1 did not influence the DAergic loss or the astrogliosis. In both brain areas, the absence of CX3CR1 increased the GFP population, in contrast to no changes in the GFP detection in the controls; (iii) after intrastriatal 6-OHDA microinjection, the absence of CX3CR1 protected the DAergic cell bodies in the substantia nigra and DAergic fibers in the striatum. However, we could not see modulation of astrocytes and microglial cells in both brain regions from CX3CR1-disrupted mice. Taken together, our data indicate that after neurotoxic stimuli to the DAergic system, the responses of CX3CR1GFP/GFP were different from those of CX3CR1-competent mice. The final effect might be influenced by the type of the neurotoxin used (MPTP or 6-OHDA), by the inoculation pathway (intranasal or intraperitoneal), and by the induced glial cell proliferation. Monoamine Metabolism and the Metabolic Rate of Monoamine Catabolism We investigated whether the absence of CX3CR1 changes monoamine levels in the brainstem and striatum of non-

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Neurotox Res Table 3 Summarized data of nigrostriatal neurodegeneration and neuroinflammation induced by intraperitoneal (i.p.) and intranasal (i.n.) MPTP, and intrastriatal 6-OHDA intoxication in CX3CR1?/GFP and CX3CR1GFP/GFP mice

Striatum

Substantia nigra ?/GFP

GFP/GFP

CX3CR1?/GFP

CX3CR1GFP/GFP

CX3CR1

CX3CR1

Daergic neuron

;

;

[

[

Astrocyte

:

:

:

[

Microglia

:

:

[

:

MPTP i.n./3d

MPTP i.n./7d Daergic neuron

;

;

;

[

Astrocyte

:

:

:

[

Microglia

[

[

[

:

Daergic neuron

;

;

;

;

Astrocyte Microglia

: [

: :

: [

: :

MPTP i.p./7d

6-OHDA Daergic neuron

;

[

;

[

Astrocyte

:

:

[

[

Microglia

[

[

[

[

The differences in the statistical analyses (p B 0.05) for 6-OHDA- or MPTP-inoculated CX3CR1?/GFP and CX3CR1GFP/GFP in comparison with saline-inoculated matched genotype are represented as ; (decrease), : (increase), or [ (no change)

intoxicated mice. Our data showed that the absence of CX3CR1 was associated with a moderate decrease (35 %) in the striatal DA levels. Our results are distinct of Thomas et al. (2008), which could not find DA striatal modification in the CX3CR1-deficient mice. The authors suggest that it may be caused by the decrease of the neurotransmitter or error variation of the measurement that might influence its detection. We found no change in the metabolite:DA ratio (turnover) in the striatum. Whether this result mean differences in the levels of the DA synthesis enzyme (tyrosine hydroxylase) or the metabolic enzymes (monoamine oxidase B or catechol-ortho-methyl-transferase) or both due to CX3CR1 absence, needs further investigation. Neurodegeneration Induced by Intranasal and Intraperitoneal MPTP Inoculation We analyzed TH, a step-limiting protein in DA biosynthesis, and also a DAergic neuronal specific marker. First, the nigrostriatal TH labeling showed no significant differences in heterozygous mutant mice (CX3CR1?/GFP) when compared with age- and gender-matched wild-type mice (CX3CR1?/?). Thus, after intranasal MPTP inoculation, we investigated whether the single (?/GFP) or double (GFP/GFP) CX3CR1 allelic inactivation was associated with a differential TH immunoreactivity. We observed decreased number of TH-positive neurons in the substantia nigra from intranasally MPTP-inoculated CX3CR1?/GFP, but no change in the CX3CR1?/? mice

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(Table 3). Interestingly, in CX3CR1GFP/GFP mice, the MPTP-induced neurotoxicity was only observed after the intraperitoneal route of intoxication the former in accordance with Cardona et al. (2006). Therefore, our study provides the new evidence that the route of MPTP inoculation affects the establishment of MPTP-induced neurotoxicity specifically in CX3CR1GFP/GFP mice. Previous data showed that intranasal-administered MPTP is converted into MPP? with similar kinetics as intraperitoneal administered MPTP (Rojo et al. 2006). However, the occurrence of different outcomes according the inoculation route was previously described (Mani et al. 1996; Weck et al. 1999). The intraperitoneal administration of B-cell-deficient mice with murine gammaherpesvirus 68 (cHV68), but not the intranasal, led to efficient establishment and reactivation from latency in the spleen (Weck et al. 1999). Additionally, the route factor also affected the outcome of neonatal herpes simplex virus infection in guinea pigs; animals inoculated on the scalp had the best outcome with no deaths or evidence of neurologic disease, while the intranasal route produced the most severe disease, with 88 % mortality (Mani et al. 1996). Many compounds administered by intranasal route are often absorbed faster and more efficiently, translating into a quick uptake of substances into the bloodstream and often resulting in a faster onset of action (Kadar et al. 2014; O’Hagan and Illum 1990). In fact, when administered intranasally drugs and/or proteins, as MPTP, they can be transported directly from the nasal epithelium into the

Neurotox Res

brain, bypassing the BBB, and avoiding peripheral effects (Graff and Pollack 2005). Contrary, the intraperitoneal MPTP injections usually cause significant inflammation in peripheral organs (liver, spleen, and kidneys). HernandezRomero et al. (2012) showed that a mild to moderate peripheral inflammation might exacerbate the degeneration of DAergic neurons caused by a harmful stimulus. Besides it is hard to directly compare both MPTP models, Rojo et al. (2006) showed that the intraperitoneal MPTP intoxication requires two- to threefold-reduced dose to induce nigrostriatal damage than the intranasal MPTP model. These findings could explain the susceptibility of DAergic neurons from CX3CR1GFP/GFP after the intraperitoneal but not after intranasal MPTP delivery. Time-Course of DAergic Neurodegeneration As the neurodegenerative and neuroinflammatory processes after the intraperitoneal MPTP intoxication are well established in the literature, we have investigated these events in a single time point after the neurotoxin inoculation, 7 days, in order to compare our data with the results showed by Cardona et al. (2006). In contrast, the utilization of intranasal route for MPTP intoxication is new, and few studies have investigated neuroinflammatory processes after intranasal MPTP delivery. In addition, available knowledge is provided only in late period after intranasal MPTP inoculation (7, 14, 21, and 28 days). Results using this route of inoculation are protocol dependent: when administered for few days, the nasal instillation of MPTP did not induce microglial activation in both striatum and substantia nigra (4 days of treatment, 1 dose/day, 65 mg/(kg dose); Tristao et al. 2014). However, a chronic intranasal MPTP administration (30 days of treatment, 1 dose/day, 60 mg/(kg dose)) increased the microglial activation in both brain regions (Rojo et al. 2006). Considering the lack in the knowledge about the neuroinflammation and neurodegenerative processes at the beginning of this experimental PD model, we evaluated parkinsonian brain at days 3 and 7 after the intranasal MPTP intoxication. Here, the microglial activation was observed only at day 3 after intranasal MPTP administration. According Sugama et al. (2004), the microglial activation, characterized by stronger CD11b immunoreactivity, larger cell bodies, and thicker processes, occurred at 1 day after intraperitoneal MPTP treatment in wild-type mice, using the following protocol: 1 day of treatment, 4 doses/day, 10 mg/(kg dose) of MPTP. This number was increased at day 3, showed intense microglial activation pattern at day 7, and was largely abated by 14 days postintraperitoneal MPTP (Sugama et al. 2004). However, the microglial activation after the intranasal MPTP inoculation seems to be dependent on the dose, experimental protocol, and brain region. In the striatum, the microglial activation may be increased (4 days of treatment, 1 dose/day, 20 mg/(kg dose);

Costa et al. 2013) or present no changes (1 day of treatment, 4 doses/day, 10 mg/(kg dose); Czlonkowska et al. 1996) according the protocol of inoculation. Similarly, in the substantia nigra, the intraperitoneal MPTP may reduce (1 day of treatment, 4 doses/day, 10–25 mg/(kg dose); Czlonkowska et al. 1996; Noelker et al. 2013; Shao et al. 2013; Vijitruth et al. 2006) or promote no alterations in the microglial activation (1 day of treatment, 4 doses/day, 10–20 mg/(kg dose); Cardona et al. 2006; Costa et al. 2013; Czlonkowska et al. 1996; Kinugawa et al. 2013; Noelker et al. 2013; Rojo et al. 2006; Shao et al. 2013). Here, we did not see microglial activation at both striatum and substantia nigra (1 day of treatment, 3 doses/day, * 22.5 mg/(kg dose)). Intranasal and Intraperitoneal MPTP-Induced Neuroinflammation The neuroprotection of cell bodies from DAergic neurons was observed in CX3CR1-deficient mice after receiving intranasal MPTP or intrastriatal microinjection of 6-OHDA. It has been reported that astrocytes and microglia participate in brain inflammation (Carpentier et al. 2005; Kim and Joh 2006). Both types of cells are activated during the process of DAergic loss (Hirsch et al. 1999), suggesting that their activation might be related to PD pathogenesis. Microglia It is indeed still discussed which markers are optimal for staining of microglial cells. For example, Iba1, F4/80, and CD11b labeling have been extensively used for identification of microglial activation. In fact, recent papers have associated an increased expression of CD11b staining with a reactive state of microglial cells (Kettenmann et al. 2011; Ghosh 2010; Rahman et al. 2014; Stott and Barker 2014), suggesting that CD11b is a useful tool for microglial investigation. Besides microglial cells play a hallmark role in brain pathology, it remains controversial whether its activation has detrimental or beneficial functions in neuropathological conditions. The nigral microgliosis in CX3CR1-deficient mice assumed a dual role, being related either to neuroprotection (intranasal MPTP) or neurotoxicity (intraperitoneal MPTP), depending on the route of neurotoxin administration. Previous works have investigated the association between microglial activation and CX3CR1 signaling, but it is still not possible to designate the outcome for this interaction in an experimental model of PD. Shan et al. (2011) injected MPP? into unilateral substantia nigra from rats, which up-regulated the CX3CL1 levels with consequent behavioral defects, DAergic cell death, and glial activation. On the other hand, the augmentation of CX3CR1 signaling may protect against microglial neurotoxicity.

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The intraperitoneal MPTP administration in mice lacking microglial CX3CR1 leads to increased loss of THimmunoreactive neurons (Cardona et al. 2006). Here, we have demonstrated that the degeneration of striatal THpositive fibers was not directly associated with microglial activation (Table 3). However, the disruption of CX3CR1 signaling increased the microglial activation in the substantia nigra from both intranasally and intraperitoneally MPTP-inoculated mice (Table 3). Interestingly, the upregulation of microglial response in CX3CR1GFP/GFP mice was associated with absence or diminished DAergic degeneration after the intranasal or intraperitoneal MPTP administration, respectively. Similarly, the neurotoxic role of microglial cells was inactivated in CX3CR1-deficient mice, preventing the neuron loss in an experimental model of Alzheimer’s disease (Fuhrmann et al. 2010).

When we investigated the inflammatory cells in the 6-OHDA-infused brain, we noticed an increased number of astrocytes in the striatum from both CX3CR1?/GFP and CX3CR1GFP/GFP mice (Table 3). Stott and Barker (2014) evaluated the microglial population using CD11b antibodies, in a 6-OHDA striatal mouse model of PD. They attempted to measure the microglial response, and the results demonstrated only a subtle, non-significant increase in CD11b expression on the lesioned side of the brain. In contrast, there was no increase of either microglial cells (Table 3; Online Resource 1) or GFAP in the substantia nigra. This observation indicates that in 6-OHDA-induced neurotoxicity, the loss of dopaminergic neurons might also be dependent of astrocyte mechanisms.

Conclusion Astrocyte Astrocytes are the major glial components of the CNS, constituting up to 20–50 % of brain volume (Tower and Young 1973). Not only are astrocytes present in all regions of the brain, but they also appear to be organized in strategic positions that are in close contact with neuronal structures, actively playing critical and integral roles in mediating the physiologic and pathologic neuronal conditions (Heneka et al. 2010; Mena and Garcia de Yebenes 2008). When astrocytes undergo a state of gliosis in response to injury or toxic insults, they can release cytokines and chemokines that are deleterious to neurons (Rappold and Tieu 2010). Here, we showed that a significant increase of GFAP cells was observed after the intraperitoneal administration in the substantia nigra from CX3CR1-deficient mice. In contrast, such proliferation was absent in the same structure in CX3CR1GFP/GFP animal receiving intranasal inoculation. It is tempting to speculate that in the CX3CR1-deficient mice, the opposite effect with respect to dopaminergic survival in the substantia nigra between intraperitoneal versus intranasal route, 7 days post-MPTP could be attributed to the differential proliferation of astrocytes. Our results suggests that the loss of dopaminergic neurons might be dependent, at least in part, by an astroglial mechanism, as previously shown by Mourlevat et al. (2003) and Michel et al. (1999). 6-OHDA-Induced Neurodegeneration and Neuroinflammation Using the 6-OHDA model, we have corroborated our data with intranasal MPTP intoxication. The hemiparkinsonian mice model also showed DAergic neuroprotection in the ipsilateral hemisphere of CX3CR1GFP/GFP mice (Table 3), which culminated in reduced number of rotations in the amphetamine test.

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In summary, the findings presented here provide evidence that the absence of CX3CL1/CX3CR1 axis prevents the TH loss in the substantia nigra from both intranasal MPTP- and 6-OHDA-intoxicated mice, in contrast to be deleterious after intraperitoneal MPTP infusion. In both protective models, there was the absence of astrocytes proliferation in the substantia nigra, highlighting that astrocytes may play a crucial role in the degeneration of the DAergic neurons. Future research should follow up whether the cytokines and chemokines released by the astrocytes increase the neurodegenerative processes. Acknowledgments The authors acknowledge Sabine Stolpe and Tanja Nilsson for their technical support. The authors gratefully thank the financial support and grants provided by Coordenaçaõ de Aperfeiçoamento de Pessoal de Nı´vel Superior (CAPES, Brazil), CAPESCOFECUB (France/Brazil; 681/2010), Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq, Brazil), Programa Cieˆncia sem Fronteiras (CsF, Brazil), Fundaçaõ de Amparo a` Pesquisa do Estado de Saõ Paulo (FAPESP, Brazil), German Academic Exchange Service DAAD-ProBral/CAPES (FK/EADB) and the Max Planck Society (WS). SM was funded through the Cluster of Excellence and DFG Research Center Nanoscale Microscopy and Molecular Physiology of the Brain. Compliance with Ethical Standards Conflict of interest The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors have no financial or personal conflicts of interest related to this study.

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