Changes in Day/Night Activity in the 6-OHDA-Induced Experimental Model of Parkinson’s Disease: Exploring Prodromal Biomarkers

fnins-14-590029 October 8, 2020 Time: 18:47 # 1 ORIGINAL RESEARCH published: 14 October 2020 doi: 10.3389/fnins.2020.590029 Edited by: Martin Witt, Centre of Transdisciplinary Neuroscience Rostock, Germany Reviewed by: Andreas Wree, University of Rostock, Germany Veronica Alexandra Antipova, Medical University of Graz, Austria Alexander Hawlitschka, University Hospital Rostock, Germany *Correspondence: Catalina Requejo [email protected] Specialty section: This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neuroscience Received: 31 July 2020 Accepted: 24 September 2020 Published: 14 October 2020 Citation: Requejo C, López-de-Ipiña K, Ruiz-Ortega JÁ, Fernández E, Calvo PM, Morera-Herreras T, Miguelez C, Cardona-Grifoll L, Cepeda H, Ugedo L and Lafuente JV (2020) Changes in Day/Night Activity in the 6-OHDA-Induced Experimental Model of Parkinson’s Disease: Exploring Prodromal Biomarkers. Front. Neurosci. 14:590029. doi: 10.3389/fnins.2020.590029 Changes in Day/Night Activity in the 6-OHDA-Induced Experimental Model of Parkinson’s Disease: Exploring Prodromal Biomarkers Catalina Requejo1,2*, Karmele López-de-Ipiña3,4, José Ángel Ruiz-Ortega5,6, Elsa Fernández3, Pilar M. Calvo3, Teresa Morera-Herreras5,6, Cristina Miguelez5,6, Laura Cardona-Grifoll1, Hodei Cepeda1, Luisa Ugedo5,6 and José Vicente Lafuente1 1LaNCE, Department of Neuroscience, University of the Basque Country (UPV/EHU), Leioa, Spain, 2Department of Neurology, Icahn School of Medicine at Mount Sinai, The Friedman Brain Institute, New York, NY, United States, 3EleKin Research Group, Department of Systems Engineering and Automation, University of the Basque Country (UPV/EHU), Donostia, Spain, 4Department of Psychiatry, University of Cambridge, Cambridge, United Kingdom, 5Department of Pharmacology, University of the Basque Country (UPV/EHU), Leioa, Spain, 6Autonomic and Movement Disorders Unit, Neurodegenerative diseases, Biocruces Health Research Institute, Barakaldo, Spain The search for experimental models mimicking an early stage of Parkinson’s disease (PD) before motor manifestations is fundamental in order to explore early signs and get a better prognosis. Interestingly, our previous studies have indicated that 6-hydroxydopamine (6-OHDA) is a suitable model to induce an early degeneration of the nigrostriatal system without any gross motor impairment. Considering our previous findings, we aim to implement a novel system to monitor rats after intrastriatal injection of 6-OHDA to detect and analyze physiological changes underlying prodromal PD. Twenty male Sprague-Dawley rats were unilaterally injected with 6-OHDA (n= 10) or saline solution (n= 10) into the right striatum and placed in enriched environment cages where the activity was monitored. After 2 weeks, the amphetamine test was performed before the sacrifice. Immunohistochemistry was developed for the morphological evaluation and western blot analysis to assess molecular changes. Home-cage monitoring revealed behavioral changes in response to 6-OHDA administration including significant hyperactivity and hypoactivity during the light and dark phase, respectively, turning out in a change of the circadian timing. A preclinical stage of PD was functionally confirmed with the amphetamine test. Moreover, the loss of tyrosine hydroxylase expression was significantly correlated with the motor results, and 6-OHDA induced early proapoptotic events. Our findings provide evidence for a novel prodromal 6-OHDA model following a customized monitoring system that could give insights to detect non-motor deficits and molecular targets to test neuroprotective/neurorestorative agents. Keywords: 6-hydroxydopamine, circadian rhythms, home-cage monitoring system, prodromal biomarkers, prodromal Parkinson’s disease symptoms, behavior, non-motor deficits, rat Abbreviations: ADN, Axodendritic network density; AKT, Protein kinase B; AUC, Area Under the Curve; CL, Contralateral; EE, Enriched environment; ERK, Extracellular signal-regulated kinase; GFAP, Glial fibrillary acidic protein; IL, Ipsilateral; NMS, Non-motor symptoms; 6-OHDA, 6-hydroxydopamine; PD, Parkinson’s disease; REM, Rapid eye movement; NREM, Non-rapid eye movement; RBD, Rapid eye movement sleep behavior disorder; ROC, Receiver Operating Characteristics; ROS, Reactive oxygen species; SN, substantia nigra; SNr, Substantia nigra reticulate; TH, Tyrosine hydroxylase; TH-ir, Tyrosine hydroxylase-immunoreactivity; Tpm, Turns per minute; VTA, Ventral tegmental area. Frontiers in Neuroscience | www.frontiersin.org 1October 2020 | Volume 14 | Article 590029 fnins-14-590029 October 8, 2020 Time: 18:47 # 2 Requejo et al. 6-OHDA Induced Prodromal PD Symptoms INTRODUCTION Parkinson’s disease (PD) is a complex neurologic disorder in which not only motor impairments occur, but also other non-motor symptoms (NMS) play a relevant role, including hyposmia, sleep disorders, depression, constipation and cognitive deficit (Schapira and Tolosa, 2010;Mantri et al., 2018;Weintraub et al., 2018). Most of them often appear earlier than the motor symptomatology, during the so-called prodromal stage and worsen following the disease’s progression (Schapira et al., 2017). Indeed, NMS may manifest several years prior to the onset of motor symptoms, even up to 20 years before the diagnosis, and the prevalence can vary between the patients (Schapira et al., 2017;Rees et al., 2019). In this context, although there are not specific NMS for PD, the presence and combination of various NMS as well as the correlation with an early dopamine depletion may be useful for early diagnosis (Berg et al., 2013;Schapira et al., 2017). Remarkably, slight motor symptoms (<40–60% of dopaminergic neuronal cell loss) have been also associated to the prodromal stage when they do not meet the criteria for the clinical diagnosis of PD (Mahlknecht et al., 2015). Thus, it is essential to establish a correlation between the early manifestations of PD (prodromal stage) with the clinical and pathological stage for an early diagnosis of PD (Mahlknecht et al., 2015;Liepelt-Scarfone et al., 2017;Sherbaf et al., 2018). Thus, studies about early events of the disease are emerging in order to find some biomarker for an early-premotor diagnosis (Miller and O’Callaghan, 2015). However, the neuroanatomical and etiological background of NMS in PD remain to be elucidated (Schapira et al., 2017). One of the first NMS that is suffered by a high percentage of the patients is the sleep disorder, such as sleep fragmentation, excessive daytime sleepiness or “REM behavior disorder” (RBD), which is a pathology based on increasing muscle tone during the REM (rapid eye movement) sleep (De Lazzari et al., 2018;De Castro Medeiros et al., 2019). Among the mechanisms underlying sleep disturbance, the disruption in the circadian system is attributed as triggering factor (Willison et al., 2013). Indeed, growing evidences support an association between circadian disruption and PD, suggesting that the dopamine depletion may lead to circadian rhythm irregularities including the alteration of the circadian control of the rest/activity rhythms (Tanaka et al., 2012;De Lazzari et al., 2018). In the search for preclinical models of PD, there is a main challenge for reproducing those functional and pathological changes that could provide the scaffold to find out about the target for the design of neuroprotective therapies before the progression of the disease (Duty and Jenner, 2011;Ko and Bezard, 2017;Lafuente et al., 2017;Iarkov et al., 2020). In this context, the well-known model of the 6-hydroxydopamine (6-OHDA) which is widely used to study motor deficits, can also be useful for identifying non-motor and early motor impairments specially when combined with advanced technological devices (Grandi et al., 2018). In this model, the site of administration and time elapsed after the injection are critical for determining the extent and time course of the lesion (Kirik et al., 2000; Deumens et al., 2002). In our hands, this model is also useful for studying the preclinical phase of PD, as intrastriatal injection of 6-OHDA in adult male rats induced an early degeneration of the nigrostriatal system without any gross motor impairment as well as upregulation of caspase-3 and downregulation of survival signaling pathways (Requejo et al., 2018a,b). Therefore, in the present study we aimed to detect prodromal changes in the behavior following an experimental model of PD based on the intrastriatal injection of 6-OHDA with a short time of evolution (2 weeks) in adult male rats housed in monitored enriched environment (EE) cages. MATERIALS AND METHODS Experimental Design and Housing Conditions Twenty adult (3-month-old) male Sprague-Dawley rats, weighting 280–300 g at the time of surgery, were obtained from Harlan Laboratories, S, A, (Barcelona, Spain). Rats were randomly assigned to the following groups: Sham group (saline-lesioned rats, n= 10) as control and 6-OHDA group (6-OHDA-lesioned rats, n= 10). After stereotaxic injection of 6-OHDA or saline solution, the animals were housed for 2 weeks in monitored enriched environment cages (10 animals per cage) consisting of large cage (790 mm ×460 mm ×640 mm) with two floors, which were connected by a plastic ramp and an external running wheel (Figure 1A and Supplementary Figure S1). A 12 h light/12 h dark cycle was established with access to food and water ad libitum. The EE cages were developed by our research group and they were designed to host up to 12 rats as well as housing food feeders, water feeding bottles, two semi-closed rooms, a wide-open area, and a ramp to access a runway situated on the upper floor. In addition to this, these EE cages also provide four gates that may be opened to connect to other cages, runways, or exercise wheels. Current EE cages were supplied with an automatic recording system. This novel system consists, basically, of an infrared camera situated above every cage and connected to a raspberry device programmed to collect pictures accurately every 30 s (Figure 1A). In fact, a Raspberry Pi (Raspberry Pi 2 Model B, Raspberry Pi products, United Kingdom) is a standalone embedded computer that measures the size of a credit card. It has enough power to run a linux operating system and connect it to several external devices and sensors. In this case, it was connected to Pi NoIR Camera V2 (Raspberry Pi products, United Kingdom) that provided a clear image of the cage even at night’s dark due to the infrared lighting that was set up throughout the room (Supplementary Figure S1). This in-house customized automatic system continuously recorded a picture every 30 s and saved it in an SD Card. At the end of the experiment, more than 60 gigabytes in pictures were collected and processed in order to measure changes in the activity (Figure 1B). Two weeks after the intrastriatal injection the amphetamine test was performed for evaluating motor deficits and with the purpose of confirming that no rat presented more than 5 turns per minute (tpm) in order to get a preclinical model (Supplementary Table S1). This criterium was established due to the fact that it has previously been shown that Frontiers in Neuroscience | www.frontiersin.org 2October 2020 | Volume 14 | Article 590029 fnins-14-590029 October 8, 2020 Time: 18:47 # 3 Requejo et al. 6-OHDA Induced Prodromal PD Symptoms FIGURE 1 | Experimental design and monitoring analysis. (A) Schematic representation illustrating the timeline of the experiments. Rats received 6-OHDA (6-OHDA group) or saline solution (Sham group) injections into the right striatum, and immediately after the lesion, they were placed in monitored EE cages for 2 weeks. These EE cages were supplied with an automatic recording system that collect pictures of the EE cages every 30 s under a regular 12 h:12 h light/dark cycle in order to detect activity changes during the light and dark phases. Infrared lighting was also set up to take clear pictures during the dark phase. Two weeks later, the amphetamine test was performed to assess motor symptoms, and then, rats were transcardially perfused or decapitated for the histological and biochemical analysis, respectively. (B) Data acquisition, pre-processing and analysis workflow. The process was divided in two phases. (1) Image pre-processing (in green): frame difference, gray level conversion, noise smoothing and binarization). (2) Image analysis (in orange): clustering analysis and centroid calculation, activity analysis. 6-OHDA, 6-hydroxydopamine; EE, enriched environment. rats rotating more than 5 tpm presented a lesion ranging from 50 to 90% which may predict motor impairment (Björklund and Dunnett, 2019). After the amphetamine test, rats were sacrificed by transcardial perfusion for the histological evaluation or by decapitation for the biochemical analysis (Figure 1A). All the experimental protocols were reviewed and approved by the Ethical Committee and Animal Welfare of the University of the Basque Country (UPV/EHU, CEBA M20/2015/024, approval date 12/28/2015), and in accordance with the European Community Council Directive on “The Protection of Animals Used for Scientific Purposes” (2010/63/EU) and Frontiers in Neuroscience | www.frontiersin.org 3October 2020 | Volume 14 | Article 590029 fnins-14-590029 October 8, 2020 Time: 18:47 # 4 Requejo et al. 6-OHDA Induced Prodromal PD Symptoms with Spanish Law (RD 53/2013) for the care and use of laboratory animals. 6-OHDA Lesions Rats were lesioned as we previously described (Requejo et al., 2018a,b). Briefly, thirty minutes before surgery, rats were pre-treated with desipramine (25 mg/kg, i.p., Sigma, St. Louis, United States) and pargyline (50 mg/kg, i.p., Sigma, St. Louis, United States) in order to preserve the noradrenergic system and the degradation of the toxin, respectively. Then, rats were deeply anaesthetized with isoflurane inhalation (1.5–2%; Esteve Química, Barcelona, Spain) and placed in the stereotaxic frame (David KopfR  Instruments). 6-OHDA (3 µg/µl, in 0.02% ascorbic acid) or saline solution (0.9% NaCl) was infused using a 10 µlHamilton syringe fitted with a 26-gauge needle at a rate of 0.5 µl/min by a single syringe infusion pump (KDS Scientific, MA, United States). To generate a mild lesion of the nigrostriatal pathway, three injections of 2.5 µl of the 6-OHDA solution (a total volume of 7.5 µl) were administered at three coordinates into the right striatum, relative to the bregma and dura, with the toothbar set at −2.4: anteroposterior (AP) +1.3 mm, mediolateral (ML) +2.8 mm, dorsoventral (DV) −4.5 mm; AP −0.2 mm, ML +3.0 mm, DV −5.0 mm and AP −0.6 mm, ML +4.0 mm, DV −5.5 mm, according to Paxinos and Watson atlas (Paxinos and Watson, 2013). The needle was left in place for an additional 5 min to allow the toxin to diffuse into the structure, and then it was slowly retracted. 6-OHDA was prepared daily for each surgery session and changed every 2–3 h. The 6-OHDA solution was kept on ice and protected from light during the surgery to avoid oxidation, which would be indicated by a color change (clear to brown-pink color). Control rats received the same volume of saline solution in the same manner. In addition, a short time of evolution (2 weeks) was elapsed before sacrifice. Monitoring Analysis The in-house customized automatic system based on the image processing with the infrared Camera Module v2 (Pi NoIR; Raspberry Pi Products, United Kingdom) was used for the analysis of the rat group behavior. A schematic diagram of the working procedure for image processing is described in Figure 1B. This process was divided in two phases: image pre-processing and image analysis. 1. In the first phase video sequences were acquired by a highquality camera and pre-processed by an own toolbox in MATLAB to create series of binary image frames of the rat group activity. The Toolbox includes: (1) function for image processing, (2) Activity analysis (centroid analysis), and (3) Period analysis among others. Thus, the activity areas (motion detection) were calculated based on a frame difference method (Manchanda and Sharma, 2016;Sengar and Mukhopadhyay, 2016). Frame absolute difference was calculated between two consecutive frames with a sampling period of 30 s. Images were converted from Red Green Blue (RGB) which is a standard format for color image, into gray scale, after that in binary images and then by a threshold filter extracted the main activity area of the system (rats, and objects moved by the rats). Finally, activity areas were defined applying morphological filters to reduce the noise and for smoothing the images. In particular, morphological filters were applied using MATLAB function bwmorph: Bwmorph (Ir,’open’);% Remove peaks Bwmorph (Ir,’close’);% Remove hole 2. During the second phase the trajectories of the animal group is generated by the system centroid (the average position of all the activity areas in the binary image) evolution. The centroid is estimated by k-means algorithm over the binarized frame series (Eguiraun et al., 2018; López-de-Ipiña et al., 2019). Thus, within each frame, the coordinates of the centers of every object were calculated, and K-means were applied to find the center of the entire group, that is the “centroid.” A centroid with two coordinates: C=(xc,yc) 3. The group trajectory consists of the evolution of that centroid from the first frame to the last one. Finally, the rat groups’ behavior was described and modeled by the following parameters: 1. Number of activity areas in the binary images. 2. Shannon entropy of the centroid trajectory. Shannon entropy is a main concept in information theory and is a measure of average uncertainty (information content). Entropy in biosignals gives information about the system evolution and behavior and can be applied to analyze pathological behaviors (Eguiraun et al., 2018;López-deIpiña et al., 2019). Velocity, speed and acceleration defined as: vcx =1xc/1t vcy =1yc/1t(1) being speed the absolute value of velocity. An acceleration: acx =1vcx/1t acy =1vcy/1t(2) Amphetamine-Induced Rotation Test Two weeks after intrastriatal injection the amphetamine test was developed following the previously described methodology for this behavioral test (Miguelez et al., 2011). Briefly, D-amphetamine (5 mg/kg in 0.9% NaCl; Sigma-Aldrich, St. Louis, United States) was intraperitoneally administered and the animals were placed in an individual circular cage (rotameter). In the present study, we used a higher dose of D-amphetamine in order to get rotational behavior due to the lesion was minor (Björklund and Dunnett, 2019). After 15 min of latency, the total number of full ipsilateral (IL) rotations was recorded during 90 min (Multicounter LE3806; Harvad Frontiers in Neuroscience | www.frontiersin.org 4October 2020 | Volume 14 | Article 590029 fnins-14-590029 October 8, 2020 Time: 18:47 # 5 Requejo et al. 6-OHDA Induced Prodromal PD Symptoms Apparatus, Holliston, MA, United States) in order to include the entire response period and avoid differences in variability due to the differences in the pharmacokinetics or in the dopamine release kinetics between rats (Miguelez et al., 2011;Björklund and Dunnett, 2019). Data were expressed as the number of turns per minute. Morphological Analysis Morphological evaluation was performed as we previously described (Requejo et al., 2015, 2018a,b). Tissue Processing for Histological Evaluation After behavioral test, five rats from each group were intraperitoneally anesthetized with chloral hydrate at 20% (Ref: 141,975, Panreac Quimica SA, Barcelona, Spain) and transcardially perfused with 0.9% sodium chloride followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate-buffered saline (PBS) pH 7.4. Brains were removed, post-fixed overnight in the same fixative solution and next transferred into a cryoprotective solution containing 30% sucrose and 0.1M PBS pH 7.4.50 µm serial coronal sections containing striatum and substantia nigra (SN) were obtained with a freezing microtome and collected following the Paxinos and Watson atlas (Paxinos and Watson, 2013) in 0.6% sodium azide in 0.1 M PBS pH 7.4 for storage and further analysis. TH Immunostaining Tyrosine hydroxylase (TH) immunohistochemical staining was developed on free-floating coronal slices. Briefly, sections were treated with 3% H202and 10% methanol in potassium phosphate buffered saline (KPBS), preincubated with 5% normal goat serum (NGS) and 1% Triton X-100 in KPBS (KPBS-T) for 1 h at room temperature (RT), and later incubated overnight at 4◦C with rabbit polyclonal anti-TH (Ref: AB-152, Millipore; 1:1,000) in KPBS/T containing 5% NGS, followed by incubation with a secondary biotinylated goat anti-rabbit IgG antibody (Ref: BA1000, Vector Laboratories, Burlingame, CA; 1:200) in 2.5% NGS KPBS/T for 2 h. Afterward, all sections were processed with the avidin-biotin-peroxidase complex for 1 h using a commercial kit (Ref. PK-6102, Elite ABC kit, Vector Laboratories, Burlingame, CA) and the reaction was shown by using 3,3diaminobenzidine (DAB). Images were visualized, captured and analyzed at 4x and 40x magnification by an Olympus BX-50 photomicroscope. Double Immunofluorescence Staining of Caspase-3 and NeuN Coronal sections were incubated with rabbit anti-caspase 3 (H-277) (Ref: sc-7148, Santa Cruz Biotechnology Inc., Spain; 1:50) and monoclonal mouse anti-NeuN (Ref: MAB377, Chemicon International, Inc., Spain; 1:100) diluted in 5% bovine serum albumin (BSA) and 0.1% Triton X-100 in PBS overnight at 4◦C. Sections were subsequently incubated with secondary antibodies conjugated to Alexa Fluor-488 (Ref: A11029; Invitrogen; 1:400) and Alexa Fluor-568 (Ref: A11036 Invitrogen; 1:400) for 1 h at room temperature in darkness. Immunostained sections were further reincubated with Hoechst for nuclear counterstaining for 10 min, then slices were washed, mounted on glass slides and coverslipped using Vectashield mounting medium for fluorescence (Ref: x-0517; Vector laboratories). Images were finally analyzed at 20x magnification using Olympus Fluoview FV500 confocal microscope. Stereological Analysis The unbiased stereological analysis was performed as previously described (Requejo et al., 2015, 2017, 2018a,b) by using a computerized image analysis system (Mercator Image Analysis system, Explora Nova, La Rochelle, France) connected to an Olympus BX-50 photomicroscope. For this purpose, a total of 7–8 sections in a 1: 8 series were analyzed to cover the entire striatum and SN for each animal. In brief, in order to evaluate the TH-immunoreactivity (ir) in the IL striatum, the volume of the preserved striatum was calculated using the Cavalieri method (Howard and Reed, 2004) available on Mercator image analysis system by delimiting the negative areas and the entire striatum at 4x magnification, and multiplying these measurements by the thickness of the slices and the intersectional distance (Requejo et al., 2015, 2018a,b). Values were expressed as the percentage of TH-ir volume of the IL striatum respect to the total volume of the IL one. Changes in the density of dopaminergic neurons and axodendritic network (ADN) were evaluated in both hemispheres through quantifying the TH-ir neuronal density in the entire SN and the TH-ir ADN density in the SN reticulata (SNr) using a stereological tool (an optical fractionator) provided by the Mercator image analysis system (West et al., 1991). Once the region of interest was outlined at 4x magnification, probes of 50 ×50 µm separated by 100 µm were launched into the SN. THir neurons and ADN inside the probe, or crossing the right side of the X–Y axis, were counted at 40x magnification in the entire SN and SNr, respectively. Data were expressed as the percentage of TH-ir neurons or ADN presents on the IL side (lesioned side) vs. the contralateral (CL) hemisphere (non-lesioned hemisphere). The study of the topological distribution was performed following the previously described approach (Requejo et al., 2015, 2017). For each animal, sections containing the striatum and the SN were evaluated at three representative rostro-caudal levels, respectively, according to Paxinos and Watson’s Atlas (Paxinos and Watson, 2013): rostral (bregma +0.70 mm), middle (bregma, −0.26 mm), and caudal (bregma, −0.80 mm) striatal sections were considered to determine the TH-ir volume; and rostral (bregma, −5.20 mm), middle (bregma, −5.60), and caudal (bregma, −6.04) nigral sections were examined to estimate the TH-ir neuronal and ADN density. Biochemical Analysis Western Blotting After the behavioral test, 5 rats of each group were anesthetized with chloral hydrate at 20% (Ref: 141,975, Panreac Quimica SA, Barcelona, Spain) and decapitated with a rodent guillotine to obtain fresh brain tissue. IL and CL striatum and SN were collected by microdissection and quickly frozen (Chiu et al., 2007). For this purpose, once brains were removed, Frontiers in Neuroscience | www.frontiersin.org 5October 2020 | Volume 14 | Article 590029 fnins-14-590029 October 8, 2020 Time: 18:47 # 6 Requejo et al. 6-OHDA Induced Prodromal PD Symptoms coronal slices containing the striatum and SN were cut out and subjected to biopsy with a small biopsy needle. Firstly, the cerebellum was separated, and the brain was cut bi-half into the right and left hemisphere, next cut was for removing the olfactory bulb at the level of the optic chiasm, and the fourth cut was at the level of the pituitary stalk, separating the two coronal slices, one of them containing the striatum and the other one the SN. The frontal cortex from the slice containing striatum was removed in the fifth cut, obtaining the striatum. The SN was collected in the sixth, separating the cortex from the slice. Samples were stored at −80◦C until the analysis. Brain slices were manually homogenized (1:20 w/v) in lysis buffer [10 mM phosphate-buffered (PB) (pH 7.4), 5 mM ethylenediaminetetraacetic acid (EDTA), 5 mM ethyleneglycolbis(2-aminoethylether)-tetraacetic acid (EGTA), 1 mM dithiotreitol (DTT)] containing a protease inhibitor cocktail (Ref: P-8340, Sigma-Aldrich, Spain). Samples were centrifuged (13,000 rpm at 4◦C for 15 min) and soluble proteins were recovered in the supernatants and quantified using the Bio-Rad Protein Assay (Ref: 500-0006, Bio-Rad Laboratories SA, Spain) based on Bradford’s method (Bradford, 1976). As previously described (Requejo et al., 2018a,b), for each sample 20 µg of proteins was loaded into polyacrylamide CRITERION TGX 12% gels (Bio-Rad Laboratories Inc., Spain) for electrophoresis and then transferred to a PVDF membrane in a Trans-Blot Turbo Transfer System (Bio-Rad, United States) for 7 min. Membranes were incubated with the following primary antibodies: rabbit anti-Phospho-protein kinase B (AKT) (Ser 473) (1:1,000), rabbit anti-AKT (1:1,000), rabbit anti-Phosphop44/42 MAPK (extracellular signal-regulated protein kinases 1 and 2 (ERK 1/2)] (Thr202/204) (1:1,000), rabbit anti-P44/42 MAPK (ERK 1/2) (1:1,000) (all of them from Cell Signaling Technology Inc., United States), rabbit anti-caspase 3 (H-277) (1:1,000) (Santa Cruz Biotechnology Inc., Spain), rabbit antiβ-Actin (1:2,000) (Sigma-Aldrich, Spain) and rabbit anti-BetaTubulin (1:1,000) (Novus Biologicals, United States) at 4◦C overnight. Afterward they were incubated with anti-rabbit IgG peroxidase conjugated secondary antibodies (1:2,000) (SigmaAldrich, Spain) for 2 h at RT and immunoblots were developed with an enhanced chemiluminescence kit (GE Healthcare Life Science, United Kingdom). The luminescence of the reaction product was detected in a personal scanner, LI-COR C-DiGit (LICOR, Bonsai Advanced Technologies SL, Spain), and visualized bands were analyzed with Image Studio Lite 4.0 software (LICOR, Bonsai Advanced Technologies SL, Spain). β-Actin and β-tubulin were used as loading controls. Statistical Analysis All results were expressed as the mean ±SEM (standard error mean). Statistical analysis was developed with GraphPad Prism (v 5; GraphPad Software, Inc., United States) and SPSS Statistics (v 20; IBM Corporation, Armonk, NY, United States). Prior to the analysis, the Shapiro–Wilk test was used to assess the normal distribution of the samples, and Levene’s test was used to determine the homogeneity of variance. Mann-Whitney U-test was performed to assess differences between groups and within groups in the monitoring analysis. The ROC (Receiver Operating Characteristics) curve was performed and the AUC (Area Under the Curve) was calculated in order to measure the accuracy of the model which are a metrics for checking classification models (Hanley and McNeil, 1982;Fawcett, 2006), in this case: Sham group and 6-OHDA group. The behavioral data, stereology and densitometry results were analyzed by means of the two-tailed unpaired Student’s t-test to compare differences between groups. A one-way analysis of variance (ANOVA) followed by Tukey’s multiple-comparisons test was used to test the differences between rostro-caudal gradients within each experimental group. The correlation was examined by Pearson product-moment correlation coefficient. Values P<0.05 were considered statistically significant. RESULTS Behavioral Evaluation The monitoring of EE cages allows obtaining data about activity and centroid features (speed, acceleration and entropy) during the light/dark cycle. Remarkably, 6-OHDA-lesioned rats decreased significantly the number of activity areas during the dark phase, which is the active period of the rodent circadian cycle, comparing to Sham group (∗∗∗p<0.0001, Mann-Whitney U-test) (Figure 2B). Moreover, the 6-OHDA group also showed a significant hyperactivity during the light phase (resting period) in comparison with control rats which may be related to a sleep behavior disruption (∗∗∗p<0.0001, Mann-Whitney U-test; Figures 2A,B). Overall, these results suggested that animals from the 6-OHDA group switched their transitional rhythms respect to the control group. Furthermore, the effect of 6-OHDA on the disruption of circadian rhythms was also supported by a significant difference in the activity between both light and dark phases which was also seen in Sham group (###p<0.0001 activity area mean in light cycle vs. activity area mean in dark phase within the same group, Mann-Whitney U-test; Figure 2B). In line with these results, although Sham group showed more remarkable changes between dark and light cycles in terms of speed and acceleration than 6-OHDA group, entropy was significantly higher in the 6-OHDA group (6-OHDA group vs. Sham group, ∗∗∗p<0.0001, Mann-Whitney U-test; Figures 2C–E). The fact that entropy was higher supported and confirmed the incipient sleep pathological condition (Figure 2E). Both groups also showed significant differences between both dark and light phases in all the examined parameters related to the centroid evolution indicating different behaviors in both phases according to the light or dark period (###p<0.0001 within the same group, Mann-Whitney U-test; Figures 2C–E). Besides, the 6-OHDA effect on the acceleration and speed in both phases pointed to a difficulty in the movement noticed in both phases. Thus, this is a model that allows integrating information about both dark and light phases. Accordingly, the efficiency of this model was confirmed by the results obtained by the ROC curve and its AUC which measure the accuracy of the model by Logistic Regression. In the ROC curve the true positive rate (Sensitivity) is represented in function Frontiers in Neuroscience | www.frontiersin.org 6October 2020 | Volume 14 | Article 590029 fnins-14-590029 October 8, 2020 Time: 18:47 # 7 Requejo et al. 6-OHDA Induced Prodromal PD Symptoms FIGURE 2 | Circadian rhythm disruption in 6-OHDA-induced rats. (A) Diagram illustrating the motion activity signal in 6-OHDA (upper panel) and Sham (bottom panel) groups during the whole manuscript. Afterward, the data are preprocessed and selected in order to obtain for the statistical analysis, appropriated balanced datasets of the light and dark periods for the two groups. Blue: motion activity signal, Green: smoothed signal for control group, Red: smoothed signal for 6-OHDA group, Yellow: light level. Graphs depict significant activity changes (B), the centroid features including the mean of centroid speed (C), the mean of centroid acceleration (D) and the entropy mean (E) between 6-OHDA and Sham groups during the light/dark cycle and within the same group. Data are expressed as mean ±SEM. General analysis was performed by considering 12 h of dark/light periods. Statistical differences appear (p<0.05) for Mann-Whitney U-test between both groups and within the same group for both light and dark phases (6-OHDA group vs. Sham group, ***p<0.001; and light cycle vs. dark cycle within the same group, ###p<0.001). of the false positive rate (1-Specificity) for different operating points, features of the system, where an AUC of 100% would represent the highest specificity and sensibility of the model to separate both groups. Our data gave a ROC curve (each point on the curve represents a sensitivity/specificity) that reached a significant AUC of 0.854 giving 85% of sensibility and specificity to this model (0.854 ±0.0047, p=0;Figure 3). On the other hand, minimal motor deficits were observed in the amphetamine test (Supplementary Table S1). Although animals increased significantly the number of IL turns per minute (tpm) respect to control group, (1.93 ±0.39 tpm vs. 0.32 ±0.076 tpm; t(17)= 4.292, p= 0.0005, Unpaired Student’s t-test), no rats turned more than 3.98 turns per minute (0.79–3.98 tpm; Supplementary Figure S2). Therefore, a prodromal stage would be supported by the mild presence of motor symptoms. Morphological Evaluation Once confirmed the functional changes displayed by 6-OHDA group 2 weeks after 6-OHDA administration, we also assessed Frontiers in Neuroscience | www.frontiersin.org 7October 2020 | Volume 14 | Article 590029 fnins-14-590029 October 8, 2020 Time: 18:47 # 8 Requejo et al. 6-OHDA Induced Prodromal PD Symptoms FIGURE 3 | ROC curve for linear regression. Modeling for 6-OHDA and Sham groups. ROC curve (each point on the curve represents a sensitivity/specificity) that reached a significant AUC of 0.854 giving 85% of sensibility and specificity to this model (0.854 ±0.0047, p= 0). ROC, Receiver Operating Characteristics; 6-OHDA, 6-hydroxydopamine; AUC, Area Under the ROC Curve. whether 6-OHDA exerted a histological effect that we had previously observed 3 weeks after 6-OHDA administration (Requejo et al., 2018a,b). 6-OHDA-Induced Mild Nigrostriatal Dopaminergic Degeneration After a Short Evolution Time Analysis of TH-immunostained sections from the striatum and SN confirmed that, at this survival time, 6-OHDA induced a moderate TH-ir fiber loss either in the striatum or in the SNr (Figure 4). In addition, dopaminergic neurons in the SN were not notably reduced (Figures 4E,G). Rostro-caudal study of TH-ir in the SN indicated that the number of the survival TH-ir neurons as well as the TH-ir axodendritic network (ADN) in 6-OHDA group increased rostro-caudally. Interestingly, one-way ANOVA followed by Turkey post hoc test indicated significant differences (*p<0.05) between rostral and caudal levels within the 6-OHDA group regarding the number of THir neurons (35.61 ±8.89% of TH-ir neuronal cells at the rostral sections vs. 70.12 ±7.03% of TH-ir neuronal cells at the caudal sections; p= 0.031). However, regarding the number of TH-ir AND no significant differences were found between rostral and caudal sections [52.24 ±6.32% of TH-ir ADN at the rostral sections vs. 76.86 ±5.29% of TH-ir of ADN at the caudal section; One-way ANOVA, F(2,9)= 3.800, p= 0.063; Figures 4G,H]. In addition to this, no topological distribution in the TH-ir striatal fibers was appreciated (Figure 4D). Correlations Between Motor and Morphological Changes The number of IL amphetamine-induced rotations was strongly correlated with the percentage of TH-ir neuronal cell loss (r=−0.94) and it was statistically significant (p≤0.05) (Figure 5B). On the other hand, the TH-ir terminal loss either in striatum or in SN also showed a correlation with the increase in the number of IL rotations per minute close to be statistically significant (r=−0.84, p= 0.073 for TH-ir IL striatal volume and r=−0.93, p= 0.065 for TH-ir AND; Figures 5A,C). Biochemical Analysis In order to assess changes in apoptosis, the expression of caspase3 in neuronal cells was morphologically evaluated by double immunostaining against NeuN (nuclear marker of neuronal cells) and caspase-3 (apoptosis marker). In addition, caspase-3 levels were also biochemically analyzed by western blot in the striatum and SN (Figure 6). Double immunofluorescence revealed higher caspase-3 immunoreactivity and lower NeuN immunoreactivity in 6-OHDA-lesioned rats compared to Sham group in the IL striatum as well as in the IL SN. In fact, 6-OHDA induced a remarkable apoptosis in neuronal cells due to a considerable amount of NeuN positive cells co-localized with caspase-3 in 6-OHDA group (Figures 6A,C). Furthermore, immunoblot results were consistent with the outcomes showed by immunostaining. The expression of Frontiers in Neuroscience | www.frontiersin.org 8October 2020 | Volume 14 | Article 590029 fnins-14-590029 October 8, 2020 Time: 18:47 # 9 Requejo et al. 6-OHDA Induced Prodromal PD Symptoms FIGURE 4 | TH-ir loss in the 6-OHDA-induced preclinical model. (A,B) Rostro-caudal distribution of coronal sections corresponding to the CL and IL striatum and SN immunostained with tyrosine hydroxylase (TH) in Sham and 6-OHDA groups. (C,D) Graphs show the effects of 6-OHDA on the loss of the TH-ir volume in the IL striatum comparing to Sham group (***p<0.001, Unpaired Student’s t-test.) and no significant changes were found along the rostro-caudal axis. (E,F) TH-ir neuronal density and TH-ir ADN density decrease significantly following 6-OHDA administration comparing to Sham group in the SN (***p<0.001, Unpaired Student’s t-test). (G,H) Topological analysis shows a selective vulnerability in the SN to 6-OHDA decreasing the TH-ir neuronal and the TH-ir ADN density rostro-caudally (*p<0.05 rostral section vs. caudal section within 6-OHDA group, One-way ANOVA). Data are presented either as the percentage of TH-ir ipsilateral striatal volume respect to the total ipsilateral one (C,D) or as the percentage of TH-ir neurons (E,G) or TH-ir ADN (F,H) remaining in the IL side respect to the CL side. Scale bar: 2 mm (A),1mm(B). CL, contralateral; IL, ipsilateral; TH-ir, tyrosine hydroxylase immunoreactivity; 6-OHDA, 6-hydroxydopamine; ADN, axodendritic network; SN, substantia nigra. FIGURE 5 | Morpho-functional correlation between amphetamine-induced rotations and dopaminergic neurons in SN in addition to dopaminergic terminals in both striatum and SN after 6-OHDA administration. (A) Amphetamine-induced IL rotations correlate negatively with the % of TH-ir striatal volume IL, (B) also with the % of TH-ir neuronal density and (C) with the % of TH-ir ADN density. Significance. p<0.05, IL rotations vs. TH-ir neurons. SN, substantia nigra; 6-OHDA, 6-hydroxydopamine; TH-ir, tyrosine hydroxylase immunoreactivity; IL, ipsilateral. caspase-3 was upregulated in 6-OHDA-lesioned rats in the striatum and in SN following a similar pattern (127.9 ±5.1% and 127.9 ±26.08%, respectively), while caspase-3 activation was barely evidenced in Sham group (103.8 ±7.6% in the striatum and 90.7 ±7.57% in the SN). However, statistically significant differences between both groups were only found in the striatum [t(7)= 2.426, p= 0.045, Unpaired Student’s t-test; Figures 6B,D]. However, no significant changes were seen in the prosurvival signaling protein levels (AKT and ERK) between 6-OHDA and Sham groups 2 weeks postinjection, despite Frontiers in Neuroscience | www.frontiersin.org 9October 2020 | Volume 14 | Article 590029