Systemic Rotenone Administration Causes Extra-Nigral Alterations in C57BL/6 Mice

Systemic administration of rotenone replicates several pathogenic and behavioural features of Parkinson’s disease (PD), some of which cannot be explained by deficits of the nigrostriatal pathway. In this study, we provide a comprehensive analysis of several neurochemical alterations triggered by systemic rotenone administration in the CNS of C57BL/6 mice. Mice injected with either 1, 3 or 10 mg/kg rotenone daily via intraperitoneal route for 21 days were assessed weekly for changes in locomotor and exploratory behaviour. Rotenone treatment caused significant locomotor and exploratory impairment at dosages of 3 or 10 mg/kg. Molecular analyses showed reductions of both TH and DAT expression in the midbrain, striatum and spinal cord, accompanied by altered expression of dopamine receptors and brain-derived neurotrophic factor (BDNF). Rotenone also triggered midbrain-restricted inflammatory responses with heightened expression of glial markers, which was not seen in extra-nigral regions. However, widespread alterations of mitochondrial function and increased signatures of oxidative stress were identified in both nigral and extra-nigral regions, along with disruptions of neuroprotective peptides, such as pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal peptide (VIP) and activity-dependent neuroprotective protein (ADNP). Altogether, this study shows that systemic rotenone intoxication, similarly to PD, causes a series of neurochemical alterations that extend at multiple CNS levels, reinforcing the suitability of this pre-clinical model for the study extra-nigral defects of PD.


Introduction
Parkinson's disease (PD) is a progressive neurodegenerative disorder that affects around 2% of the population over the age of 60 [1,2]. It clinically manifests with motor impairments, including bradykinesia, tremor, rigidity and postural instability [3]. These classical parkinsonian motor symptoms are caused by the degeneration of dopamine neurons in the substantia nigra pars compacta (SNpc), resulting in a lack of dopamine in the striatum [4]. Notwithstanding these pathological hallmarks, PD patients also develop a myriad of non-motor symptoms, including depression, anxiety, cognitive deficits and autonomic dysfunction, suggesting that damage might also occur in other regions of the central nervous system (CNS) [5].
The driving force triggering the neurodegeneration observed in PD remains unknown; however, several factors contribute to disease pathogenesis, including genetics, environmental factors, oxidative stress and neuroinflammation [6].
Rotenone is a naturally occurring compound used commercially as a pesticide and piscicide, which reproduces some of the major clinical and behavioural features of PD in rodents [7][8][9]. Since its discovery as a PD-mimetic, the rotenone intoxication model has become a popular preclinical model of PD based on the high lipophilic nature of the compound, which enables it to cross biological membranes allowing systemic administration, but also due to its ability to trigger several pathological mechanisms, including oxidative stress, protein aggregation and CNS inflammation, as well as its capability of reproducing a PD-like behavioural phenotype [10]. This ability to reproduce key pathological and phenotypic features of PD has made rotenone a valuable tool to study drug-mediated neuroprotection [11].
Rotenone administered at low dosages induces degeneration of the dopaminergic nigrostriatal pathway and causes motor deficits [12][13][14][15][16]. Rotenone-mediated dopaminergic degeneration occurs via the inhibition of the mitochondria electron transport chain complex I, which results in the formation and accumulation of reactive oxygen species, leading to oxidative stress [9,17]. Studies of human post-mortem brain tissues indicate that oxidative stress and accumulation of reactive oxygen species are critical events in the pathogenesis of PD [18,19].
Chronic daily intraperitoneal injections of rotenone induce l-3,4-dihydroxyphenylalanine (L-DOPA) responsive locomotor deficits and neurochemical abnormalities characteristic of PD in both rats and mice [15,16,20,21]. However, most of these investigations have been performed using non-standardised rotenone injection regimens, which often produce a certain degree of variability-mainly depending on the route of administration, concentration, animal strain and experimental regimes, making it difficult to compare studies [22]. Additionally, most of these investigations focus on the analyses of neurochemical alterations in the midbrain and striatum (i.e., the nigrostriatal pathway), partly overlooking for any possible neurochemical changes in other regions of the CNS [23].
Based on the evidence indicating the existence of common clinical and pathological features shared by the rotenone toxicity model and PD patients, in the present study we aimed at further characterising the range of neurochemical alterations triggered by systemic rotenone administration in the CNS of C57BL/6 mice. Once we established the suitability of the animal model by behavioural assessments, we determined if rotenone toxicity disrupted the expression of dopamine, oxidative stress and inflammatory markers, as well as neuropeptides and trophic factors in at least six distinct CNS sites. Our results indicate that rotenone triggers widespread neurochemical changes that extend beyond the nigrostriatal dopaminergic system. Altogether, these findings suggest that the neurodegenerative process triggered by rotenone intoxication, similarly to PD, affects multiple central and perhaps peripheral systems, offering a viable model to investigate both nigral and extra-nigral defects triggered by PD.

Animal Experiments
Twenty-four 7-week-old male C57BL/6 mice were purchased from ARC (Perth, WA, Australia). Mice were allowed to acclimate for one-week and experimental regimen commenced when mice were 8 weeks old. Mice were housed in individually ventilated cages (4 mice per cage), under normal 12:12 h light/dark cycle, with access to food and water ad libitum. All experiments were conducted in line with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, and in compliance with the ARRIVE guidelines. The University of Technology Sydney Animal Care and Ethics Committee (ETH19-3322) approved all animal experiments.

Rotenone Experimental Protocol
Mice were randomly assigned to one of the four treatment groups: control (vehicle), rotenone 1 mg/kg, 3 mg/kg or 10 mg/kg. Rotenone was prepared as a stock solution in 0.1% DMSO diluted in 0.1% saline. Mice underwent behaviour testing every 7 days to assess locomotor and exploratory behaviour. Mice in each group were daily injected with intraperitoneal injections as indicated for 21 days and monitored for 2 h post injection. On day 22, mice were sacrificed and brains and spinal cord were collected. Tissue was snap frozen and used to perform molecular analysis. The brains were subsequently micro dissected into the following regions: prefrontal cortex, striatum, hippocampus, amygdala and midbrain with one hemisphere used for RNA analyses and the other for protein analyses.

Open Field Behaviour Test
The open field (OF) test was conducted every 7 days in the light cycle between 08:00 h and 12:00 h. Animals were acclimated in the testing rooms for 30 min for habituation. The OF was conducted in the dark. The OF consisted of a square box (40 × 40 cm ) and 50 cm tall made of grey plexiglass. Mice were placed individually in the centre of the area and allowed to explore the environment freely for 5 min while being recorded. The OF was cleaned thoroughly between each mouse to eliminate any odour cues with 70% ethanol. The FIJI/ImageJ Plugin MouBeAt software [24] was used to analyse videos. MouBeAt quantifies the distance and time spent in the central and peripheral areas, number of entries to centre region and average speed. An entry into an area was counted when at least 70% of the mouse body had completely entered the area. For this type of analyses, data was computed using a repeated measures 2-way ANOVA, factoring both treatment and time as independent variables.

Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR)
Briefly, total RNA was extracted using TRI-reagent (Sigma-Aldrich, Castle Hill, NSW, Australia) and chloroform and precipitated with ice-cold 2-propanol following established protocols [25,26]. RNA concentration was determined using spectrophotometry (Nanodrop ND-1000 ® spectrophotometer, Wilmington, DE, USA). Single-stranded cDNA was synthesized using Tetro cDNA synthesis kit (Bioline, Sydney, NSW, Australia). Real-time qPCR was performed to analyse the steady-state mRNA levels of twelve genes ( Table 1). The ribosomal protein 18S was used as the housekeeping gene. Each reaction consisted of 3 µL cDNA (final concentration 100 ng), 5 µL iTaq Universal SYBR Green Master Mix (BioRad, VIC, Australia), 0.8 µL of forward and reverse primers. To examine changes in expression, the mean fold changes of each sample was calculated using the ∆∆Ct method as previously described [27,28]. PCR product specificity was evaluated by melting curve analysis, with each gene showing a single peak (data not shown). Forward and reverse primers were selected from the 5 and 3 region of each gene mRNA. The expected length of each amplicon is indicated in the right column. #: number.

Protein Extraction and Western Blot
Protein was extracted by homogenizing tissues in radioimmunoprecipitation assay (RIPA) buffer containing a protease inhibitor to preserve protein integrity (cOmplete TM , Mini, EDTA-free Protease Inhibitor Cocktail, Sigma-Aldrich, Castle Hill, NSW, Australia) [29]. Tissues were sonicated and then cleared by centrifugation at 12,000× g for 10 min. Protein quantification was determined with bicinchoninic acid assay (Pierce BCA Protein Assay Kit, ThermoFisher Scientific, North Ryde, NSW, Australia). Equal amounts of protein (30 µg) was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using 4-20% Mini-PROTEAN TGX Stain-Free Gels (15 well, BioRad, South Granville, NSW, Australia). The Precision Plus Protein Prestained Standard in All Blue (BioRad, South Granville, NSW, Australia) was included for comparison. Transfer to a PVDF membrane was performed using the semi-dry method (BioRad Trans-Blot Turbo Transfer System). Incubation with primary antibodies was performed overnight in 5% skim milk in TBST blocking solution at 4 • C. Antibodies and dilutions are summarized in Table 2. Western blots were visualized using chemiluminescence BioRad Clarity Western ECL Blotting Substrate Solution. Images were acquired using the BioRad ChemiDoc MP System. Images were analysed using Fiji ImageJ and ratios normalized to GAPDH which was used as a loading control.

Immunohistochemistry
Brains were fixed in 4% paraformaldehyde for 48 h before dehydration and embedding in paraffin. 5 µm thick coronal sections were cut with using a microtome and mounted on glass slides. The sections were deparaffinised in xylene and rehydrated through decreasing concentrations of ethanol. Dopaminergic neurons were labeled with tyrosine hydroxylase (TH) (1:500, ab112, Abcam). The immunoreactivity of the antibody was revealed using the Rabbit specific HRP/DAB (ABC) Detection IHC Kit (ab64261, Abcam, VIC, Australia) according to manufacturer's protocol. Hematoxylin was used to counterstain nuclei and to appreciate the gross architecture of the examined CNS region. Sections were dehydrated in increasing concentrations of ethanol and xylene before being mounted. Images were taken on a ZEISS AxioScan.Z1 at ×20 magnification. ImageJ software was used to quantify TH-staining intensity in the region of interest [30].

Statistical Analysis
All data are reported as mean ± S.E.M. Statistical analyses were performed using GraphPad Prism software ver. 9.0.2. (GraphPad Software, La Jolla, CA, USA). For behavioural analyses, in order to determine a possible relationship between treatment (drug dosage) and duration, two-way repeated measures ANOVA followed by Tukey post hoc test was performed, factoring both treatment regime and time as independent variables. All the other comparisons involving only two groups were analysed by unpaired Student's t-test. Comparisons between multiple groups were analysed in function of a single independent variable (rotenone treatment), where results attained from mice receiving increasing dosages of rotenone were compared with vehicle-treated mice. Therefore, unless otherwise specified, these data were analyzed by one-way ANOVA followed by Dunnett's post hoc test. All p-values ≤ 0.05 were considered statistically significant.

Rotenone Intoxication Impairs Locomotion and Exploratory Behaviour in Mice
To assess if rotenone induced changes in locomotor and/or exploratory behaviour, mice were subjected to the Open Field (OF) test as outlined in the experimental design ( Figure 1a).
Mice were weighed daily prior to injection (Figure 1b). Analyses of body weight revealed that mice receiving 3 mg/kg and 10 mg/kg of rotenone gained significantly less weight compared with saline-injected controls (** p = 0.01 and * p = 0.0247 vs. saline, respectively; Figure 1c).
MouBeAt, an ImageJ plugin, was used to track and quantify general locomotor (i.e., average speed and total distance travelled) and exploratory behaviours of mice in the OF (i.e., number of entries and total time spent in the centre quadrant) [24], which allowed us to generate representative heat maps depicting the locomotor pattern of tested mice ( Figure 1d). Drug-induced deficits in locomotor activity were assessed by comparing the total distance travelled (F (9, 6) = 2.723, ** p = 0.0099; Figure 1e) and average speed (F (9, 60) = 1.631, p = 0.1269; Figure 1f) at 7, 14 and 21 days with respect to baselinemeasurements taken prior to exposure to rotenone. To examine exploratory behaviour, we measured the number of times (F (9, 60) = 3.648, ** p = 0.0011; Figure 1g) and the total time spent by each mouse in the centre quadrant (F (9, 60) = 3.062, ** p = 0.0044; Figure 1h) of the OF. Mice display a natural aversion to open areas and preferentially stay close to the walls of the field (thigmotaxis). In contrast, they display an instinctive drive to explore a perceived threatening environment [31]. Therefore, the overall number of times a mouse enters the centre quadrant and the total time spent with respect to the periphery are indicative of the exploratory behaviour of mice ( Figure 1a).
We report that only mice administered with 3 mg/kg or 10 mg/kg of rotenone developed deficits in locomotor and exploratory behaviours as compared with baseline controls. In more detail, at 3 mg/kg, rotenone significantly reduced the total distance travelled in the OF on day 14 (* p = 0.0207), and this was further reduced by day 21 (** p = 0.0028) (Figure 1e), although this was not associated with an overall reduction in speed, except on day 7 (* p = 0.0442). In contrast, at this rotenone dosage, reductions in exploratory behaviour were only recorded on day 21 (* p = 0.0398; Figure 1h). Rotenone impairs locomotor and exploratory behaviour. Experimental timeline for injections and behavioural assessments (a). The Open Field Test mice was used to assess for locomotor and exploratory behaviour in rotenone Vs baseline measurements every 7 days. The centre quadrant was defined as a central square with a surface area that is 25% smaller than the total area (red box), whereas the peripheral area was defined as the surface area between the centre quadrant the walls of the Open Field box. Mice received an intraperitoneal injection of indicated treatment daily for 21 days. On day 22, mice were humanely sacrificed and the brain and spinal cord was collected. Mean daily weight per treatment group (b) and the average weight loss/gain (c) was calculated using mean daily weights of day 1 vs. day 22. Representative heat maps from MouBeAt Software that tracks the movement of mice during the open field (d). Locomotor and behaviour measurements were determined by MouBeAt software. Heat map uses colour to represent how often a mouse spent in an area. The longer a mouse spent in an area, the colour would shift from blue to red to yellow. Comparisons were made within the same treatment group compared to baseline measurements. An entry into an area was counted when at least 7% of the mouse body had completely entered the area. Total distance travelled (cm) in 5 min (e). Average speed reported as cm/s (f). Number of entries in centre (g). Time spent in centre(s) (h). Data shown represents means of n = 6-10 mice per group. * p < 0.05 or ** p < 0.01 as determined by one-way ANOVA followed by Dunnett's post hoc test (weight change) or two-way repeated measures ANOVA followed by Tukey post hoc test (behavioural measurements).
As expected, at the highest rotenone dosage tested in this study (10 mg/kg), the drug severely impaired both locomotor and exploratory behaviours already after 14 days of rotenone treatment. Specifically, there was a significant reduction in the total distance travelled by these mice at both days 14 (* p = 0.0214 vs. baseline) and 21 (* p = 0.0207 vs. baseline; * p = 0.0201 vs. Day 7) (Figure 1e). This was correlated with significantly slower average speed at days 14 (* p = 0.0160 vs. baseline) and 21 (** p = 0.0093 vs. baseline; * p = 0.0324 vs. Day 7) (Figure 1f). Additionally, mice exhibited reduced exploratory behaviour, as demonstrated by the reduction in the number of entries in the centre of the OF at day 21 (* p = 0.0458) ( Figure 1g) and the reduction in the time spent in the centre vs. the periphery, recorded both at day 14 (* p = 0.0317 vs. baseline; ** p = 0.0094 vs. Day 7) and at day 21 (* p = 0.0342 vs. baseline; * p = 0.0111 vs. Day 7) (Figure 1h).

Rotenone Reduces the Expression of Dopaminergic Markers in the Midbrain, Striatum and Spinal Cord
PD is characterized by the loss of dopamine neurons in the midbrain, which results in a loss of dopamine availability in the striatum [32]. In addition, there is evidence that tyrosine hydroxylase positive (TH) + and dopamine receptors'-expressing neurons are also localized in the spinal cord [33,34], implicating a role of the spinal dopaminergic system in motor control. As such, we sought to analyse the expression of two main dopamine markers, TH and dopamine transporter (DAT), in the midbrain, striatum and spinal cord of mice to determine if rotenone caused a similar loss of dopaminergic neurons throughout the CNS (Figure 2).
The expression of dopamine markers in the spinal cord revealed a significant reduction of TH transcripts across all treatment groups (**** p < 0.0001; F (3,28) = 41.55; Figure 2g), including rotenone 1 mg/kg (*** p = 0.0002 vs. vehicle), with both higher doses-3 mg/kg and 10 mg/kg-able to further reduce TH transcript levels (**** p < 0.0001; Figure 2g Our rationale to test multiple dosages of the insecticide was to identify a dosage that reliably induced PD-like pathophysiology and associated behavioural deficits. Both behaviour ( Figure 1) and dopaminergic markers ( Figure 2) confirmed that 10 mg/kg rotenone reliably reproduced the pathological and clinical features of PD. These data were further supported by immunohistochemical evidence of reduced TH + staining in the midbrain of mice exposed to increasing dosages of rotenone compared with controls (*** p < 0.001 [1 mg/kg rotenone vs. vehicle], **** p < 0.0001 [3 and 10 mg/kg rotenone vs.  were sacrificed and midbrains, striata and spinal cords were collected for molecular analyses. TH mRNA and protein expression in the midbrain (a-c). DAT mRNA and protein expression in the striatum (d-f). TH and DAT mRNA and protein expression in the spinal cord (g-k). Representative photomicrographs showing TH-immunoreactivity and semi-quantitative measurement of staining intensity in the midbrain of vehicle-, 1, 3 and 10 mg/kg rotenone-treated mice (l-p). At least two sections from n = 3 mice per group were used for semi-quantitative analyses of staining intensity. Scale bar = 200 µm. Gene expression was measured using real-time qPCR and quantified using the ∆∆Ct method after normalization to s18 (ribosomal protein s18 gene), the housekeeping gene. Real-time qPCR results are presented as mean fold changes with respect to vehicle-treated mice (control). In Western blots, protein expression was normalized to GAPDH, used as the loading control. Densitometry results are expressed as mean ± S.E.M. Data in qPCR, Western blots and immunohistochemistry represents means of n = 4 samples for each group. * p < 0.05, ** p < 0.01, *** p < 0.001 or **** p < 0.0001 as determined by one-way ANOVA followed by Dunnett's post hoc test. Ctl: control; R1, 3, 10: Rotenone (1, 3, 10 mg/kg); TH: tyrosine hydroxylase; DAT: dopamine transporter; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; kDa: Kilodalton; VTA: ventral tegmental area; SNpc: Substantia Nigra pars compacta.

Rotenone Triggers CNS Region-Specific Changes in the Expression of Dopamine Receptors and Brain-Derived Neurotrophic Factor
Dopamine is the main neurotransmitter affected in PD pathogenesis as a result of the loss of dopaminergic neurons. To examine potential disturbances of the dopaminergic system in nigral and extra-nigral regions, we analysed the mRNA expression of all dopamine receptors both in vehicle-and 10 mg/kg rotenone-treated mice.
Interestingly, compared with extra-nigral regions, nigrostriatal regions reported little disruptions to dopamine receptors expression despite using the highest rotenone dosage (10 mg/kg) ( Figure 3).

Rotenone-Induced Neuroinflammation Is Confined to the Midbrain and Inhibited in Extra-Nigral CNS Regions
In order to define whether chronic rotenone treatment caused diffuse neuroinflammation, we interrogated the gene expression of inflammatory mediators as well as the gene and protein expression of glial activation markers in the same CNS regions indicated above. To our surprise, we report that rotenone-induced neuroinflammation is restricted to the midbrain. In contrast, a global down-regulation of the expression of the pro-inflammatory cytokine IL-1β and of two distinct glial activation markers was seen in extra-nigral CNS regions. (Figure 5).

Rotenone Intoxication Causes a Global Disruption in the Expression of Neuropeptides in the CNS
In view of the protective role elicited by endogenous neuropeptides in preventing neuronal deterioration in PD and other neurodegenerative disorders [39][40][41], we sought to determine if rotenone intoxication interfered with the expression of well-established neuropeptides/neurotrophic molecules. As such, we measured transcript levels of ADNP, PACAP and VIP in the CNS of mice that received increasing dosages of rotenone. In addition, for both PACAP and VIP neuropeptides, we also measured protein expression.

Discussion
In this study, we sought to provide a comprehensive analysis of the behavioural and neurochemical alterations caused by rotenone intoxication in C57BL/6 mice, which are summarized in Table 3. For this purpose, animals were injected daily with rotenone at increasing dosages for a period of 21 days via intraperitoneal route. We opted to utilize this route of administration as this has been found to be the most effective to achieve a PD-like phenotype, at least in mice. Whereas a large bulk of studies have demonstrated that rotenone intoxication reliably mimics human PD pathology [12][13][14], several discrepancies have been identified when comparing studies using different administration routes. For instance, a study reported that dopamine loss and behavioural deficits were seen in mice that were administered with 5 mg/kg rotenone by oral gavage for 12 weeks [42]. Another study using the same route of administration reported that dosages as high as 30 mg/kg rotenone for periods of 28 or 56 days were required to obtain similar nigrostriatal degeneration and locomotor impairment [10]. In contrast, studies in which rotenone was administered via intraperitoneal route have shown that lower dosages of rotenone (0.75-3 mg/kg) and relatively shorter exposure periods (up to 3 weeks) were sufficient to achieve similar outcomes [15,43]. These results suggest that, at least in mice, rotenone adsorption via the gastrointestinal tract might not be as efficient as in rats. Dopaminergic markers ↓ n/a n/a n/a n/a ↓ TH ↓ n/a n/a n/a n/a ↓ n/a ↓ n/a n/a n/a ↓ DAT n/a ↓ n/a n/a n/a ↓ Dopamine receptors D1 - A large portion of our investigations was to assess if rotenone toxicity disrupted the expression of dopamine, oxidative stress and inflammatory markers, as well as neuropeptides and trophic factors in the midbrain and in extra-nigral CNS regions. The reason behind these investigations was based on the ever-growing repertoire of behavioural alterations that are seen in patients with PD, which are not limited to the locomotor impairment associated with nigro-striatal degeneration. Our data indicates that rotenone toxicity causes a broad spectrum of neurochemical alterations, such as changes in the expression of dopamine, oxidative stress and inflammatory markers, as well as neurotrophic factors/neuropeptides, some of which extend beyond the canonical nigrostriatal path-way. These results provide evidence of distinct and CNS region-specific neurochemical alterations at multiple CNS levels in rotenone intoxicated mice.

Toxic Effects of Rotenone in Extra-Nigral Regions-General Considerations
Several CNS regions are altered in PD pathogenesis; however, most PD studies focus on the nigral-striatal pathway and pay little attention to extra-nigral changes. This is surprising as non-motor symptoms of PD are known to clinically manifest much earlier than the classical motor symptoms associated with degeneration of dopaminergic neurons in the SNpc. For example, neuroimaging studies revealed structural and functional changes in the limbic cortico-striato-thalamocortical circuits that correlated with the high prevalence of anxiety in PD [44]. Further imaging studies demonstrated atrophy in the gray matter, caudate, putamen, nucleus accumbens and amygdala and associated atrophy of these regions with the cognitive impairment observed in PD [45]. Villar-Conde and colleagues revealed synaptic changes in the hippocampus of PD patients [46], aligning with an increased dementia risk in PD patients. Apathy is one of the most common PD-related mood changes and has been linked to alterations in the medial and lateral prefrontal cortex and the limbic system [47]. Finally, the spinal cord, an often neglected part of the CNS in the context of PD, has been shown to exhibit alpha-synucleinopathy and could be the origin of several non-motor symptoms, including urinary, sexual, gastrointestinal dysfunctions and pain [48]. PD is a very heterogeneous disease that presents differently in each patient. Accordingly, PD research should not be restricted to the nigro-striatal pathway, as this study and others have contributed to reveal a unique profile of extra-nigral regions that could help inform early diagnosis and clinical management of disease.

Rotenone Impairs Locomotor and Exploratory Behaviours
First, we evaluated the effects of rotenone on body weight and locomotor and exploratory behaviour. Mouse rotenone models have previously shown that the toxicant does not induce significant weight loss in rats. As shown by Indel et al., they found that there was no difference in weight when comparing C57BL/6 mice that received 30 or 100 mg/kg rotenone for 56 days with controls [2]. These results align with our study, as rotenone did not cause any significant weight loss; however, mice gained less weight compared to saline-injected controls. Assessments of locomotor and exploratory behaviours unveiled a dose-dependent deterioration of both behavioural domains, although these were best appreciated by measures of locomotor behaviour. A similar outcome has been highlighted in several studies, where rotenone reliably reduced the total distance travelled and number of times mice moved between areas of the OF [38,49]. This finding is consistent with previous studies demonstrating that intraperitoneal injections of 3 mg/kg rotenone daily for 21 days induced locomotor impairment in C57BL/6 mice [43]. Furthermore, studies report that at least a 3-week intoxication protocol is required to attain behavioural deficits [37], in agreement with our study, where the most consistent induction of locomotor and exploratory impairments was seen on day 21.

Rotenone Reduces the Expression of Dopaminergic Markers
Next, we confirmed if the behavioural impairments triggered by rotenone were associated with the deterioration of dopaminergic pathway. As expected, our study reproduced the selective decline of dopaminergic neurons within the midbrain and striatum seen in other studies [37,43]. However, rotenone dosages in our study were about ten times lower than that used in orally administered mice to achieve similar outcomes [37]. Investigations were also extended to the spinal cord, another CNS region that also seem to be vulnerable to the effects of rotenone intoxication [50]. In this region, TH and DAT transcripts and protein levels were reduced, especially with the highest rotenone dosage. This result is, to some extent, similar to that reported in mutant A53T mice (a genetic model of PD), where axonal degeneration and motor neuron cell loss also extended to the spinal cord of these mice [51]. This is particularly important, as some studies have already reported some degree of association between spinal cord damage and the autonomic dysfunctions seen in PD [52][53][54]. Interestingly, we report that extra-nigral regions such as the prefrontal cortex, amygdala, hippocampus and spinal cord were the only CNS regions that demonstrated significant changes in the expression of the dopamine receptors. These receptors were analysed due to their known alterations during PD pathogenesis [55]. Apparently, it can be inferred that damage to the dopaminergic system in PD may not be limited to the nigrostriatal pathway, although more in-depth investigations are needed to understand the involvement of extra-nigral circuits and spinal dopaminergic system damage in the development of motor and non-motor symptoms. Additionally, the expression of brain-derived neurotrophic factor (BDNF) was reduced in several CNS regions, including the midbrain, amygdala and spinal cord, suggesting neurotoxic damage in these areas. However, it should be highlighted that BDNF mRNA and protein levels (as well as other markers interrogated in this study) produced some discordant results. We believe this can be attributed to several factors including: post-transcriptional and post-translational modifications, short half-life of proteins in vivo, different turnover of mRNA vs. protein at time of tissue harvesting and other factors not mentioned here.

Systemic Rotenone Triggers Widespread Signs of Oxidative Stress
Rotenone is a known inhibitor of mitochondrial complex I [13,56], which can trigger oxidative stress damage in the CNS. Here, we interrogated OPA1, a mitochondrial fusion protein and SOD1, an antioxidant enzyme, as two markers of mitochondrial function and oxidative stress in the CNS [57,58]. Our results revealed disturbances in both OPA1 and SOD1 expression in all regions tested, suggesting that rotenone toxicity causes redox dysfunctions that extended to various CNS regions. Systemic neurotoxicity of rotenone has been reported in numerous studies [10,50,59]. Furthermore, clinical imaging studies in PD patients have identified the coexistence of structural and functional alterations in extra-nigral regions such as the hippocampus [60], amygdala and prefrontal cortex [44]. The evidence of widespread increase in oxidative stress markers in regions outside of the nigrostriatal pathway in our PD model support the idea that such neurochemical changes might be the consequence (or cause) of structural changes. This could be clinically relevant, as there is some evidence suggesting that non-motor symptoms often precede motor symptoms in the prodromal stages of the disease [5].

Rotenone-Induced Inflammation Is Restricted to the Midbrain
Neuroinflammation has become a well-established player in PD pathogenesis [61,62]. Rotenone has previously been shown to induce inflammation, with many studies utilizing rotenone reporting an association between oxidative stress and neuroinflammation as co-conspirators in PD pathogenesis [63]. Zhang and colleagues provided evidence of microglial activation in hippocampal and cortical regions of mice and suggested that this contributed to the cognitive decline seen in their rotenone-induced mouse PD model [16]. Evidence of neuroinflammation in PD patients has been found in several brain regions, including the SNpc, striatum, hippocampus and cerebral cortex, as well as in bodily fluids such as the cerebrospinal fluid [63]. Surprisingly, in our study rotenone-induced inflammation was confined primarily to the midbrain. This is intriguing, especially given the findings reported above from other research groups. To make the scenario even more complex, we also found that the expression of most inflammatory and glial markers were globally down-regulated in all the other CNS structures tested. Whilst the latter could be considered as a direct effect of systemic toxicity, we cannot rule out that the stress response triggered by treatments (and behavioural tests) may have contributed to dampening the overall activity of the immune system, hence explaining why only a restricted pattern of inflammation was found in the midbrain. Other aspects to consider are the existence of conflicting data available in the literature on the ability of rotenone as an inducer of neuroinflammation. In fact, this seems to depend on several factors, including the route of administration and duration of treatment. In one study, oral administration of rotenone caused dopaminergic degeneration in the absence of marked changes in glial activation [37]. Conversely, rotenone infusion via an osmotic pump strongly activated both astrocytes and microglia in the SNpc and striatum [64]. A more recent study that included a 2-week period where neuroinflammation was allowed to progress after rotenone administration concluded that activation of glial cells appeared to drive neuronal loss following neurotoxic exposure to rotenone [65]. Altogether, these results suggest that variations to the experimental protocols used in rotenone intoxication models may produce signs of inflammation ranging from no inflammation to widespread inflammation in several CNS sites.

Alterations of Neurotrophic Factors and Neuropeptides Expression Profiles in the Rotenone Mouse Model
Lastly, we analysed the expression of neuropeptides known to play critical roles in the modulation of neurological processes within the CNS. PACAP and VIP are two related neuropeptides that are expressed throughout the CNS that exert essential neuroprotective and immune modulatory roles [66]. PACAP knockout mice display age-related degenerative signs earlier than wild type animals, including increased neuronal vulnerability, systemic degeneration and increased inflammation, suggesting the importance of this peptide in maintaining a healthy and functional CNS [67]. VIP has been shown to prevent PD pathogenesis in several preclinical models of disease [68]. These peptides were analysed as we have previously shown that both peptides reduce microglial polarization in vitro [69], demonstrating their potential as therapeutic targets in PD. In a study by de Souza and collaborators, the authors describe these two peptides as neuroprotective and anti-inflammatory against experimental PD, acting mainly by reducing neuroinflammation, promoting dopaminergic neuronal survival and preserving cognitive functions [70]. Moreover, other evidence indicates that PACAP and BDNF [71] share similar neuroprotective pathways, as do VIP and ADNP [72]. Our results demonstrate these pathways are globally down-regulated in our rotenone model, suggesting that one of the modalities through which rotenone imparts damage to the CNS is via reducing the endogenous neuroprotective potential of these peptides. This correlates with studies that provide evidence on the therapeutic potential of PACAP, VIP, BDNF and ADNP in PD [39,41,73]. However, more research is needed to elucidate the exact role of these factors in PD pathogenesis.
Like most models of neurodegenerative diseases, it is difficult to model PD, as we still do not have a defined clinical diagnostic criteria that enables early identification, with confirmation of clinical PD occurring from postmortem analyses of brain tissue. However, comparisons between genetic, neurotoxic and inflammatory models indicate that regardless of the initial trigger, the CNS is subjected to a cascade of pathological events that include inflammation, mitochondrial dysfunction, oxidative stress and dopaminergic neuronal loss [62]. However, the neurochemical changes reported here correlate with other PD models as well as clinical studies [48,60,74,75]. Altogether, our results suggest that rotenone induces multiple neurochemical alterations across different CNS sites, suggesting that this disease model may be useful to study certain PD domains, thereby providing an excellent scaffold to study the efficacy of novel compounds to target non-motor and motor symptoms of PD.

Data Availability Statement:
The raw data generated and/or analysed during the current study are available upon reasonable request.