Pharmacological Reports Copyright © 2012 2012, 64, 10811090 by Institute of Pharmacology ISSN 1734-1140 Polish Academy of Sciences
Characterization of motor, depressive-like and neurochemical alterations induced by a short-term rotenone administration
Lívia H. Morais1, Marcelo M.S. Lima2, Bruno J. Martynhak1, Ronise Santiago1, Tatiane T. Takahashi1, Deborah Ariza1, Janaína K. Barbiero1, Roberto Andreatini1, Maria A.B.F. Vital1
1Department of Pharmacology, 2Department of Physiology, Federal University of Parana, Av. Francisco H. dos Santos s/n, ZIP 81.531-990, Curitiba, PR, Brazil
Correspondence: Marcelo M.S. Lima, e-mail: [email protected]
Abstract: Background: Rotenone exposure in rodents provides an interesting model for studying mechanisms of toxin-induced dopaminergic neuronal injury. However, several aspects remain unclear regarding the effects and the accuracy of rotenone as an animal model of Parkinson’s disease (PD). In this study, we investigated the motor and depressive-like behaviors associated to neurochemical altera- tions induced by a novel protocol of rotenone administration. Methods: In the first experiment, we adopted the paw test to characterize an effective dose of rotenone able to promote nigrostriatal toxicity. In the second experiment, control and rotenone 2.5 mg/kg groups were injected (ip) for 10 consecutive days. Results: This test indicated that intraperitonial (ip) rotenone at 2.5 and 5.0 mg/kg promoted a significant neurotoxicity to striatum and nucleus accumbens. However, only 2.5 mg/kg of rotenone was associated to a negligible mortality rate. Open-field tests were conducted on 1, 7, 14 and 21 day after the last day of treatment and showed an important locomotor impairment, confined to 1 and 7 day. Besides, rotenone affected dopamine levels and increased its turnover in the striatum. Modified forced swim test (performed on 22 day) and sucrose preference test (performed on 14 and 21 day) demonstrated that rotenone produced impairments in the swim- ming and immobility. In parallel, increments in the serotonin and noradrenaline turnovers were observed in the striatum and hippo- campus of the rotenone group. Conclusions: These data suggest important participations of serotonin and noradrenaline in depressive-like behaviors induced by rotenone. Thus, it is proposed that the current rotenone protocol provides an improvement regarding the existing rotenone models of PD.
Key words: dopamine, depression, Parkinson’s disease, serotonin, substantia nigra pars compacta, turnover
Abbreviations: 5-HIAA – 5-hydroxyindoleacetic acid, 6-OHDA Introduction – 6-hydroxydopamine, DA – dopamine, DHPG – dihydroxy- phenylglycol, DOPAC – 4-dihydroxyphenylacetic acid, EDTA Parkinson’s disease (PD) is the second most common – ethylenediaminetetraacetic acid, FRT – forelimb retraction neurodegenerative disease affecting about 1% of peo- time, HPLC – high performance liquid chomatography, HRT – ple over 65 years old and 4–5% of people over 85% hind limb retraction time, HVA – homovanillic acid, MPTP – 1-methyl-4-phenyl-1,2,3,4-tetrahydropyridine, NA – noradrena- [27, 28]. Major clinical features at presentation in- line, PD – Parkinson’s disease, 5-HT – serotonin, SNpc – sub- clude motor (asymmetric onset of bradykinesia, rigid- stantia nigra pars compacta ity, resting tremor) and non-motor (depression, sleep
Pharmacological Reports, 2012, 64, 10811090 1081 disturbances, memory deficits) complications. These Materials and Methods are mainly the outcome of neurodegeneration of the substantia nigra pars compacta (SNpc), which leads to subsequent reduction of dopaminergic input to the Animals striatum [31]. A range of factors are thought to be implicated in- Male Wistar rats from our breeding colony weighing cluding ageing, oxidative stress, inflammation, mito- 200–230 g at the beginning of the experiments were chondrial dysfunction and environmental toxins. The used. The animals were randomly housed in groups of first indication that exogenous toxins could lead to five in polypropylene cages with wood shavings as parkinsonism was described following the accidental bedding and maintained in a temperature-controlled exposure of drug abusers to 1-methyl-4-phenyl- room (22 ± 2°C) on a 12-h light-dark cycle (lights on 1,2,3,4-tetrahydropyridine (MPTP) which developed at 7:00 a.m.). The animals had free access to water an acute and irreversible PD-like symptoms [29, 30]. and food throughout the experiment. The studies were Since that, numerous studies have linked environ- carried out in accordance with the guidelines of the mental factors such as rural living, farming, drinking Committee on the Care and Use of Laboratory Ani- well-water and exposure to toxins and herbicides trig- mals, United States National Institutes of Health (pro- gering the neurodegeneration [1, 4, 9, 34, 43, 54, 55]. tocol # 470). In addition, the protocol complies with In view of that, one of the most promising models of the recommendations of the Universidade Federal do PD to have emerged in recent years is one in which Paraná and was approved by the University Ethics the pesticide rotenone is administered to rodents [22, Committee. 24]. The environmental exposure to rotenone and its subsequent parkinsonism is apparently underesti- mated as it is revealed by recent reports [13, 23]. Rotenone administration and experimental design Thus, epidemiological studies have been provided strong evidence of a potential role for rotenone in In the first experiment, rats were randomly distributed some cases of PD [22]. in the following groups: control (n = 10), rotenone In this model, systemic inhibition of mitochondrial 2.5 mg/kg (n = 10) and rotenone 5 mg/kg (n = 10). complex I produces selective degeneration of the ni- Rotenone (Sigma-Aldrich, Germany) or vehicle – ad- grostriatal dopamine (DA) system and reproduces key ministered in the control group – (sunflower oil pathological features of clinical PD. Indeed, rotenone 1 ml/kg) was single intraperitoneally (ip) adminis- administration affects many of the pathogenic path- tered and 1 h after the treatment, limb retraction ways including: oxidative stress, a-synuclein phospho- behavior was assessed through the paw test. Comple- rylation and aggregation and Lewy pathology, DJ-1 mentarily, 24 h after the injections, locomotor activity acidification and translocation, proteasomal dysfunc- was measured through the open-field test. tion and nigral iron accumulation [5]. Rotenone as In the second experiment, we adopted only the dos- a PD-induced model is still relatively new compared to age of 2.5 mg/kg of rotenone that demonstrated to be 6-hydroxydopamine (6-OHDA) and MPTP models; the most effective in promoting motor disability asso- both of which are still undergoing refinement in an at- ciated to negligible mortality rate. The animals were tempt to more closely mimic the motor and non-motor randomly distributed in 2 groups: control (n = 10) and features of the disease [2, 3, 21, 31, 33, 44, 47, 52]. rotenone 2.5 mg/kg (n = 10). Rotenone or vehicle – However, there is still a lack of knowledge regarding administered in the control group – (sunflower oil and connecting the motor and non-motor disruptions 1 ml/kg) was injected (ip) daily for 10 consecutive promoted by these neurotoxins, particularly rotenone. days (from 9:00 to 10:00 a.m.). Open-field tests were Considering the environmental causality associated conducted 1, 7, 14 and 21 days after the last day of ro- to PD and rotenone exposure, we investigated in the tenone 2.5 mg/kg treatment. In addition, modified present study, the motor and depressive-like altera- forced swim test was performed on 21 day (training tions, one of the most relevant and incapacitating session) and 22 day (test session) after the neurotoxin non-motor deficit associated to PD [7, 10, 42]. We or vehicle administration. Another set of animals – present herein experimental details of the rotenone control (n = 10) and rotenone 2.5 mg/kg (n = 10) – administration protocol adopted and the characteriza- underwent the same rotenone treatment, although, tion of its behavioral and neurochemical aspects. they were conducted to the sucrose preference test
1082 Pharmacological Reports, 2012, 64, 10811090 Behavioral and neurochemical alterations induced by rotenone Livia H. Morais et al.
that was performed at 14 and 21 day after rotenone. ity (defined as the lack of motion of the whole body Subsequently, on day 23 after, the animals from both consisting only of the small movements necessary to sets were decapitated followed by dissection of the keep the animal’s head above the water); climbing striatum and hippocampus structures (for neuro- (vigorous movements with forepaws in and out of the chemical purposes). water, usually directed against the wall of the tank) and swimming (was coded when large forepaw move- Paw test ments displacing the body around the cylinder, more than necessary to merely keep the head above the wa- The paw test has been shown to model both the an- ter). The water was changed after each animal to tipsychotic efficacy as well as the extrapyramidal avoid the influence of substance and temperature. side-effect liability of neuroleptic drugs [16, 17, 40]. Each rat was placed on a platform with two holes for Sucrose preference test forelimbs (40 mm) and other two for hindlimbs (50 mm). The dependent variables were the forelimb Sucrose preference is frequently used as a measure of retraction time (FRT) and the hind limb retraction anhedonia in rodents [39, 53]. The animals were time (HRT). The rationale for this test is that the stria- transferred into single housing cages with free access tum and the nucleus accumbens play differential roles to food. Each rat was provided with two bottles of wa- in regulating forelimb and hind limb rigidity [14, 15]. ter, pre-weighed, on the extreme sides of the cage dur- In view of that, it was investigated whether rotenone ing the 24 h training phase to adapt the rats to drink could increase differentially the HRT, in detriment to from two bottles. After training, one bottle was ran- FRT, indicating a more pronounced toxicity to the domly switched to contain 1% sucrose solution for striatum. 1 h, as described previously [51], and 24 h later, the bottles were reversed, and provided for 1 h, to avoid Open-field test perseveration effects. The sum of water consumption and sucrose solution consumption was defined as the The apparatus consists of a rectangular box (40 × 50 total intake. The percentage of sucrose intake was cal- × 63 cm) with a floor divided into 20 (10 × 10 cm) culated by using the following equation: rectangular units [47]. The animals were gently % sucrose preference = sucrose intake placed in the right corner of the open-field and were × 100/total intake allowed to freely explore the area for 5 min. Three motor parameters were quantfiied throughout this All the tests were carried out weekly (each Tues- test: locomotion frequency (number of crossings from day) between 9:00 and 11:00 a.m., beginning one one rectangle to the other), rearing frequency (number week prior to the neurotoxins exposure (to provide of times the animals stood on their hind paws) and baseline values) and finishing 21 days after that. After immobility time (rats kept without any movement). the sucrose preference test, all the rats received free The open-field was cleaned with a 5% water-ethanol access to food and water. solution before behavioral testing to eliminate possi- ble bias due to odors left by previous rats. Determination of DA, noradrenaline (NA), serotonin (5-HT) and metabolites concentrations Modified forced swim test The striatum and hippocampus structures were The procedure used was a modification of the method rapidly dissected and stored at –80°C until the neuro- proposed by [35, 41, 46]. The test was conducted in chemical quantification. The endogenous concentra- two sessions. First, in the training session, the rats tions of DA, 3,4-dihydroxyphenylacetic acid (DOPAC), were placed in a tank (20 × 20 × 40 cm) containing ho- movanillic acid (HVA), NA, dihydroxyphenylgly- water at a temperature of 24 ± 1°C at a depth of 15 cm col (DHPG), 5-HT and 5-hydroxyindoleacetic acid for 15 min. Twenty four hours after the training ses- (5-HIAA) were assayed by reverse phase high per- sion, the rats were subjected to the forced swim test formance liquid chomatography (HPLC) with electro- for 5 min, which was video taped for subsequent chemical detection. Briefly, the system consisted of quantification of the following parameters: immobil- a Synergi Fusion-RP C-18 reverse-phase column
Pharmacological Reports, 2012, 64, 10811090 1083 (150 × 4.6 mm i.d., 4 µm particle size) fitted with After centrifugation at 10,000 × g for 30 min at 4°C, a 4 × 3.0 mm pre-column (Security Guard Cartridges 20 µl of the supernatant was injected into the chroma- Fusion-RP); an electrochemical detector (ESA Cou- tograph. The mobile phase, used at a flow rate of lochem III Electrochemical Detector) equipped with 1 ml/min, had the following composition: 20 g citric a guard cell (ESA 5020) with the electrode set at 350 acid monohydrated (Merck), 200 mg octane-1-sulfonic mV and a dual electrode analytical cell (ESA 5011A); acid sodium salt (Merck), 40 mg ethylenediaminete- a LC-20AT pump (Shimadzu) equipped with a man- traacetic acid (EDTA) (Sigma), 900 ml HPLC-grade ual Rheodyne 7725 injector with a 20 µl loop. The water. The pH of the buffer running solution was ad- column was maintained inside in a temperature- justed to 4.0, then filtered through a 0.45 µm filter. controlled oven (25°C; Shimadzu). The cell contained Methanol (Merck) was added to give a final composi- two chambers in series: each chamber including a po- tion of 10% methanol (v/v). The neurotransmitters and rous graphite coulometric electrode, a double counter metabolites concentrations were calculated using stan- electrode and a double reference electrode. Oxidizing dard curves that were generated by determining in tripli- potentials were set at 100 mV for the first electrode cate the ratios between three different known amounts of and at 450 mV for the second electrode. The tissue the internal standard. The unit was expressed as ng/g of samples were homogenized with an ultrasonic cell wet weight. Turnovers were calculated according to the disrupter (Sonics) in 0.1 M perchloric acid containing equation: metabolite(s)/neurotransmitter. The values sodium metabisulfite 0.02% and internal standard. were expressed without units.
Fig. 1. Comparison of behavioral motor effects elicited by two doses of acute ip rotenone (2.5 and 5.0 mg/kg). The data were obtained one day after the neurotoxin administration. (A) Paw test; (B) open-field test locomotion frequency; (C) open-field test rearing frequency; (D) open- field test immobility time. Values are expressed as the mean ± SEM (n = 10/group). * p < 0.05; ** p < 0.01 and *** p < 0.001 compared to the control group. One-way ANOVA followed by Newman-Keuls test
1084 Pharmacological Reports, 2012, 64, 10811090 Behavioral and neurochemical alterations induced by rotenone Livia H. Morais et al.
Statistical analysis
Differences between groups in the first experiment were analyzed by one-way analysis of variance (ANOVA) followed by the Newman-Keuls test. In the second experiment, open-field and sucrose preference tests were analyzed by one-way analysis of variance (ANOVA) with repeated measures followed by the Newman-Keuls test. Forced swim test and the neuro- chemical data were analyzed by Student’s t-test. The results are reported as the mean ± SEM. Differences were considered statistically significant when p £ 0.05.
Results
Paw test and open-field test
As can be seen in Figure 1A, the groups treated with rotenone 2.5 and 5.0 mg/kg presented significant in- creases in the FRT (p < 0.05), (p< 0.001) compared to the control group, respectively [F (2, 24) = 4.21; p < 0.001]. Moreover, considering the HRT, both rote- none doses (2.5 and 5.0 mg/kg) inflicted similar in- creases (p < 0.05) in this parameter when compared to the control group [F (2, 24) = 1.57; p < 0.001]. In ad- dition, the open-field test indicated a robust decrease (p < 0.001) in the locomotion frequency restricted to the rotenone 5.0 mg/kg group in comparison to the control group [F (2, 24) = 16.71; p < 0.001] (Fig. 1B). Similarly, the rearing frequency showed that the rote- none 5.0 mg/kg group presented a significant de- crease (p < 0.01) when compared to the control group [F (2, 24) = 5.819; p < 0.01] (Fig. 1C). In contrast, re- garding the immobility time, rotenone administration caused a vigorous increment in this parameter for the both doses tested – 2.5 mg/kg (p < 0.01) and 5.0 mg/ kg (p < 0.001) – in comparison to the control group [F (2, 24) = 13.77; p < 0.001] (Fig. 1D). The administration of rotenone 2.5 mg/kg for 10 consecutive days produced a significant decrease in the locomotion frequency in the first two time-points analyzed: 1 (p < 0.05) and 7 (p < 0.01) days after the conclusion of the treatment, in comparison to the con- trol group. Moreover, it is worth noting that 7 days Fig. 2. Effects of the 10-day protocol of rotenone (2.5 mg/kg) admini- after rotenone was detected, a significant reduction stration on the open-field test. (A) locomotion frequency; (B) rearing (p < 0.05) in the locomotion frequency compared to frequency; (C) immobility time. * p < 0.05; ** p < 0.01 compared to the control group, # p < 0.05; ## p < 0.01 compared to the same group the same group at 1 day [F (7, 63) = 5.84; p < 0.001] at 1 day. One-way ANOVA with repeated measures followed by (Fig. 2A) was observed. Furthermore, rotenone was Newman-Keuls test
Pharmacological Reports, 2012, 64, 10811090 1085 able to decrease the rearing frequency only at 7 days 3A). In opposite, rotenone was able to produce an in- (p < 0.05), in comparison to the respective control crease (t = 2.87; df = 22; p < 0.05) in the immobility group. Also, a significant reduction (p < 0.05) of rear- time in comparison to the control group (Fig. 3B). ing was detected for the rotenone group at 7 days Complementarily, the analysis of the climbing pa- when compared to the same group at 1 day [F (7, 63) rameter demonstrated that rotenone did not exert sig- = 3.53; p < 0.01] (Fig. 2B). In opposite, rotenone nificant effects in this parameter (t = 0.98; df = 22; p = could evoke a significant increase in the immobility 0.336) (Fig. 3C). In addition, rotenone promoted time 1 (p < 0.05) and 7 days (p < 0.01) after its treat- a significant decrease (p < 0.05) in the sucrose prefer- ment in comparison to the respective time-points of ence only at 21 day, when compared to the control the control group. However, a significant increase group at the same time point [F (7, 63) = 3.60; p < (p < 0.01) in the immobility was detected at 7 day com- 0.05] (Fig. 3D). pared to 1 day [F (7, 63) = 7.90; p < 0.001] (Fig. 2C). Determination of DA, NA, 5-HT and metabolites Modified forced swim test and sucrose concentration preference test Table 1 shows the neurochemical study performed in The examination of the modified forced swim test re- the striatum and hippocampus. Rotenone produced vealed that the rotenone group exhibited a significant a significant (t = 2.54; df = 15; p < 0.05) decrease in decrease (t = 2.76; df = 22; p < 0.001) in the swim- the levels of DA within the striatum, compared to ming parameter, compared to the control group (Fig. the control group. However, it is observed the occur-
Fig. 3. Depressive-like behaviors elicited by the 10-day protocol of rotenone (2.5 mg/kg) administration. (A) swimming time; (B) immobility time; (C) climbing; (D) sucrose preference. * p < 0.05; ** p < 0.01 compared to the control group. Forced swim test data were analyzed by Stu- dents t-test. Sucrose preference was analyzed by one-way ANOVA with repeated measures followed by Newman-Keuls test
1086 Pharmacological Reports, 2012, 64, 10811090 Behavioral and neurochemical alterations induced by rotenone Livia H. Morais et al.
Tab. 1. Neurotransmitters and its turnovers quantified 23 days after the last day of rotenone 2.5 mg/kg prolonged treatment Discussion
Control Rotenone
Striatum The present study sought to develop a novel protocol DA 4732 ± 552.3 3011 ± 410.6* of rotenone ip administration mainly characterizing key points that characterize an attractive model of PD, DOPAC 1816 ± 129.0 2045 ± 206.7 such as motor and non-motor deficits, low operational HVA 728.5 ± 63.19 805.1 ± 36.40 cost and reproducibility. The neurotoxin dose was DOPAC + HVA/DA 0.5528 ± 0.02038 1.095 ± 0.1483** selected in the first experiment, considering that rote- NA 438.0 ± 38.29 426.8 ± 14.78 none 5.0 mg/kg was far more potent to cause a mas- DHPG 312.4 ± 18.27 250.7 ± 32.40 sive locomotor impairment, as indicated by the paw DHPG/NA 0.7240 ± 0.02434 0.8129 ± 0.04866 and open-field tests. However, such feature is not nec- essarily desired because this amount of disruption is 5-HT 1059 ± 103.6 844.5 ± 55.90 more related to the late onset of PD, such as observed 5-HIAA 414.3 ± 10.51 603.0 ± 49.79 with 6-OHDA when injected within the SNpc [25]. 5-HIAA/5-HT 0.7467 ± 0.08393 1.022 ± 0.05620* Besides, the paw test indicated that rotenone, in both Hippocampus doses, generated a substantial increase in the FRT and DA 137.4 ± 5.287 132.6 ± 13.29 HRT, suggesting that this pesticide induced a signifi- DOPAC 146.4 ± 6.779 143.1 ± 14. 70 cant neurotoxic effect on the striatum and nucleus accumbens. HVA 149.7 ± 5.642 150.8 ± 15.84 It is postulated that the ability of drugs to affect the DOPAC + HVA/DA 2.161 ± 0.05808 2.209 ± 0.02370 rat’s FRT is associated with the liability of the drug to NA 389.6 ± 13.47 387.2 ± 15.80 induce so-called extra-pyramidal side-effects in man. DHPG 136.0 ± 7.971 184.2 ± 19.89* Likewise, the ability of drugs to affect the rat’s HRT is DHPG/NA 0.3498 ± 0.01797 0.5165 ± 0.05815** associated with the antipsychotic efficacy of the drug. 5-HT 1034 ± 93.09 867.3 ± 54.36 Accordingly, the paw test opened the perspective that the FRT is analogue of parkinsonian side-effects, and 5-HIAA 414.3 ± 10.51 603.0 ± 49.79** that the HRT is analogue of antipsychotic effects [14, 5-HIAA/5-HT 0.9466 ± 0.03639 1.490 ± 0.1756* 16, 17]. In view of that, it is noticeable that the higher dose of rotenone was more harmful to the striatum The values are expressed as the mean ± SEM (n = 10 rats/group). * p < 0.05, ** p < 0.01; Students t-test. Turnovers were calculated than the lower dose; however, rotenone at 2.5 mg/kg according to the equation: metabolite(s)/neurotransmitter. For the elicited a moderate motor alteration that encompasses neurotransmitters and their metabolites, the unit was expressed as ng/g of wet weight, whereas for the turnovers, the values were with an early phase model of PD. expressed without units The administration of different neurotoxins such as MPTP and 6-OHDA, which classically destroy dopa- minergic neurons, remains the most widely used ani- mal models of PD. Although these models have un- rence of a significant increase (t = 3.41; df = 15; p < doubtedly contributed to a better understanding of 0.01) in the DA turnover of the rotenone group in many features of PD, most studies have focused on comparison to the control group. Still, considering the the ability of these toxins to induce nigrostriatal dam- striatum, 5-HT turnover demonstrated to be increased age and motor alterations associated with advanced (t = 2.72; df = 15; p < 0.01) in the rotenone group, phases of PD. Research is now emerging on the use of compared to the control group. The analysis of hippo- these and other substances (e.g., rotenone, paraquat, campus indicated that rotenone provoked an increase salsolinol, leukoaminochrome-o-semiquinone, iron) of the DHPG levels (t = 2.149; df = 15, p < 0.05) and [8, 49] to induce a moderate loss of nigral neurons, re- also in the NA turnover (t = 2.74; df = 15; p < 0.01), sulting in motor and non-motor deficits, thus model- compared to the control group. Besides, 5-HIAA ing the early phase of PD. Therefore, we choose to fo- showed to be increased (t = 3.4999; df = 15) and in cus on both: motor and non-motor effects of ip rote- the same way rotenone treatment produced 5-HT none (2.5 mg/kg) administration during 10 days turnover increase (t = 2.86; df = 15; p < 0.05). (second experiment). We found that this regimen of
Pharmacological Reports, 2012, 64, 10811090 1087 treatment caused an important locomotor impairment, ming, climbing and immobility behaviors in the although, confined to a restricted time lag (1 and 7 forced swimming test [12, 46, 47]. Indeed, the impair- days). ments in the swimming and immobility reflect the in- The apparent improvement on the open-field pa- crement in the 5-HT and NA turnovers, respectively. rameters (detected on 14 and 21 day) could be due to Besides, the reduction in the striatal DA content could the habituation effect observed in the control group, also be correlated to the increased immobility time. since the rats were re-exposed to the test. Neverthe- The anhedonic state was confined at 21 days after ro- less, the rotenone group was also equally re-exposed tenone, possibly due to the characteristic of the lesion and did not shown additional deficits compared to the which is less abrupt than that promoted by MPTP or previous time-points. Moreover, the neurochemical 6-OHDA [8, 47, 49, 52]. This result is in accordance findings indicated that rotenone affected DA levels with previous data that compared intranigral injec- and increased its turnover within the striatum. This in- tions of several neurotoxins (including rotenone) and dicates the existence of a compensatory neurochemi- detected a prominent anhedonic state at 21 day after cal effect that possibly explains the motor reparation, the exposure [26, 47]. probably as a result of plasticity events that balanced Previous studies have shown that rotenone, when the neuronal loss. Such adaptation is a well docu- administered systemically [18, 50] or into the SNpc mented characteristic of the early phase models of PD [47, 48, 56] or striatum [38] can cause nigrostriatal [6, 11, 19, 31, 32, 44, 45]. degeneration. However, to our knowledge, this is the Depression is a frequent non-motor feature of PD first report that verified the motor and depressive-like and exerts a signficant impact on patient’s quality of alterations promoted by this novel, low cost and eas- life and may complicate treatment. The etiology of ily reproducible protocol of rotenone ip exposure. depression in PD is complex and may result from In conclusion, our data indicate that rotenone, ac- changed monoaminergic brain chemistry that is re- cording to our proposed protocol of administration, lated with the central dopaminergic deficiency associ- was able to produce motor alterations followed by ated with PD motor symptoms [36, 37]. Thus, obser- depressive-like behaviors accompanied by an increase vations of pathological features in the SNpc of de- in the hippocampal 5-HT and NA turnovers. These pressed PD patients bolsters the notion that the data suggest an important participation of these neu- nigrostriatal circuit is implicated in the depression of rotransmitters in anhedonia and behavioral despair PD [20]. Even with such importance there is a lack of induced by our protocol of rotenone administration. studies concerning animal models of PD and Besides, rotenone also impacted on striatal DA neuro- depressive-like behaviors. A recent study from our transmission, supporting the notion of its relevance as laboratory reported that the intranigral injections of an interesting model of PD. In fact, more studies are MPTP, 6-OHDA and rotenone, were able to produce needed to examine the participation of these neuro- depressive-like behaviors accompanied primarily by transmitters in the depressive-like behaviors found in hippocampal 5-HT reductions [47]. Whereas our cur- the present study. rent results demonstrated that the striatal (p = 0.197) and hippocampal (p = 0.132) contents of 5-HT were Acknowledgment: not directly affected by rotenone, it is noticeable that This work was supported by grants from CNPq, CAPES and REUNI 5-HT turnovers were increased in both structures. that had no further role in the study design; in the collection, Hence, it is suggested the occurrence of a compensa- analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication. RA and MABFV tory mechanism elicited by rotenone that impacts on are recipients of CNPq fellowship. MMSL is recipient of Fundação 5-HT neurotransmission. Similarly, NA turnover was Araucária - Governo do Estado do Paraná fellowship. found increased within the hippocampus caused by the rotenone treatment. The aforementioned results are closely related to the depressive-like behaviors alterations assessed References: through the forced swim test and the sucrose prefer- ence test. In this line, our results are in accordance 1. Abbott RD, Ross GW, White LR, Sanderson WT, with others that associate serotoninergic, noradrener- Burchfiel CM, Kashon M, Sharp DS et al.: Environmen- gic and dopaminergic neurotransmissions with swim- tal, life-style, and physical precursors of clinical Parkin-
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