Assessment of neurobehavioral parameters in zebrafish larvae in a rotenone-induced Parkinson’s-like disease model

Assessment of neurobehavioral parameters in zebrafish larvae in a rotenone-induced Parkinson’s-like disease model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Assessment of neurobehavioral parameters in zebrafish larvae in a rotenone-induced Parkinson’s-like disease model Renatta Priscilla Ferreira Silva, Aline Amanda da Silva, Jadson Freitas da Silva, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8270673/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The development of animal models that mimic the behavioral and symptomatic aspects of Parkinson’s Disease (PD) is essential for advancing new therapies. This study aimed to assessment of neurobehavioral parameters in zebrafish larvae in a rotenone-induced Parkinson’s-like disease model. Zebrafish larvae at 72 hours post-fertilization, when dopaminergic projections are fully developed, were exposed to rotenone (RT: 5.00–15.00 µg/L), a compound known to selectively damage dopaminergic neurons, thus inducing PD-like symptoms. Levodopa treatment was also evaluated. Survival and neurobehavioral endpoints were evaluated using a series of tests: thigmotaxis, touch sensitivity, optomotor response (OMR), and bouncing balls. Larval survival significantly decreased at RT concentrations above 12.00 µg/L. Thigmotaxis and touch sensitivity were impaired at concentrations greater than 11.00 µg/L. In the OMR test, all measured parameters showed significant changes from 10.00 µg/L onward. Similarly, RT at concentrations above 10.00 µg/L reduced permanence in the non-stimulated area, while the clustering response of larvae was notably affected starting at 11.00 µg/L. These findings demonstrate that RT exposure induces specific and measurable neurobehavioral alterations in zebrafish larvae. The neurobehavioral parameters in this study is both detailed and reproducible, providing a valuable tool for future research and the screening of potential therapeutic compounds targeting Parkinson’s Disease. Animal Behavior Neurobiology of Disease Toxicology Neurodegenerative disease Danio rerio animal behavior animal model Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Parkinson's disease (PD) is a progressive neurodegenerative condition that results in the loss of dopaminergic neurons in the substantia nigra compacta of the midbrain. In addition to classic motor symptoms, such as resting tremors, bradykinesia, muscle rigidity, and postural instability, PD may also present with non-motor symptoms, including cognitive impairment, sleep disturbances, hyposmia, depression, and anxiety [ 1 , 2 ]. The disease predominantly affects men over the age of 65 years, with an estimated prevalence of 1–2% [ 3 ]. Currently, there is no definitive treatment for PD, and existing treatments are only aimed at alleviating motor symptoms, with no ability to regress the condition and often causing long-term adverse effects such as levodopa [ 4 ]. The complexity of pathological mechanisms hinders the development of effective therapies [ 5 ]. Therefore, animal models that reproduce PD-like symptoms, known as PD-like models, are essential for the study of the pathophysiology of the disease and the development of new therapeutic approaches [ 6 – 8 ]. The zebrafish ( Danio rerio ) has stood out as an animal model for studies of several diseases due to its advantages, such as small size, easy to maintain, low cost, high reproduction rate, and external fertilization. Its rapid maturation and short life allow for large-scale experiments, ensuring statistically reliable results. In addition, zebrafish share genetic, physiological, anatomical, and behavioral homology with humans, making them an effective tool for studying human diseases [ 9 , 10 ]. In the neurological context, the nervous system of zebrafish has anatomical similarities with humans, including the division into the forebrain, middle, and hindbrain, with structures such as the diencephalon, telencephalon, cerebellum, and spinal cord. Studies suggest that the zebrafish ventral diencephalon is functionally homologous to the human substantia nigra, which is involved in the pathogenesis of PD. The dopaminergic projections of zebrafish are fully formed between 72- and 96-hours post-fertilization [ 11 , 12 ], and it can be considered a good model for studying PD at this level of development. Dopaminergic neurons are detected early in zebrafish embryos, enabling the study of diseases such as PD [ 5 , 11 – 14 ]. The zebrafish has been used as a PD-like model using neurotoxins to generate relevant neurochemical and neurobehavioral phenotypes. Substances such as MPTP, 6-OHDA, paraquat, and rotenone are commonly used to induce PD-like symptoms in zebrafish [ 6 , 12 , 15 ]. These studies highlight the potential of zebrafish as a model to understand the pathophysiology of PD and develop new therapeutic approaches, contributing significantly to advances in the field of neuroscience. Rotenone (RT), a naturally derived and lipophilic pesticide, is widely used as a PD-inducing agent due to its ability to cross the blood-brain barrier and cell membranes, affecting various organelles, including mitochondria. Its action mainly inhibits mitochondrial complex I, recapitulating clinical and pathological characteristics of PD [ 8 , 16 , 17 ]. The use of zebrafish as a model for RT-induced PD-like by immersion exposure has made it possible to reduce invasive procedures, such as intracerebral injections of 6-OHDA used for induction in adult zebrafish [ 18 , 19 ]. In addition, the evaluation of behavioral and locomotor parameters in zebrafish exposed to RT allows the presence of PD-like symptoms to be verified, as established of the related phenotypes of the disease [ 6 , 7 ]. The zebrafish exhibit complex repertories of behaviors, including social behavior, anxiety, learning, and memory, as well as defense behavior, similar to that of humans. Adults and zebrafish larvae exhibit behaviors relevant to neuropharmacology, making them useful for modeling neurological and psychiatric diseases [ 10 ]. Behavioral tests performed with zebrafish can be important tools for the study of behavioral disorders, providing relevant information about normal and pathological brain function [ 7 , 10 , 12 , 20 ]. Additionally, due to the toxicity of the substance used in the present study to induce behavioral phenotypes pertinent to PD, it is worth adding that the threshold between RT concentrations capable of causing physiological effects and ineffective concentrations is quite narrow, as observed by Melo, Oliveira [ 21 ]. Therefore, it is essential to use different RT concentrations with small variations between them, in order to induce observable changes without causing the death of the animals. In addition, the protocol for exposure to PD-like inducing substances in zebrafish [ 12 ] suggested that the lack of standardization at the time of onset of exposure to these substances produced "conflicting" results. It is recommended that the time of onset of exposure be selected, thus avoiding inhibition of zebrafish neurogenesis. Consequently, it is necessary to study the best concentration of RT that can be used to induce PD-like symptoms for the screening of molecules for new therapeutic approaches and which neurobehavioral tests can be used as endpoints to evaluate the effect of these molecules. In this framework, this study aimed to assessment of neurobehavioral parameters in zebrafish larvae in a rotenone-induced Parkinson’s-like disease model. Because, at this level of development, RT can cause PD-like symptoms in zebrafish embryos. We also used levodopa treatment as a standard. Since, the ventral diencephalon of zebrafish demonstrates functional homology with the human substantia nigra, a site associated with the development of PD pathogenesis [ 13 ]. Thus, this exposure approach was adopted to ensure advanced dopaminergic development and to enable the assessment of behavioral changes resulting from RT-induced dopaminergic neurodegeneration. 2. Material and methods 2.1. Maintenance and reproduction of animals The experiments were carried out at the Laboratório de Ecofisiologia e Comportamento Animal (LECA) of the Universidade Federal Rural de Pernambuco (UFRPE), a vivarium registered in the CIUCA Platform of the Brazilian National Council for the Control of Animal Experimentation (CONCEA). The methods were previously approved by the Ethics Committee on the Use of Animals (CEUA) (License No. 3581030221) of UFRPE. Adult animals of the species Danio rerio (WT strain, nine months old) were reared and housed in the vivarium. These animals were maintained in 80-liter aerated aquariums with continuous maintenance of abiotic conditions such as dissolved oxygen, nitrite, nitrate, and ammonia. The photoperiod was controlled at 10/14 h (light/dark), the temperature at 26 ± 1°C, and the pH at 7.0 ± 0.5 [ 22 , 23 ]. They were fed three times a day, twice with extruded commercial feed (≈ 30% crude protein) and once with brine shrimp saline. To obtain the eggs, adult animals were separated according to sex in breeding tanks for zebrafish (Zebclean, Alesco), using males and females in an 8:4 ratio [ 23 ]. After spawning, viable eggs were collected and evaluated using an optical light microscope (with LED lamp) at 1-hour post-fertilization (hpf) (Cadena et al., 2020a). Only eggs whose spawning was more than 90% viable were used [ 22 ]. The eggs were randomly placed in sterile polystyrene chambers [ 22 ] with a capacity of 80 mL, kept in an incubator with the same physicochemical parameters described above and considered optimal for the species. 2.2 Preparation of the rotenone solution, exposure, and survival of the animals A 0.02 mg/mL aqueous solution of rotenone (RT) (CAS# 83-79-4, purity ≥ 95%, R8875, SIGMA, St. Louis MO, USA) in 0.1% (v/v) dimethyl sulfoxide (DMSO) was prepared. This concentrated solution was used for dilution in 80 mL of water in the polystyrene chambers. Final nominal concentrations of 5.00 (RT5), 10.00 (RT10), 11.00 (RT11), 11.25 (RT11.25), 12.00 (RT12), and 15.00 µg/L (RT15) were obtained, where the larvae, from 72 hpf, were exposed to RT forming 7 experimental groups containing 15 eggs per chamber at each replication. Levodopa (LD) (CAS# 59-92-7) and benserazide hydrochloride (CAS# 10035-04-8) were purchased from commercial suppliers. 100 mg LD and 25 mg CB tablets were macerated to obtain nominal concentrations of 0.20 mg/L LD (1000 mM) and 0.05 mg/L CB (170 mM), respectively, and then diluted to 750.0 mM and 127.5 mM with 0.1% (v/v) DMSO aqueous solution. We used 2 more experimental groups to evaluate the toxicity of LD (LD 750.0 group) and if LD protected against RT toxic effects (LD 750.0 + RT 11.25).The final concentration of DMSO was always less than 0.001% (v/v) after dilution in water, which is 500 times lower than that reported in the literature, which can affect the behavior of animals [ 24 ]. RT exposure occurred from 72 hpf to 144 hpf with daily renewal of solutions in semi-static conditions [ 22 ]. Animal mortality was verified daily by the absence of movement and heartbeat [ 22 ]. The experimental design is shown in Fig. 1 . 2.3 Neurobehavioral tests At 144 hpf, visibly healthy larvae, without the presence of morphological defects [ 20 ], were transferred from polystyrene pots to 48-well plates (one larva per well) to undergo neurobehavioral tests in a controlled low-light environment. These tests were always performed between 10:00 and 12:00 h (AM) to avoid influences on the locomotor activity and visual sensitivity of the animals [ 25 ]. Prior to each test, the larvae were acclimatized for 15 minutes to avoid behavioral changes from handling and transport [ 26 ]. As these are non-invasive methods, sequential neurobehavioral tests based on Gomes et al. [ 27 ] were performed: Thigmotaxis, Touch Sensitivity, Optomotor response, and Bouncing Balls (Fig. 1 ). The thigmotaxis test (TH test) was used to assess anxiety-like behaviors, as evidenced by previous studies [ 10 , 20 ]. The 48-well plates containing the larvae were arranged horizontally on a monitor (LCD, Dell E2211H), and the response was recorded to assess their preference for the edges of the wells. During the TH test, larvae were acclimatized for 15 minutes, and it was recorded whether the larvae showed a tendency to stay close to or away from the walls of the plates [ 20 ]. During this test, the scan sampling method was used, with a dichotomous response [ 28 ] being "yes" for positive thigmotaxis when the larva was close to the wall and "no" for affected when the larva was away from the wall. Next, the touch sensitivity test (TS test) was performed, which was used to assess the larvae's response to mechanical stimuli [ 20 ]. The scan sampling method [ 28 ] was also used, with a dichotomous response. The stimuli were applied to the tail or head region of the larvae, and the positive response was recorded if the larvae demonstrated an escape behavior. Pictures of TH and TS tests were recorded using a camera (Canon EOS 6D DSLR; lens: Canon EF 75-300mm f/4-5.6) with a top view and a distance of 150 cm from the plates arranged on the monitor, while the monitor displayed a white background. Optomotor response (OMR) was used to assess the visual impairments of zebrafish larvae [ 20 , 27 , 29 ]. For this test, a new methodology was developed where the larvae of each group were relocated in an acrylic rectangular aquarium (dimensions: 15 x 5 x 5 cm, length, width, and height, respectively) with a wall positioned 3 cm from one end. The wall prevented the larvae from moving throughout the aquarium ahead of time. The aquarium with the larvae was positioned horizontally on a Full HD monitor (Dell E2211Hc), and then the larvae were exposed to a video with a total duration of 75 s, with black and white lines moving to the right and left [ 29 ]. The video simulates a stream of water to count the number of larvae that followed the direction of the stimulus [ 29 ]. The video consisted of 5 s of white (acclimatization) for the initial time (IT) and 30 s of alternating lines in the right direction. Simultaneously with the beginning of the animation, the wall of the aquarium was removed, allowing the larvae to swim freely so that they could follow the direction of the stimulus. The response was recorded by 200 seconds video recording by a camera (Canon EOS 6D DSLR; lens: Canon EF 75–300 mm f/4-5.6) with a top view and a distance of 150 cm from the plate placed on the monitor. From this test, data such as the percentage of grouped animals that swam the aquarium at each interval of 4 cm, average speed (cm/s) of zebrafish larvae, and percentage of animals that swam until the end of the aquarium (cm) were obtained. Finally, the bouncing balls test was used to assess spatial interaction responses, social cohesion, cognitive ability, and larval escape from visual stimuli [ 27 , 30 , 31 ]. The larvae were transferred to 6-well plates, each well containing 5 animals from each group, totaling 3 wells per group. The plates were placed on the monitor, and after 1.53 minutes of acclimatization on a dark screen, an animation with visual stimuli created in Microsoft PowerPoint (Office 365) was shown. The animation contained red balls (diameter 1.35 cm) that moved from left to right in a straight line with a trajectory of 2 cm in the stimulus area (lower half of the well) for 5 minutes. The number of larvae in the same quadrant of the well-provided data regarding the level of grouping and social cohesion among them. The response was recorded by 7 minutes video recording by the same camera described above. An analysis was performed every 30 s [ 27 , 30 , 31 ]. 2.4. Statistical analysis Normality was carried out using the Komogorov Smirnov test (p < 0.05). The data from the TH, TS, OMR, and Bouncing Balls tests and the data regarding the survival of the animals were analyzed by one-way ANOVA parametric test. When the difference was significant, the means were compared using Tukey's test with p < 0.05. For statistical analyses, the Origin Pro Academic 2015 software (Origin Lab. Northampton, MA, USA) was used. 3. Results The results of the survival of the animals are shown in Fig. 2 . It was observed that the survival percentage of zebrafish larvae was significantly reduced in the groups exposed to RT concentrations > 12.00 µg/L. However, LD did not induce toxicity and prevented RT toxic effects (Fig. 2 ). Regarding the neurobehavioral assessments, the TH test was used to evaluate behaviors analogous to anxiety and is presented in Fig. 3 A. The DMSO group had approximately 90% of the larvae near the edges of the wells, which is the expected behavior for this group. RT affected the thigmotaxis in the RT11 to RT15 groups, indicating that in concentrations > 11.00 µg/L the toxic effect was observed. The TS test was used to verify the responsiveness to mechanical stimuli and significant reductions were observed in the RT11 to RT15 groups indicating that the escape behavior was affected (Fig. 3 B). Again, contractions > 11.00 µg/L induced toxic effects. In addition, LD was not toxic as expected and reduced RT toxicity (Fig. 3 A and 3 B). The results of the OMR test are shown in Fig. 4 . Significant changes were observed in all neurobehavioral parameters analyzed in this test when the animals were exposed to RT. In Fig. 4 A, it was found that RT significantly reduced the total distance traveled by the larvae in almost all groups, except in the group exposed to the lowest concentration of RT used (5 µg/L). It was observed that 71% of the animals swam the entire course of the aquarium in the control group, while 27% of the animals did so in the RT15 group. In Fig. 4 B, it was observed in relation to the mean velocity of the animals that the concentration of 5 µg/L of RT was also not able to cause significant changes in this parameter, only the concentrations > 5 µg/L. The data related to the ability of the larvae to reach the finish line in the OMR test are presented in Fig. 4 C, where it was observed that this parameter was affected in all groups exposed to RT. Finally, we observed the concentration-dependent effect on all parameters evaluated in the OMR test (Fig. 4 ), i.e., as the RT concentration increased, the larvae's ability to respond to the OMR test decreased. This was probably because RT at higher concentrations induced greater toxicity in zebrafish larvae. Regarding LD effects on OMR studied parameters, LD did not affect and protected against RT toxic effects in parameters of the total distance traveled (Fig. 4 A) and percentage of arrival of animals (Fig. 4 C). However, LD reduced average speed of animals (Fig. 4 B). The results of the bouncing balls test showed that RT affected the percentage in non-stimuli areas in concentrations > 10.00 µg/L (Fig. 5 A). The same was observed in relation to the responsiveness of larval clustering at concentrations ≥ 11.00 µg/L (Fig. 5 B). Regarding LD effects, the percentage of animals in non-stimuli areas decreased in both LD groups (Fig. 5 A) and exposure to LD 750.0 mM increased the percentage of grouping of animals. Interestingly, LD protected against RT toxic effects (Fig. 5 B). Thus, our findings using an embryo-larval model with exposure from 72 hpf showed us concentration-dependent results, i.e., as the RT concentration increased, it directly affected the neurobehavioral response. Our results also corroborated with the work of Andrade et al. [ 33 ] where the concentration of 11.25 µg/L proved to be the best to standardize the PD-Like neurobehavioral model for screening pharmacologically active molecules. Finally, as expected LD protected against RT toxic effects in most behavioral data studied which validated our study. 4. Discussion The zebrafish has been employed as a PD-Like model, playing a significant role in the study of neurodegenerative diseases, as it has specific brain regions homologous to those found in humans [ 32 ], showing that dopaminergic projections are fully formed between 72 and 96 hpf [ 11 , 12 ]. Our study standardized a sequence of neurobehavioral tests in a PD-like zebrafish model that can be used for the screening of new drugs using RT as an inducing molecule. Our findings showed a significant reduction in the survival percentage of zebrafish larvae in the groups exposed to the highest RT concentrations. This is in agreement with the study by Andrade et al. [ 33 ], who observed a high mortality rate in the zebrafish embryo-larval toxicity model at RT highest concentrations (15 and 20 µg/L). The same authors highlighted that conducting tests on animals exposed to these higher concentrations may be difficult due to the significant reduction in survival. In addition, in the embryo-larval model used by Andrade et al. [ 33 ], mortality was even higher than in the larval model used in our study, since they started exposing the animals with 2 hpf. This makes the larval model more suitable for PD-related neurobehavioral studies. The TH test was used to evaluate behaviors analogous to anxiety, which showed that RT affected TH in the groups with higher concentrations (> 11.00 µg/L). Andrade et al. [ 33 ] also used the TH test to assess anxiety-like behaviors in an embryo-larval zebrafish model exposed to RT. Exposure of zebrafish embryos was initiated from 2 hpf at concentrations of 5, 10, 15, and 20 µg/L. In this study, changes in larval TH test were observed at the highest RT concentrations (15 and 20 µg/L). The authors concluded that these toxic effects in the TH test may indicate the ability of RT to modify neuronal activity, resulting in behavioral changes similar to anxiety, corroborating the findings of our study. While studies investigating anxiety-like behaviors in zebrafish larvae exposed to neurotoxins for PD-Like are still limited, some studies have examined this type of behavior in adult fish. Wang, Liu [ 6 ] evaluated anxiety-like behavior in an adult PD-like zebrafish model exposed to RT, using the light/dark preference test. In this study, a concentration of 2 µg/L was able to affect the behavior of the animals. The same authors suggested that these findings were mainly related to dopamine depletion in the brain caused by the action of RT, which inhibits mitochondrial complex I of dopaminergic neurons, resulting in their death. In addition, just like adult fish, zebrafish larvae also have a developed dopaminergic system, with dopaminergic projections fully formed between 72 and 96 hpf [ 11 , 12 ]. Therefore, it is suggested that RT may have acted similarly in our study, causing dopaminergic loss and consequent changes in the anxiety-like behavior of zebrafish larvae. In the TS test, significant reductions in escape behavior were observed as a response to touch in the groups exposed to the highest RT concentrations. Andrade et al. [ 33 ] also used zebrafish exposed to RT to investigate responses to mechanical stimuli through TS in an embryo-larval toxicity model. They observed a significant reduction of TS in larvae exposed to the highest concentration of RT (20 µg/L), which may be associated with the neurotoxic action of RT. In a study carried out by Lam, Korzh [ 14 ], the neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) was administered to induce neurodegeneration in zebrafish embryos at 24 hpf, at which time dopaminergic neurons are first detected in the diencephalon. A test similar to ours was used to verify responses to tactile stimuli at 72 hpf. The authors observed that the larvae of the control group showed strong reflexes by trunk movement, while the larvae treated with MPTP produced weak and brief tactile responses. This suggests that exposure to MPTP resulted in deficits in the larval' swimming response. Therefore, in addition to sensory impairment, the inability or difficulty of the larvae to respond to the TS test may be related to locomotion defects induced by the neurotoxin used to induce PD-like symptoms. As RT also induces dopaminergic neurodegeneration and consequent reduction of dopamine levels, it is possible that it affected the ability of the larvae to present motor responses when subjected to the TS test, in the same way as observed in a PD-like model exposed to MPTP. This can be explained by the intrinsic role of the neurotransmitter dopamine in motor control, and insufficient dopamine production results in locomotor deficits, which may also have been observed in the larvae in our study. Thus, the results of our work confirm that RT can affect the responsiveness to the TS test of animals. We have developed a new OMR method specific to detect PD-like symptoms in our larval model. This was necessary because the OMR test [ 20 ] was previously carried out but did not show efficient results, since the methodology adopted only provided us with data referring only to the high and low alignment of the animals. However, we adopted a new approach to this test, by a study model that provides us with information regarding not only the alignment of the animals but also the distance traveled, speed, and percentage of animals that traveled the entire distance of the swimming board. This provides a more robust and information-rich model regarding the motor effects caused by RT in our animal model. Thus, we performed the OMR test to investigate possible visual and also motor changes in zebrafish larvae exposed to RT. This test is based on the fish's response to swimming in the direction of a visual stimulus, usually a stripe pattern [ 34 ]. Evaluating the OMR in PD-like animal models is relevant because, in addition to motor deficits, patients affected by the PD disease may present visual impairments [ 35 ]. OMR tests were poorly studied in PD-like animal models. Benvenutti, Marcon [ 36 ] analyzed OMR in zebrafish larvae exposed to 6-hydroxydopamine (6-OHDA), a compound that causes the death of dopaminergic neurons, as well as RT. The exposure of the animals occurred from 72 hpf at a concentration of 250 µM of 6-OHDA. The authors observed that larvae exposed to 6-OHDA showed a reduction in time spent in the stimulus zone (the region where the stripe pattern moved), indicating optomotor damage. In addition, as in our study, Benvenutti, Marcon [ 36 ] evaluated locomotor parameters such as mean speed and total distance traveled, however, unlike our methodology, these parameters were evaluated in isolation using the OMR test. It was found that 6-OHDA was able to cause a decrease of approximately 80% in the total distance traveled and average velocity of the larvae. Regarding these results, the authors suggested, based on other studies, that because 6-OHDA causes the death of dopaminergic neurons of important pathways related to movement, exposure to this compound resulted in the motor deficits found. This may also be applicable to our study since RT acts similarly to 6-OHDA causing motor deficits due to dopaminergic neuronal death. However, although a direct comparison is not possible due to the difference in strains between the larvae (WT in our study and AB in the study by Benvenutti, Marcon [ 36 ]), the results of the other tests in our study we performed involving the locomotor activity of animals (touch sensitivity and bouncing balls) corroborated with results of the OMR test. Also, in addition to motor deficits, RT is capable of causing visual changes, as was previously discussed in the bouncing balls test. Therefore, the changes in the OMR test may not have been exclusively motor, as animals needed to visualize the stimulus in order to present the motor response. Hence, it is necessary to consider possible alterations in visual perception and to confirm these alterations, additional endpoints are needed in which the visual part is isolated from the motor part. RT influenced the clustering responsiveness of the larvae at higher concentrations in the bouncing balls test. The literature on the effects of RT on zebrafish social behavior is still scarce. However, a recent study demonstrated that RT alters this behavior in adult zebrafish [ 37 ]. In addition, no studies were found in the literature evaluating the ability to cluster as a response to aversive stimuli in zebrafish larvae exposed to neurotoxins used to induce PD-like symptoms. Therefore, it is necessary to consider that social behavior involves several aspects, including cognitive capacity, perception of environmental factors, and the manifestation of appropriate spatial and social interaction responses by larvae. In addition, the cognitive response is related to visual and locomotor capacity, which includes the escape movement and the approximation between conspecifics. In the study by Kalyn, Hua [ 12 ], zebrafish larvae exposed to RT from 72 hpf had locomotion defects, resulting in a significant reduction in the total distance traveled during swimming. There were also decreases in gene expression of th1 (tyrosine hydroxylase), an enzyme involved in dopamine synthesis, and a significant loss of dopaminergic neurons in the ventral diencephalon of the larvae. These results indicated that RT could cause locomotor deficits and neurochemical changes, which may have an impact on the behavior of the larvae in our bouncing balls test. In addition, the bouncing balls test involves visual stimuli and interaction between conspecifics. Considering that zebrafish larvae have dopaminergic neurons in the retina, exposure to RT can lead to visual changes due to the degeneration of these neurons. Studies in other models, such as rats and mice, have shown retinal damage and visual changes under the administration of RT and MPTP, related to oxidative stress and dopaminergic deficiency. Therefore, in addition to the locomotor changes observed in our study, the inability of the larvae to respond to the bouncing balls test may be related to visual changes resulting from the degeneration of dopaminergic neurons of the retina by the action of RT. These results highlighted the complexity of RT effects on the behavior of zebrafish larvae and the importance of considering multiple aspects in the interpretation of experimental results [ 38 , 39 ]. Given the above, our findings raise the hypothesis that RT affected the visual capacity of the larvae, leading them to present difficulty in seeing the stimuli, or even difficulty in moving to the conspecifics. This leaves room for future studies. As observed throughout this study, the RT concentrations that caused behavioral effects in zebrafish larvae ranged between 11.00 and 15.00 µg/L. However, concentrations of 12.00 and 15.00 µg/L resulted in a significant reduction in animal survival. Therefore, considering a higher percentage of survival and the presence of behavioral defects in all tests, the most suitable concentrations to observe the behavioral effects in this model were 11.00 and 11.25 µg/L. LD is the most efficacious drug in the therapeutic of PD [ 4 ] and because of this, we chose as the gold standard of our tests. LD is associated with benserazide hydrochloride due to the last one preventing the decarboxylation of LD increasing the therapeutic effect [ 40 ]. We maintained this association in our study because this therapeutic approach is used in humans. LD protected against RT toxic effects in most behavioral data studied. However, reduced the average speed of animals was observed in our study. This result is in agreement of literature because LD may be neurotoxic in neurons that are related to the locomotor activity in zebrafish larvae [ 15 ]. Finally, the literature presents several classical neurotoxins used in zebrafish to mimic the pathophysiological aspects of PD, such as MPTP/MPP⁺, 6-hydroxydopamine (6-OHDA), and paraquat, each reproducing specific mechanisms related to dopaminergic degeneration. However, most studies employ these neurotoxins in zebrafish via injection methods [ 41 , 42 ], not immersion, as in our model. The injection route is an invasive technique, typically performed by intraperitoneal administration, which does not align with the principles of the 3Rs, especially regarding Refinement [ 43 ], thereby compromising animal welfare. In a study by Kalyn et al. [ 12 ], neurotoxins mentioned above were used at different concentrations, including RT. However, in the MPTP model, at the lowest concentration (0.25 mM), teratogenic effects were observed, resulting in increased mortality rate between 4 and 5 dpf, which interfered in neurobehavioral tests carried out at 7 dpf in this study. In the paraquat model, although this compound is associated with an increased risk of PD in humans, Kalyn et al. [ 12 ] did not observe significant changes in larval locomotion. In addition, there was only a slight reduction in the expression of dopaminergic genes, and thus, the observed toxicity level was insufficient to produce a measurable locomotor activity. The same authors described that 6-OHDA has a limited ability to cross the blood-brain barrier, resulting in a lack of specificity in targeting only dopaminergic neurons. In conclusion, exposure to RT appeared promising for the assessment of behaviors associated with locomotor response, as shown in our study. Conclusion With our embryo-larval model using zebrafish larvae with 72 hpf, it was possible to evaluate behavioral parameters in larvae exposed to RT, evidencing their suitability to analyze Parkinson's-like symptoms. It was found that the onset and duration of exposure to RT, especially at its high concentrations, resulted in consistent behavioral effects, mimicking Parkinsonian phenotypes in all tests performed. Considering the survival of the animals and the behavioral effects observed in the tests, the most appropriate concentrations to investigate the behavioral effects were ≈ 11.25 µg/L of RT. In addition, LD treatment was useful for a standard in our behavioral tests. Therefore, our behavioral tests proved to be appropriate for application in screening potential compounds that may contribute to the development of new therapies for PD, aiming to improve the quality of life of affected individuals. Declarations Funding: This work was supported by CAPES (Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for postgraduate scholarships, CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for a productivity scholarship for Pabyton Cadena (302399/2023-2) and FACEPE (Fundação de Amparo a Ciência e Tecnologia do Estado de Pernambuco) for postgraduate scholarships for Renatta Silva (IBPG-0837-2.12/22) and junior postdoctoral scholarships for Samara Gomes (BFP-0043-2.10/24). Acknowledgments: The authors thank members of the Laboratório de Ecofisiologia e Comportamento Animal – LECA (Brazil). Conflicts of Interest: The authors declare no conflict of interest. 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Neurotox Res 32:496–508 Cadena PG, Cadena MRS, Sarmah S, Marrs JA (2020) Folic acid reduces the ethanol-induced morphological and behavioral defects in embryonic and larval zebrafish (Danio rerio) as a model for fetal alcohol spectrum disorder (FASD). Reprod Toxicol 96:249–257 Melo KM, Oliveira R, Grisolia CK, Domingues I, Pieczarka JC, de Souza Filho J et al (2015) Short-term exposure to low doses of rotenone induces developmental, biochemical, behavioral, and histological changes in fish. Environ Sci Pollut Res Int 22:13926–13938 OECD236 (2013) Education at a Glance 2013: OECD Indicators. OECD Publishing Westerfield M (2000) The zebrafish book: A guide for the laboratory use of zebrafish (Danio rerio), 4th edn. University of Oregon, Eugene, OR Hedge JM, Hunter DL, Sanders E, Jarema KA, Olin JK, Britton KN et al (2023) Influence of Methylene Blue or Dimethyl Sulfoxide on Larval Zebrafish Development and Behavior. 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Chemosphere 214:330–340 Brastrom LK, Scott CA, Dawson DV, Slusarski DC (2019) A High-Throughput Assay for Congenital and Age-Related Eye Diseases in Zebrafish. Biomedicines. 7 Nery LR, Eltz NS, Hackman C, Fonseca R, Altenhofen S, Guerra HN et al (2014) Brain intraventricular injection of amyloid-beta in zebrafish embryo impairs cognition and increases tau phosphorylation, effects reversed by lithium. PLoS ONE 9:e105862 Bridi D, Altenhofen S, Gonzalez JB, Reolon GK, Bonan CD, Glyphosate (2017) Roundup((R)) alter morphology and behavior in zebrafish. Toxicology 392:32–39 Manasa K, Chitra V, Tamilanban T (2020) Teleost model as an alternative in parkinson's disease. Neurol India 68:979–984 Andrade ALC et al (2025) Rotenone-treated in the Early Life Stages of Zebrafish (Danio rerio) as a Model for Parkinson-like Motor and Non-motor Symptoms and Anxiety-like Behaviour. Archives Curr Res Int v 25, n. 7, p. 676–691 LeFauve MK, Rowe CJ, Crowley-Perry M, Wiegand JL, Shapiro AG (2021) Connaughton, V. P. Using a variant of the optomotor response as a visual defect detection assay in zebrafish. J Biol Methods 8:e144 Nieto-Escamez F, Obrero-Gaitán E, Cortés-Pérez I (2023) Visual Dysfunction in Parkinson’s Disease. Brain Sci 13:1173 Benvenutti R, Marcon M, Reis CG, Nery LR, Miguel C, Herrmann AP et al (2018) N-acetylcysteine protects against motor, optomotor and morphological deficits induced by 6-OHDA in zebrafish larvae. PeerJ 6:e4957 Ilie O-D, Duta R, Balmus I-M, Savuca A, Petrovici A, Nita I-B et al (2022) Assessing the Neurotoxicity of a Sub-Optimal Dose of Rotenone in Zebrafish (Danio rerio) and the Possible Neuroactive Potential of Valproic Acid, Combination of Levodopa and Carbidopa, and Lactic Acid Bacteria Strains. Antioxidants 11:2040 Biehlmaier O, Alam M, Schmidt WJ (2007) A rat model of Parkinsonism shows depletion of dopamine in the retina. Neurochem Int 50:189–195 Sasaoka M, Ota T, Kageyama M (2020) Rotenone-induced inner retinal degeneration via presynaptic activation of voltage-dependent sodium and L-type calcium channels in rats. Sci Rep. 10 Hauser RA, Levodopa (2009) Past, present, and future. European Neurology, v. 62, n. 1, p. 1–8 Babu SN et al (2016) 1-Methyl‐4‐phenyl‐1, 2, 3, 6‐tetrahydropyridine induced Parkinson's disease in zebrafish. Proteom v 16:1407–1420 Bagwell E et al (2024) 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-treated adult zebrafish as a model for Parkinson’s Disease. Neurosci Lett 842:137991 Cassar S et al (2019) Use of zebrafish in drug discovery toxicology. Chem Res Toxicol v 33(1):95–118 Additional Declarations The authors declare no competing interests. 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09:24:11","extension":"html","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":111350,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8270673/v1/583a6304429d1448a11c3206.html"},{"id":97421320,"identity":"397f7b2c-647b-4a92-8252-e0650f76fd36","added_by":"auto","created_at":"2025-12-04 08:37:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":25008749,"visible":true,"origin":"","legend":"\u003cp\u003eA scheme of the neurobehavioral test sequence using the zebrafish as an experimental model for Parkinson's-like disease. Legend: RT – Rotenone; LD - Levodopa.\u003c/p\u003e","description":"","filename":"Figure1Cor.png","url":"https://assets-eu.researchsquare.com/files/rs-8270673/v1/8f9e99a5d623f40601983d15.png"},{"id":97421305,"identity":"71ec48ce-8887-45e3-9107-82a728ae6546","added_by":"auto","created_at":"2025-12-04 08:37:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":873838,"visible":true,"origin":"","legend":"\u003cp\u003eZebrafish larvae were exposed to rotenone from 72 hpf. The percentage of larval survival was significantly reduced (p \u0026lt; 0.05) in the groups exposed to higher RT concentrations (12.00 and 15.00 μg/L). In addition, LD did not induce toxicity and prevented RT toxic effects. Each experimental group was compared with the DMSO group by one-way ANOVA (F (8, 58) = 4.21, p \u0026lt; 0.05) followed by Tukey's test (p \u0026lt; 0.05). Legend: DMSO - Dimethylsulfoxide; RT – Rotenone (μg/L); LD – Levodopa (µM). *Statistically significant difference from DMSO group (p \u0026lt; 0.05) by Tukey test.\u003c/p\u003e","description":"","filename":"Figure2Cor.png","url":"https://assets-eu.researchsquare.com/files/rs-8270673/v1/160ad2401080b7d8c58bc3fc.png"},{"id":97666556,"identity":"66d446e9-4980-48e8-9abd-fe0a7810ff2d","added_by":"auto","created_at":"2025-12-08 09:21:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3565047,"visible":true,"origin":"","legend":"\u003cp\u003eZebrafish larvae were induced to Parkinson's-like disease from 72 hpf with increasing concentrations of RT with thigmotaxis (A) and touch sensitivity (B) evaluated at 144 hpf. A decrease in the neurobehavioral response (%) was observed in a concentration-dependent manner in both tests. In addition, LD did not induce toxicity and prevented RT toxic effects. Thigmotaxis (F (9, 61) = 6.15, p \u0026lt; 0.05) and touch sensitivity (F (8, 61) = 11.08, p \u0026lt; 0.05) were significantly affected (p \u0026lt; 0.05) at concentrations higher than 11.00 μg/L RT. Legend: DMSO - Dimethylsulfoxide; RT – Rotenone (μg/L) LD – Levodopa (µM). *Statistically significant difference from DMSO group (p \u0026lt; 0.05) by Tukey test.\u003c/p\u003e","description":"","filename":"Figure3Cor.png","url":"https://assets-eu.researchsquare.com/files/rs-8270673/v1/09c8eb7e530654f26a21a2c0.png"},{"id":97421312,"identity":"330f0609-d017-46e9-8ea2-9b119e76e956","added_by":"auto","created_at":"2025-12-04 08:37:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":7017755,"visible":true,"origin":"","legend":"\u003cp\u003eZebrafish larvae were induced to Parkinson's-like disease from 72 hpf with increasing concentrations of RT and optomotor responses were evaluated at 144 hpf. Three parameters were evaluated: Percentage of grouped animals that swam the aquarium at each interval of 4 cm (A) (8 - 12 cm (F (8, 47) = 6.99, p \u0026lt; 0.001); (4 - 8 cm (F (8, 47) = 0.06, p \u0026gt; 0.05); (0 - 4 cm (F (8, 47) = 3.21, p \u0026lt; 0.01); Average speed (cm/s) of zebrafish larvae (B) (F (8, 289) = 45.50, p \u0026lt; 0.001); Percentage of animals that swam until the end of the aquarium (C) (F(8, 47) = 26.49, p \u0026lt; 0.001). In Figure A, an increase in the percentage of animals that travel a shorter distance was observed with increased RT concentration; LD did not induce toxicity and prevented RT toxic effects. The average velocity was reduced in a concentration-response manner with the increased RT concentration; LD exposure also reduced the average velocity and did not protect against RT. In C, a reduction in the percentage of arrival of animals exposed to concentrations higher than 11.00 μg/L of RT was observed; LD did not induce toxicity and prevented RT toxic effects. Legend: DMSO - Dimethylsulfoxide; RT – Rotenone (μg/L); Levodopa (µM). *Statistically significant difference from DMSO group (p \u0026lt; 0.05) by Tukey test.\u003c/p\u003e","description":"","filename":"Figure4Cor.png","url":"https://assets-eu.researchsquare.com/files/rs-8270673/v1/e198279f0bd7387422b21499.png"},{"id":97668076,"identity":"2c78c50d-bc10-497a-ad9c-8a237e9571b9","added_by":"auto","created_at":"2025-12-08 09:24:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":5168631,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage of zebrafish larvae with 72 hpf that exhibited the behavior of escape from visual stimuli and remained in the upper region of the well, an area without stimulus, during the Bouncing Balls (A) test (F (8, 98) = 76.51, p \u0026lt; 0.001) at RT concentrations higher than and equal to 11.00 μg/L. LD, in both groups, decreased percentage of animals in the non-stimuli area. Percentage of grouping and ungrouping response (B) of zebrafish larvae with 144 hpf during the Bouncing Balls test. LD increased the percentage of grouping animals and protected against RT toxic effects (Grouping: F (8, 61) = 24.46, p \u0026lt; 0.001; Ungrouping: F (8, 61) = 24.50, p \u0026lt; 0.001). Legend: DMSO - Dimethylsulfoxide; RT – Rotenone (μg/L); Levodopa (µM). *Statistically significant difference from DMSO group (p \u0026lt; 0.05) by Tukey test.\u003c/p\u003e","description":"","filename":"Figure5Cor.png","url":"https://assets-eu.researchsquare.com/files/rs-8270673/v1/720924aca76d1c2cbd3375f3.png"},{"id":97893252,"identity":"3edd2154-8d57-46ef-b633-8eba7f868506","added_by":"auto","created_at":"2025-12-10 15:29:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":29299391,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8270673/v1/90676d72-d06f-4adf-8771-c36cefbf2955.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eAssessment of neurobehavioral parameters in zebrafish larvae in a rotenone-induced Parkinson’s-like disease model\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eParkinson's disease (PD) is a progressive neurodegenerative condition that results in the loss of dopaminergic neurons in the substantia nigra compacta of the midbrain. In addition to classic motor symptoms, such as resting tremors, bradykinesia, muscle rigidity, and postural instability, PD may also present with non-motor symptoms, including cognitive impairment, sleep disturbances, hyposmia, depression, and anxiety [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The disease predominantly affects men over the age of 65 years, with an estimated prevalence of 1\u0026ndash;2% [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Currently, there is no definitive treatment for PD, and existing treatments are only aimed at alleviating motor symptoms, with no ability to regress the condition and often causing long-term adverse effects such as levodopa [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The complexity of pathological mechanisms hinders the development of effective therapies [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Therefore, animal models that reproduce PD-like symptoms, known as PD-like models, are essential for the study of the pathophysiology of the disease and the development of new therapeutic approaches [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe zebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e) has stood out as an animal model for studies of several diseases due to its advantages, such as small size, easy to maintain, low cost, high reproduction rate, and external fertilization. Its rapid maturation and short life allow for large-scale experiments, ensuring statistically reliable results. In addition, zebrafish share genetic, physiological, anatomical, and behavioral homology with humans, making them an effective tool for studying human diseases [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In the neurological context, the nervous system of zebrafish has anatomical similarities with humans, including the division into the forebrain, middle, and hindbrain, with structures such as the diencephalon, telencephalon, cerebellum, and spinal cord. Studies suggest that the zebrafish ventral diencephalon is functionally homologous to the human substantia nigra, which is involved in the pathogenesis of PD. The dopaminergic projections of zebrafish are fully formed between 72- and 96-hours post-fertilization [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], and it can be considered a good model for studying PD at this level of development. Dopaminergic neurons are detected early in zebrafish embryos, enabling the study of diseases such as PD [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe zebrafish has been used as a PD-like model using neurotoxins to generate relevant neurochemical and neurobehavioral phenotypes. Substances such as MPTP, 6-OHDA, paraquat, and rotenone are commonly used to induce PD-like symptoms in zebrafish [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. These studies highlight the potential of zebrafish as a model to understand the pathophysiology of PD and develop new therapeutic approaches, contributing significantly to advances in the field of neuroscience.\u003c/p\u003e\u003cp\u003eRotenone (RT), a naturally derived and lipophilic pesticide, is widely used as a PD-inducing agent due to its ability to cross the blood-brain barrier and cell membranes, affecting various organelles, including mitochondria. Its action mainly inhibits mitochondrial complex I, recapitulating clinical and pathological characteristics of PD [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The use of zebrafish as a model for RT-induced PD-like by immersion exposure has made it possible to reduce invasive procedures, such as intracerebral injections of 6-OHDA used for induction in adult zebrafish [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn addition, the evaluation of behavioral and locomotor parameters in zebrafish exposed to RT allows the presence of PD-like symptoms to be verified, as established of the related phenotypes of the disease [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The zebrafish exhibit complex repertories of behaviors, including social behavior, anxiety, learning, and memory, as well as defense behavior, similar to that of humans. Adults and zebrafish larvae exhibit behaviors relevant to neuropharmacology, making them useful for modeling neurological and psychiatric diseases [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Behavioral tests performed with zebrafish can be important tools for the study of behavioral disorders, providing relevant information about normal and pathological brain function [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Additionally, due to the toxicity of the substance used in the present study to induce behavioral phenotypes pertinent to PD, it is worth adding that the threshold between RT concentrations capable of causing physiological effects and ineffective concentrations is quite narrow, as observed by Melo, Oliveira [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Therefore, it is essential to use different RT concentrations with small variations between them, in order to induce observable changes without causing the death of the animals. In addition, the protocol for exposure to PD-like inducing substances in zebrafish [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] suggested that the lack of standardization at the time of onset of exposure to these substances produced \"conflicting\" results. It is recommended that the time of onset of exposure be selected, thus avoiding inhibition of zebrafish neurogenesis.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eConsequently, it is necessary to study the best concentration of RT that can be used to induce PD-like symptoms for the screening of molecules for new therapeutic approaches and which neurobehavioral tests can be used as endpoints to evaluate the effect of these molecules. In this framework, this study aimed to assessment of neurobehavioral parameters in zebrafish larvae in a rotenone-induced Parkinson\u0026rsquo;s-like disease model. Because, at this level of development, RT can cause PD-like symptoms in zebrafish embryos. We also used levodopa treatment as a standard. Since, the ventral diencephalon of zebrafish demonstrates functional homology with the human substantia nigra, a site associated with the development of PD pathogenesis [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Thus, this exposure approach was adopted to ensure advanced dopaminergic development and to enable the assessment of behavioral changes resulting from RT-induced dopaminergic neurodegeneration.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Maintenance and reproduction of animals\u003c/h2\u003e\u003cp\u003eThe experiments were carried out at the \u003cem\u003eLaborat\u0026oacute;rio de Ecofisiologia e Comportamento Animal\u003c/em\u003e (LECA) of the \u003cem\u003eUniversidade Federal Rural de Pernambuco\u003c/em\u003e (UFRPE), a vivarium registered in the CIUCA Platform of the Brazilian National Council for the Control of Animal Experimentation (CONCEA). The methods were previously approved by the Ethics Committee on the Use of Animals (CEUA) (License No. 3581030221) of UFRPE. Adult animals of the species \u003cem\u003eDanio rerio\u003c/em\u003e (WT strain, nine months old) were reared and housed in the vivarium. These animals were maintained in 80-liter aerated aquariums with continuous maintenance of abiotic conditions such as dissolved oxygen, nitrite, nitrate, and ammonia. The photoperiod was controlled at 10/14 h (light/dark), the temperature at 26\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, and the pH at 7.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. They were fed three times a day, twice with extruded commercial feed (\u0026asymp;\u0026thinsp;30% crude protein) and once with brine shrimp saline.\u003c/p\u003e\u003cp\u003eTo obtain the eggs, adult animals were separated according to sex in breeding tanks for zebrafish (Zebclean, Alesco), using males and females in an 8:4 ratio [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. After spawning, viable eggs were collected and evaluated using an optical light microscope (with LED lamp) at 1-hour post-fertilization (hpf) (Cadena et al., 2020a). Only eggs whose spawning was more than 90% viable were used [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The eggs were randomly placed in sterile polystyrene chambers [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] with a capacity of 80 mL, kept in an incubator with the same physicochemical parameters described above and considered optimal for the species.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Preparation of the rotenone solution, exposure, and survival of the animals\u003c/h2\u003e\u003cp\u003eA 0.02 mg/mL aqueous solution of rotenone (RT) (CAS# 83-79-4, purity\u0026thinsp;\u0026ge;\u0026thinsp;95%, R8875, SIGMA, St. Louis MO, USA) in 0.1% (v/v) dimethyl sulfoxide (DMSO) was prepared. This concentrated solution was used for dilution in 80 mL of water in the polystyrene chambers. Final nominal concentrations of 5.00 (RT5), 10.00 (RT10), 11.00 (RT11), 11.25 (RT11.25), 12.00 (RT12), and 15.00 \u0026micro;g/L (RT15) were obtained, where the larvae, from 72 hpf, were exposed to RT forming 7 experimental groups containing 15 eggs per chamber at each replication. Levodopa (LD) (CAS# 59-92-7) and benserazide hydrochloride (CAS# 10035-04-8) were purchased from commercial suppliers. 100 mg LD and 25 mg CB tablets were macerated to obtain nominal concentrations of 0.20 mg/L LD (1000 mM) and 0.05 mg/L CB (170 mM), respectively, and then diluted to 750.0 mM and 127.5 mM with 0.1% (v/v) DMSO aqueous solution. We used 2 more experimental groups to evaluate the toxicity of LD (LD 750.0 group) and if LD protected against RT toxic effects (LD 750.0\u0026thinsp;+\u0026thinsp;RT 11.25).The final concentration of DMSO was always less than 0.001% (v/v) after dilution in water, which is 500 times lower than that reported in the literature, which can affect the behavior of animals [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. RT exposure occurred from 72 hpf to 144 hpf with daily renewal of solutions in semi-static conditions [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Animal mortality was verified daily by the absence of movement and heartbeat [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The experimental design is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Neurobehavioral tests\u003c/h2\u003e\u003cp\u003eAt 144 hpf, visibly healthy larvae, without the presence of morphological defects [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], were transferred from polystyrene pots to 48-well plates (one larva per well) to undergo neurobehavioral tests in a controlled low-light environment. These tests were always performed between 10:00 and 12:00 h (AM) to avoid influences on the locomotor activity and visual sensitivity of the animals [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Prior to each test, the larvae were acclimatized for 15 minutes to avoid behavioral changes from handling and transport [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. As these are non-invasive methods, sequential neurobehavioral tests based on Gomes et al. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] were performed: Thigmotaxis, Touch Sensitivity, Optomotor response, and Bouncing Balls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe thigmotaxis test (TH test) was used to assess anxiety-like behaviors, as evidenced by previous studies [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The 48-well plates containing the larvae were arranged horizontally on a monitor (LCD, Dell E2211H), and the response was recorded to assess their preference for the edges of the wells. During the TH test, larvae were acclimatized for 15 minutes, and it was recorded whether the larvae showed a tendency to stay close to or away from the walls of the plates [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. During this test, the scan sampling method was used, with a dichotomous response [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] being \"yes\" for positive thigmotaxis when the larva was close to the wall and \"no\" for affected when the larva was away from the wall. Next, the touch sensitivity test (TS test) was performed, which was used to assess the larvae's response to mechanical stimuli [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The scan sampling method [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] was also used, with a dichotomous response. The stimuli were applied to the tail or head region of the larvae, and the positive response was recorded if the larvae demonstrated an escape behavior. Pictures of TH and TS tests were recorded using a camera (Canon EOS 6D DSLR; lens: Canon EF 75-300mm f/4-5.6) with a top view and a distance of 150 cm from the plates arranged on the monitor, while the monitor displayed a white background.\u003c/p\u003e\u003cp\u003eOptomotor response (OMR) was used to assess the visual impairments of zebrafish larvae [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. For this test, a new methodology was developed where the larvae of each group were relocated in an acrylic rectangular aquarium (dimensions: 15 x 5 x 5 cm, length, width, and height, respectively) with a wall positioned 3 cm from one end. The wall prevented the larvae from moving throughout the aquarium ahead of time. The aquarium with the larvae was positioned horizontally on a Full HD monitor (Dell E2211Hc), and then the larvae were exposed to a video with a total duration of 75 s, with black and white lines moving to the right and left [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The video simulates a stream of water to count the number of larvae that followed the direction of the stimulus [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The video consisted of 5 s of white (acclimatization) for the initial time (IT) and 30 s of alternating lines in the right direction. Simultaneously with the beginning of the animation, the wall of the aquarium was removed, allowing the larvae to swim freely so that they could follow the direction of the stimulus. The response was recorded by 200 seconds video recording by a camera (Canon EOS 6D DSLR; lens: Canon EF 75\u0026ndash;300 mm f/4-5.6) with a top view and a distance of 150 cm from the plate placed on the monitor. From this test, data such as the percentage of grouped animals that swam the aquarium at each interval of 4 cm, average speed (cm/s) of zebrafish larvae, and percentage of animals that swam until the end of the aquarium (cm) were obtained.\u003c/p\u003e\u003cp\u003eFinally, the bouncing balls test was used to assess spatial interaction responses, social cohesion, cognitive ability, and larval escape from visual stimuli [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The larvae were transferred to 6-well plates, each well containing 5 animals from each group, totaling 3 wells per group. The plates were placed on the monitor, and after 1.53 minutes of acclimatization on a dark screen, an animation with visual stimuli created in Microsoft PowerPoint (Office 365) was shown. The animation contained red balls (diameter 1.35 cm) that moved from left to right in a straight line with a trajectory of 2 cm in the stimulus area (lower half of the well) for 5 minutes. The number of larvae in the same quadrant of the well-provided data regarding the level of grouping and social cohesion among them. The response was recorded by 7 minutes video recording by the same camera described above. An analysis was performed every 30 s [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Statistical analysis\u003c/h2\u003e\u003cp\u003eNormality was carried out using the Komogorov Smirnov test (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The data from the TH, TS, OMR, and Bouncing Balls tests and the data regarding the survival of the animals were analyzed by one-way ANOVA parametric test. When the difference was significant, the means were compared using Tukey's test with p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. For statistical analyses, the Origin Pro Academic 2015 software (Origin Lab. Northampton, MA, USA) was used.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003eThe results of the survival of the animals are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. It was observed that the survival percentage of zebrafish larvae was significantly reduced in the groups exposed to RT concentrations\u0026thinsp;\u0026gt;\u0026thinsp;12.00 \u0026micro;g/L. However, LD did not induce toxicity and prevented RT toxic effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eRegarding the neurobehavioral assessments, the TH test was used to evaluate behaviors analogous to anxiety and is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA. The DMSO group had approximately 90% of the larvae near the edges of the wells, which is the expected behavior for this group. RT affected the thigmotaxis in the RT11 to RT15 groups, indicating that in concentrations\u0026thinsp;\u0026gt;\u0026thinsp;11.00 \u0026micro;g/L the toxic effect was observed. The TS test was used to verify the responsiveness to mechanical stimuli and significant reductions were observed in the RT11 to RT15 groups indicating that the escape behavior was affected (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Again, contractions\u0026thinsp;\u0026gt;\u0026thinsp;11.00 \u0026micro;g/L induced toxic effects. In addition, LD was not toxic as expected and reduced RT toxicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe results of the OMR test are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Significant changes were observed in all neurobehavioral parameters analyzed in this test when the animals were exposed to RT. In Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, it was found that RT significantly reduced the total distance traveled by the larvae in almost all groups, except in the group exposed to the lowest concentration of RT used (5 \u0026micro;g/L). It was observed that 71% of the animals swam the entire course of the aquarium in the control group, while 27% of the animals did so in the RT15 group. In Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, it was observed in relation to the mean velocity of the animals that the concentration of 5 \u0026micro;g/L of RT was also not able to cause significant changes in this parameter, only the concentrations\u0026thinsp;\u0026gt;\u0026thinsp;5 \u0026micro;g/L. The data related to the ability of the larvae to reach the finish line in the OMR test are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, where it was observed that this parameter was affected in all groups exposed to RT. Finally, we observed the concentration-dependent effect on all parameters evaluated in the OMR test (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), i.e., as the RT concentration increased, the larvae's ability to respond to the OMR test decreased. This was probably because RT at higher concentrations induced greater toxicity in zebrafish larvae. Regarding LD effects on OMR studied parameters, LD did not affect and protected against RT toxic effects in parameters of the total distance traveled (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) and percentage of arrival of animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). However, LD reduced average speed of animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe results of the bouncing balls test showed that RT affected the percentage in non-stimuli areas in concentrations\u0026thinsp;\u0026gt;\u0026thinsp;10.00 \u0026micro;g/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The same was observed in relation to the responsiveness of larval clustering at concentrations\u0026thinsp;\u0026ge;\u0026thinsp;11.00 \u0026micro;g/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Regarding LD effects, the percentage of animals in non-stimuli areas decreased in both LD groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) and exposure to LD 750.0 mM increased the percentage of grouping of animals. Interestingly, LD protected against RT toxic effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThus, our findings using an embryo-larval model with exposure from 72 hpf showed us concentration-dependent results, i.e., as the RT concentration increased, it directly affected the neurobehavioral response. Our results also corroborated with the work of Andrade et al. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] where the concentration of 11.25 \u0026micro;g/L proved to be the best to standardize the PD-Like neurobehavioral model for screening pharmacologically active molecules. Finally, as expected LD protected against RT toxic effects in most behavioral data studied which validated our study.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe zebrafish has been employed as a PD-Like model, playing a significant role in the study of neurodegenerative diseases, as it has specific brain regions homologous to those found in humans [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], showing that dopaminergic projections are fully formed between 72 and 96 hpf [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Our study standardized a sequence of neurobehavioral tests in a PD-like zebrafish model that can be used for the screening of new drugs using RT as an inducing molecule. Our findings showed a significant reduction in the survival percentage of zebrafish larvae in the groups exposed to the highest RT concentrations. This is in agreement with the study by Andrade et al. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], who observed a high mortality rate in the zebrafish embryo-larval toxicity model at RT highest concentrations (15 and 20 \u0026micro;g/L). The same authors highlighted that conducting tests on animals exposed to these higher concentrations may be difficult due to the significant reduction in survival. In addition, in the embryo-larval model used by Andrade et al. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], mortality was even higher than in the larval model used in our study, since they started exposing the animals with 2 hpf. This makes the larval model more suitable for PD-related neurobehavioral studies.\u003c/p\u003e\u003cp\u003eThe TH test was used to evaluate behaviors analogous to anxiety, which showed that RT affected TH in the groups with higher concentrations (\u0026gt;\u0026thinsp;11.00 \u0026micro;g/L). Andrade et al. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] also used the TH test to assess anxiety-like behaviors in an embryo-larval zebrafish model exposed to RT. Exposure of zebrafish embryos was initiated from 2 hpf at concentrations of 5, 10, 15, and 20 \u0026micro;g/L. In this study, changes in larval TH test were observed at the highest RT concentrations (15 and 20 \u0026micro;g/L). The authors concluded that these toxic effects in the TH test may indicate the ability of RT to modify neuronal activity, resulting in behavioral changes similar to anxiety, corroborating the findings of our study. While studies investigating anxiety-like behaviors in zebrafish larvae exposed to neurotoxins for PD-Like are still limited, some studies have examined this type of behavior in adult fish. Wang, Liu [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] evaluated anxiety-like behavior in an adult PD-like zebrafish model exposed to RT, using the light/dark preference test. In this study, a concentration of 2 \u0026micro;g/L was able to affect the behavior of the animals. The same authors suggested that these findings were mainly related to dopamine depletion in the brain caused by the action of RT, which inhibits mitochondrial complex I of dopaminergic neurons, resulting in their death. In addition, just like adult fish, zebrafish larvae also have a developed dopaminergic system, with dopaminergic projections fully formed between 72 and 96 hpf [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Therefore, it is suggested that RT may have acted similarly in our study, causing dopaminergic loss and consequent changes in the anxiety-like behavior of zebrafish larvae. In the TS test, significant reductions in escape behavior were observed as a response to touch in the groups exposed to the highest RT concentrations. Andrade et al. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] also used zebrafish exposed to RT to investigate responses to mechanical stimuli through TS in an embryo-larval toxicity model. They observed a significant reduction of TS in larvae exposed to the highest concentration of RT (20 \u0026micro;g/L), which may be associated with the neurotoxic action of RT.\u003c/p\u003e\u003cp\u003eIn a study carried out by Lam, Korzh [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], the neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) was administered to induce neurodegeneration in zebrafish embryos at 24 hpf, at which time dopaminergic neurons are first detected in the diencephalon. A test similar to ours was used to verify responses to tactile stimuli at 72 hpf. The authors observed that the larvae of the control group showed strong reflexes by trunk movement, while the larvae treated with MPTP produced weak and brief tactile responses. This suggests that exposure to MPTP resulted in deficits in the larval' swimming response. Therefore, in addition to sensory impairment, the inability or difficulty of the larvae to respond to the TS test may be related to locomotion defects induced by the neurotoxin used to induce PD-like symptoms. As RT also induces dopaminergic neurodegeneration and consequent reduction of dopamine levels, it is possible that it affected the ability of the larvae to present motor responses when subjected to the TS test, in the same way as observed in a PD-like model exposed to MPTP. This can be explained by the intrinsic role of the neurotransmitter dopamine in motor control, and insufficient dopamine production results in locomotor deficits, which may also have been observed in the larvae in our study. Thus, the results of our work confirm that RT can affect the responsiveness to the TS test of animals.\u003c/p\u003e\u003cp\u003eWe have developed a new OMR method specific to detect PD-like symptoms in our larval model. This was necessary because the OMR test [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] was previously carried out but did not show efficient results, since the methodology adopted only provided us with data referring only to the high and low alignment of the animals. However, we adopted a new approach to this test, by a study model that provides us with information regarding not only the alignment of the animals but also the distance traveled, speed, and percentage of animals that traveled the entire distance of the swimming board. This provides a more robust and information-rich model regarding the motor effects caused by RT in our animal model. Thus, we performed the OMR test to investigate possible visual and also motor changes in zebrafish larvae exposed to RT. This test is based on the fish's response to swimming in the direction of a visual stimulus, usually a stripe pattern [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Evaluating the OMR in PD-like animal models is relevant because, in addition to motor deficits, patients affected by the PD disease may present visual impairments [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOMR tests were poorly studied in PD-like animal models. Benvenutti, Marcon [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] analyzed OMR in zebrafish larvae exposed to 6-hydroxydopamine (6-OHDA), a compound that causes the death of dopaminergic neurons, as well as RT. The exposure of the animals occurred from 72 hpf at a concentration of 250 \u0026micro;M of 6-OHDA. The authors observed that larvae exposed to 6-OHDA showed a reduction in time spent in the stimulus zone (the region where the stripe pattern moved), indicating optomotor damage. In addition, as in our study, Benvenutti, Marcon [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] evaluated locomotor parameters such as mean speed and total distance traveled, however, unlike our methodology, these parameters were evaluated in isolation using the OMR test. It was found that 6-OHDA was able to cause a decrease of approximately 80% in the total distance traveled and average velocity of the larvae. Regarding these results, the authors suggested, based on other studies, that because 6-OHDA causes the death of dopaminergic neurons of important pathways related to movement, exposure to this compound resulted in the motor deficits found. This may also be applicable to our study since RT acts similarly to 6-OHDA causing motor deficits due to dopaminergic neuronal death. However, although a direct comparison is not possible due to the difference in strains between the larvae (WT in our study and AB in the study by Benvenutti, Marcon [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]), the results of the other tests in our study we performed involving the locomotor activity of animals (touch sensitivity and bouncing balls) corroborated with results of the OMR test. Also, in addition to motor deficits, RT is capable of causing visual changes, as was previously discussed in the bouncing balls test. Therefore, the changes in the OMR test may not have been exclusively motor, as animals needed to visualize the stimulus in order to present the motor response. Hence, it is necessary to consider possible alterations in visual perception and to confirm these alterations, additional endpoints are needed in which the visual part is isolated from the motor part.\u003c/p\u003e\u003cp\u003eRT influenced the clustering responsiveness of the larvae at higher concentrations in the bouncing balls test. The literature on the effects of RT on zebrafish social behavior is still scarce. However, a recent study demonstrated that RT alters this behavior in adult zebrafish [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In addition, no studies were found in the literature evaluating the ability to cluster as a response to aversive stimuli in zebrafish larvae exposed to neurotoxins used to induce PD-like symptoms. Therefore, it is necessary to consider that social behavior involves several aspects, including cognitive capacity, perception of environmental factors, and the manifestation of appropriate spatial and social interaction responses by larvae. In addition, the cognitive response is related to visual and locomotor capacity, which includes the escape movement and the approximation between conspecifics. In the study by Kalyn, Hua [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], zebrafish larvae exposed to RT from 72 hpf had locomotion defects, resulting in a significant reduction in the total distance traveled during swimming. There were also decreases in gene expression of th1 (tyrosine hydroxylase), an enzyme involved in dopamine synthesis, and a significant loss of dopaminergic neurons in the ventral diencephalon of the larvae. These results indicated that RT could cause locomotor deficits and neurochemical changes, which may have an impact on the behavior of the larvae in our bouncing balls test. In addition, the bouncing balls test involves visual stimuli and interaction between conspecifics. Considering that zebrafish larvae have dopaminergic neurons in the retina, exposure to RT can lead to visual changes due to the degeneration of these neurons. Studies in other models, such as rats and mice, have shown retinal damage and visual changes under the administration of RT and MPTP, related to oxidative stress and dopaminergic deficiency. Therefore, in addition to the locomotor changes observed in our study, the inability of the larvae to respond to the bouncing balls test may be related to visual changes resulting from the degeneration of dopaminergic neurons of the retina by the action of RT. These results highlighted the complexity of RT effects on the behavior of zebrafish larvae and the importance of considering multiple aspects in the interpretation of experimental results [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eGiven the above, our findings raise the hypothesis that RT affected the visual capacity of the larvae, leading them to present difficulty in seeing the stimuli, or even difficulty in moving to the conspecifics. This leaves room for future studies. As observed throughout this study, the RT concentrations that caused behavioral effects in zebrafish larvae ranged between 11.00 and 15.00 \u0026micro;g/L. However, concentrations of 12.00 and 15.00 \u0026micro;g/L resulted in a significant reduction in animal survival. Therefore, considering a higher percentage of survival and the presence of behavioral defects in all tests, the most suitable concentrations to observe the behavioral effects in this model were 11.00 and 11.25 \u0026micro;g/L.\u003c/p\u003e\u003cp\u003eLD is the most efficacious drug in the therapeutic of PD [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] and because of this, we chose as the gold standard of our tests. LD is associated with benserazide hydrochloride due to the last one preventing the decarboxylation of LD increasing the therapeutic effect [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. We maintained this association in our study because this therapeutic approach is used in humans. LD protected against RT toxic effects in most behavioral data studied. However, reduced the average speed of animals was observed in our study. This result is in agreement of literature because LD may be neurotoxic in neurons that are related to the locomotor activity in zebrafish larvae [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFinally, the literature presents several classical neurotoxins used in zebrafish to mimic the pathophysiological aspects of PD, such as MPTP/MPP⁺, 6-hydroxydopamine (6-OHDA), and paraquat, each reproducing specific mechanisms related to dopaminergic degeneration. However, most studies employ these neurotoxins in zebrafish via injection methods [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], not immersion, as in our model. The injection route is an invasive technique, typically performed by intraperitoneal administration, which does not align with the principles of the 3Rs, especially regarding Refinement [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], thereby compromising animal welfare. In a study by Kalyn et al. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], neurotoxins mentioned above were used at different concentrations, including RT. However, in the MPTP model, at the lowest concentration (0.25 mM), teratogenic effects were observed, resulting in increased mortality rate between 4 and 5 dpf, which interfered in neurobehavioral tests carried out at 7 dpf in this study. In the paraquat model, although this compound is associated with an increased risk of PD in humans, Kalyn et al. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] did not observe significant changes in larval locomotion. In addition, there was only a slight reduction in the expression of dopaminergic genes, and thus, the observed toxicity level was insufficient to produce a measurable locomotor activity. The same authors described that 6-OHDA has a limited ability to cross the blood-brain barrier, resulting in a lack of specificity in targeting only dopaminergic neurons. In conclusion, exposure to RT appeared promising for the assessment of behaviors associated with locomotor response, as shown in our study.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWith our embryo-larval model using zebrafish larvae with 72 hpf, it was possible to evaluate behavioral parameters in larvae exposed to RT, evidencing their suitability to analyze Parkinson's-like symptoms. It was found that the onset and duration of exposure to RT, especially at its high concentrations, resulted in consistent behavioral effects, mimicking Parkinsonian phenotypes in all tests performed. Considering the survival of the animals and the behavioral effects observed in the tests, the most appropriate concentrations to investigate the behavioral effects were \u0026asymp;\u0026thinsp;11.25 \u0026micro;g/L of RT. In addition, LD treatment was useful for a standard in our behavioral tests. Therefore, our behavioral tests proved to be appropriate for application in screening potential compounds that may contribute to the development of new therapies for PD, aiming to improve the quality of life of affected individuals.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This work was supported by CAPES (Funda\u0026ccedil;\u0026atilde;o Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior) for postgraduate scholarships, CNPq (Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico) for a productivity scholarship for Pabyton Cadena (302399/2023-2) and FACEPE (Funda\u0026ccedil;\u0026atilde;o de Amparo a Ci\u0026ecirc;ncia e Tecnologia do Estado de Pernambuco) for postgraduate scholarships for Renatta Silva (IBPG-0837-2.12/22) and junior postdoctoral scholarships for Samara Gomes (BFP-0043-2.10/24).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e The authors thank members of the Laborat\u0026oacute;rio de Ecofisiologia e Comportamento Animal \u0026ndash; LECA (Brazil).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e The authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is available on request from the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBucur M, Papagno C (2023) Deep Brain Stimulation in Parkinson Disease: A Meta-analysis of the Long-term Neuropsychological Outcomes. 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Int J PharmTech Res 8:614\u0026ndash;621\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVijayanathan Y, Lim FT, Lim SM, Long CM, Tan MP, Majeed ABA et al (2017) 6-OHDA-Lesioned Adult Zebrafish as a Useful Parkinson's Disease Model for Dopaminergic Neuroregeneration. Neurotox Res 32:496\u0026ndash;508\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCadena PG, Cadena MRS, Sarmah S, Marrs JA (2020) Folic acid reduces the ethanol-induced morphological and behavioral defects in embryonic and larval zebrafish (Danio rerio) as a model for fetal alcohol spectrum disorder (FASD). Reprod Toxicol 96:249\u0026ndash;257\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMelo KM, Oliveira R, Grisolia CK, Domingues I, Pieczarka JC, de Souza Filho J et al (2015) Short-term exposure to low doses of rotenone induces developmental, biochemical, behavioral, and histological changes in fish. 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Neurosci Lett 842:137991\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCassar S et al (2019) Use of zebrafish in drug discovery toxicology. Chem Res Toxicol v 33(1):95\u0026ndash;118\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"8d591de9-991c-4c1b-a12b-825fbe30dd55","identifier":"10.13039/501100002322","name":"Coordenação de Aperfeiçoamento de Pessoal de Nível Superior","awardNumber":"Postgraduate scholarships","order_by":0},{"identity":"dd486b14-efec-4153-a159-1e5598337627","identifier":"10.13039/501100003593","name":"Conselho Nacional de Desenvolvimento Científico e Tecnológico","awardNumber":"302399/2023-2","order_by":1},{"identity":"eeb8d53c-59dc-4621-94e4-4fe6534e0a32","identifier":"10.13039/501100006162","name":"Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco","awardNumber":"IBPG-0837-2.12/22","order_by":2},{"identity":"2d064229-7941-4359-bbc4-c2ca2f5caf67","identifier":"10.13039/501100006162","name":"Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco","awardNumber":"BFP-0043-2.10/24","order_by":3}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Universidade Federal Rural de Pernambuco","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Neurodegenerative disease, Danio rerio, animal behavior, animal model","lastPublishedDoi":"10.21203/rs.3.rs-8270673/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8270673/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe development of animal models that mimic the behavioral and symptomatic aspects of Parkinson\u0026rsquo;s Disease (PD) is essential for advancing new therapies. This study aimed to assessment of neurobehavioral parameters in zebrafish larvae in a rotenone-induced Parkinson\u0026rsquo;s-like disease model. Zebrafish larvae at 72 hours post-fertilization, when dopaminergic projections are fully developed, were exposed to rotenone (RT: 5.00\u0026ndash;15.00 \u0026micro;g/L), a compound known to selectively damage dopaminergic neurons, thus inducing PD-like symptoms. Levodopa treatment was also evaluated. Survival and neurobehavioral endpoints were evaluated using a series of tests: thigmotaxis, touch sensitivity, optomotor response (OMR), and bouncing balls. Larval survival significantly decreased at RT concentrations above 12.00 \u0026micro;g/L. Thigmotaxis and touch sensitivity were impaired at concentrations greater than 11.00 \u0026micro;g/L. In the OMR test, all measured parameters showed significant changes from 10.00 \u0026micro;g/L onward. Similarly, RT at concentrations above 10.00 \u0026micro;g/L reduced permanence in the non-stimulated area, while the clustering response of larvae was notably affected starting at 11.00 \u0026micro;g/L. These findings demonstrate that RT exposure induces specific and measurable neurobehavioral alterations in zebrafish larvae. The neurobehavioral parameters in this study is both detailed and reproducible, providing a valuable tool for future research and the screening of potential therapeutic compounds targeting Parkinson\u0026rsquo;s Disease.\u003c/p\u003e","manuscriptTitle":"Assessment of neurobehavioral parameters in zebrafish larvae in a rotenone-induced Parkinson’s-like disease model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-04 08:37:02","doi":"10.21203/rs.3.rs-8270673/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"607b3f5d-4849-4ea9-a17f-8526722d3805","owner":[],"postedDate":"December 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":59032453,"name":"Animal Behavior"},{"id":59032454,"name":"Neurobiology of Disease"},{"id":59032455,"name":"Toxicology"}],"tags":[],"updatedAt":"2025-12-04T08:37:02+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-04 08:37:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8270673","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8270673","identity":"rs-8270673","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}