Evaluation of behavioral and neurochemical changes induced by carbofuran in zebrafish (Danio rerio).

Carbofuran (CF) is a carbamate class pesticide, widely used in agriculture for pest control in crops. This pesticide has high toxicity in non-target organisms, and its presence in the environment poses a threat to the ecosystem. Research has revealed that this pesticide acts as an inhibitor of acetylcholinesterase (AChE), inducing an accumulation of acetylcholine in the brain. Nonetheless, our understanding of CF impact on the central nervous system remains elusive. Therefore, this study explored how CF influences behavioral and neurochemical outcomes in adult zebrafish. The animals underwent a 96-hour exposure protocol to different concentrations of CF (5, 50, and 500 µg/L) and were subjected to the novel tank (NTT) and social preference tests (SPT). Subsequently, they were euthanized, and their brains were extracted to evaluate neurochemical markers associated with oxidative stress and AChE levels. In the NTT and SPT, CF did not alter the evaluated behavioral parameters. Furthermore, CF did not affect the levels of AChE, non-protein sulfhydryl groups, and thiobarbituric acid reactive species in the zebrafish brain. Nevertheless, further investigation is required to explore the effects of environmental exposure to this compound on non-target organisms.

Locomotor Behavior and Memory Dysfunction Induced by 3-Nitropropionic Acid in Adult Zebrafish: Modulation of Dopaminergic Signaling.

Huntington's disease (HD) is a progressive neurodegenerative disease characterized by neuropsychiatric disturbance, cognitive impairment, and locomotor dysfunction. In the early stage (chorea) of HD, expression of dopamine D2 receptors (D2R) is reduced, whereas dopamine (DA) levels are increased. Contrary, in the late stage (bradykinesia), DA levels and the expression of D2R and dopamine D1 receptors (D1R) are reduced. 3-Nitropropionic acid (3-NPA) is a toxin that may replicate HD behavioral phenotypes and biochemical aspects. This study assessed the neurotransmitter levels, dopamine receptor gene expression, and the effect of acute exposure to quinpirole (D2R agonist) and eticlopride (D2R antagonist) in an HD model induced by 3-NPA in adult zebrafish. Quinpirole and eticlopride were acutely applied by i.p. injection in adult zebrafish after chronic treatment of 3-NPA (60 mg/kg). 3-NPA treatment caused a reduction in DA, glutamate, and serotonin levels. Quinpirole reversed the bradykinesia and memory loss induced by 3-NPA. Together, these data showed that 3-NPA acts on the dopaminergic system and causes biochemical alterations similar to late-stage HD. These data reinforce the hypothesis that DA levels are linked with locomotor and memory deficits. Thus, these findings may suggest that the use of DA agonists could be a pharmacological strategy to improve the bradykinesia and memory deficits in the late-stage HD.

Classical Hereditary galactosemia: findings in patients and animal models.

Classic galactosemia is a rare inborn error of metabolism that affects the metabolism of galactose, a sugar derived from milk and derivates. Classic galactosemia is caused by variants of the GALT gene, which lead to absent or misfolded forms of the ubiquitously present galactose-1-phosphate uridylyltransferase enzyme (GALT) driving galactose metabolites to accumulate, damaging cells from neurons to hepatocytes. The disease has different prevalence around the world due to different allele frequencies among populations and its symptoms range from cognitive and psychomotor impairment to hepatic, ophthalmological, and bone structural damage. The practice of newborn screening still varies among countries, dairy restriction treatment is a consensus despite advances in preclinical treatment strategies. Recent clinical studies in Duarte variant suggest dairy restriction could be reconsidered in these cases. Despite noteworthy advances in the classic galactosemia understanding, preclinical trials are still crucial to fully understand the pathophysiology of the disease and help propose new treatments. This review aims to report a comprehensive analysis of past studies and state of art research on galactosemia screening, its clinical and preclinical trials, and treatments with the goal of shedding light on this complex and multisystemic innate error of the metabolism.

Biological Pathways Associated with Vitamins in Autism Spectrum Disorder.

Autism spectrum disorder (ASD) is characterized by early-appearing social communication deficits, with genetic and environmental factors potentially playing a role in its etiology, which remains largely unknown. During pregnancy, certain deficiencies in critical nutrients are mainly associated with central nervous system impairment. The vitamin B9 (folate) is primarily related to one-carbon and methionine metabolism, participating in methyl donor generation. In addition, supplementation with folic acid (FA) is recommended by the World Health Organization (WHO) in the first three gestational months to prevent neural tube defects. Vitamin B12 is related to folate regeneration, converting it into an active form. Deficiencies in this vitamin have a negative impact on cognitive function and brain development since it is involved in myelin synthesis. Vitamin D is intimately associated with Ca2+ levels, acting in bone development and calcium-dependent signaling. This vitamin is associated with ASD at several levels since it has a relation with ASD genes and oxidative stress environment. This review carries the recent literature about the role of folate, vitamin B12, and vitamin D in ASD. In addition, we discuss the possible impact of nutrient deficiency or hypersupplementation during fetal development. On the other hand, we explore the biases of vitamin supplementation studies such as the loss of participants in retrospective studies, as well as multiple variants that are not considered in the conclusion, like dietary intake or auto-medication during pregnancy. In this regard, we aim to contribute to the discussion about the role of vitamins in ASD currency, but also in pregnancy and fetal development as well. Furthermore, stress during pregnancy can be an ASD predisposition, with cortisol as a regulator. In this view, we propose that cortisol is the bridge of susceptibility between vitamin disorders and ASD prevalence.

Role of the nucleoside-metabolizing enzymes on pain responses in zebrafish larvae.

Purinergic signaling is a pathway related to pain underlying mechanisms. Adenosine is a neuromodulator responsible for the regulation of multiple physiological and pathological conditions. Extensive advances have been made to understand the role of adenosine in pain regulation. Here we investigated the effects of purinergic compounds able to modulate adenosine production or catabolism on pain responses induced by Acetic Acid (AA) in zebrafish larvae. We investigated the preventive role of the ecto-5'-nucleotidase inhibitor adenosine 5'-(α,ß-methylene)diphosphate (AMPCP) and adenosine deaminase inhibitor erythro-9-(2-Hydroxy-3-nonyl)-adenine (EHNA) on the AA-pain induced model. The pain responses were evaluated through exploratory and aversive behaviors in zebrafish larvae. The exploratory behavior showed a reduction in the distance covered by animals exposed to 0.0025% and 0.050% AA. The movement and acceleration were reduced when compared to control. The treatment with AMPCP or EHNA followed by AA exposure did not prevent behavioral changes induced by AA for any parameter tested. There were no changes in aversive behavior after the AA-induced pain model. After AA-induced pain, the AMP hydrolysis increased on zebrafish larvae. However, the AMPCP or EHNA exposure did not prevent changes in AMP hydrolysis induced by the AA-induced pain model in zebrafish larvae. Although AMPCP or EHNA did not show differences in the AA-induced pain model, our results revealed changes in AMP hydrolysis, suggesting the involvement of the purinergic system in zebrafish larvae pain responses.

Modulation of adenosine signaling reverses 3-nitropropionic acid-induced bradykinesia and memory impairment in adult zebrafish.

Huntington's disease (HD) is a neurodegenerative disorder, characterized by motor dysfunction, psychiatric disturbance, and cognitive decline. In the early stage of HD, occurs a decrease in dopamine D2 receptors and adenosine A2A receptors (A2AR), while in the late stage also occurs a decrease in dopamine D1 receptors and adenosine A1 receptors (A1R). Adenosine exhibits neuromodulatory and neuroprotective effects in the brain and is involved in motor control and memory function. 3-Nitropropionic acid (3-NPA), a toxin derived from plants and fungi, may reproduce HD behavioral phenotypes and biochemical characteristics. This study investigated the effects of acute exposure to CPA (A1R agonist), CGS 21680 (A2AR agonist), caffeine (non-selective of A1R and A2AR antagonist), ZM 241385 (A2AR antagonist), DPCPX (A1R antagonist), dipyridamole (inhibitor of nucleoside transporters) and EHNA (inhibitor of adenosine deaminase) in an HD pharmacological model induced by 3-NPA in adult zebrafish. CPA, CGS 21680, caffeine, ZM 241385, DPCPX, dipyridamole, and EHNA were acutely administered via i.p. in zebrafish after 3-NPA (at dose 60 mg/kg) chronic treatment. Caffeine and ZM 241385 reversed the bradykinesia induced by 3-NPA, while CGS 21680 potentiated the bradykinesia caused by 3-NPA. Moreover, CPA, caffeine, ZM 241385, DPCPX, dipyridamole, and EHNA reversed the 3-NPA-induced memory impairment. Together, these data support the hypothesis that A2AR antagonists have an essential role in modulating locomotor function, whereas the activation of A1R and blockade of A2AR and A1R and modulation of adenosine levels may reduce the memory impairment, which could be a potential pharmacological strategy against late-stage symptoms HD.

Pannexin channel 1, P2×7 receptors, and Dimethyl Sulfoxide mediate pain responses in zebrafish.

The zebrafish has been considered an ideal model for studies of complex behaviors since its behavioral repertoire is well described. Therefore, this study evaluated the perceived pain through behavioral changes in zebrafish larvae. Here we investigated the Acetic Acid (AA) effects on zebrafish larvae exposed in a short-time period (60 s) and the preventive effect from routinely used compounds, Dimethyl Sulfoxide (DMSO), Ethanol (EtOH), Ibuprofen (IBP), and Paracetamol (PAR). In addition, the effect of P2×7 antagonist, A740003, and pannexin channel 1 (PANX-1) inhibitor Probenecid (PROB) on AA-induced behavioral changes were evaluated. AA impaired the distance covered, acceleration, movement, and latency to the first entry in the center from 5 dpf exposed larvae. At 0.050% AA, PAR prevented alterations from the distance covered, acceleration, and movement. Surprisingly, 0.3% DMSO prevented behavioral changes induced by AA. However, the effects from 0.2% DMSO were not prominent. We used 0.2% DMSO as a PROB diluent. PROB prevented the changes in distance and movement observed at both AA concentrations (0.0025% and 0.05%) tested. Since EtOH had no analgesic properties, we used it as an A740003 vehicle to observe the analgesic effects of this compound. As noted, A740003 did not prevent the behavioral changes in the AA-induced pain model. In contrast, 0.2% DMSO and PROB prevented AA-induced behavioral changes. These data enforce that zebrafish could be used in translational studies since this species has behavioral responses related to pain in the early stages of development and responses to analgesics similar to observed in mammals.

Oxytetracycline induces anxiety-like behavior in adult zebrafish.

Oxytetracycline (OTC) is one of the broad-spectrum antibiotics widely used for the treatment of fish-farm infection. Considering that behavior is directly related to reproduction, individual fitness, and survival, it is important to evaluate the impact of antibiotics on the behavioral repertoire in fish. Zebrafish (Danio rerio) presents a well-described behavioral repertoire to reliably demonstrate complex responses to chemical compound exposure. This work aims to identify the role of OTC in comprehensive behavioral parameters and whole-body cortisol levels in adult zebrafish. Here we report that OTC exposure (10, 20, and 100 mg/L) induces an anxiogenic-like phenotype in the novel tank test. OTC exposure also changes the behavior of social interaction with a shoal of unknown zebrafish - characterized as a stimulus group. Zebrafish exposed to OTC (10 mg/L) remains a longer period in the stimulus zone when compared to the control group. Clonazepam (0.006 mg/L) was able to reverse anxiogenic-like behavior and the changes in social behavior induced by OTC. We also demonstrated that cortisol levels were significantly decreased after exposure to OTC (10, 20, and 100 mg/L), which were not reversed by clonazepam. These findings highlight the growing utility of zebrafish as a model to understand the impact of antibiotics on behavior and their underlying mechanisms.

Effects of anesthetic MS-222 on stress and reproduction of South American silver catfish (Rhamdia quelen) males.

Anesthesia is a common practice used in fish research and aquaculture. It is important to understand anesthetic effects on the animal and tissues of interest to ensure validity of data and to improve animal welfare in research and fish production endeavors. The production of some captive fish species is only possible by imposing artificial reproduction procedures, and manipulation of fish for these purposes is a stressor. The purpose of this study, therefore, was to evaluate effects of different concentrations (100, 200, and 300 mg/L) of the anesthetic MS-222 (tricaine methanesulfonate) on cortisol concentrations and effects on sperm quality in Rhamdia quelen. After hormonal induction of gamete production, 28 sexually mature males were randomly assigned to treatments, and milt and blood samples were collected. Anesthesia induction time, motility rate, sperm concentration and morphology, plasma cortisol concentrations, and reproductive hormone concentrations (testosterone, 17-α-hydroxyprogesterone, and estradiol) were evaluated. Sperm motility was greater in the control than 300 mg/L treatment group but did not differ among the control, 100, and 200 mg/L groups. The estradiol concentration was greater in non- anesthetized than anesthetized Rhamdia quelen, but plasma cortisol concentrations did not differ among treatment groups (182.50 ±â€¯42.03 ng/mL). The anesthetic MS-222 at concentrations of 100, 200, and 300 mg/L did not inhibit the stress response due to handling of Rhamdia quelen males. In addition, treatment with MS-222 was not effective in inhibiting detrimental effects on sperm quality because this treatment was associated with impaired sperm motility and lesser concentrations of plasma estradiol.

Pyriproxyfen Exposure Impairs Cognitive Parameters and Alters Cortisol Levels in Zebrafish.

Pyriproxyfen is one of the most used larvicides and insecticides; it acts as an analog of juvenile insect hormone (a growth regulator). It is highly toxic during all stages of mosquito development, suppresses metamorphosis, and interferes in insect reproduction and proliferation. Pyriproxyfen and its main metabolite have been shown to affect brain development in rodents. This compound is employed mainly to eliminate outbreaks of the genus Aedes, even in potable water. Despite the increasing number of toxicological studies about larvicides and insecticides-with an indication of continuous use-there have been few studies about the effects of pyriproxyfen in non-target species such as fish. This study evaluated the effects of pyriproxyfen on behavioral, cognitive, and endocrine parameters in zebrafish. We exposed adult zebrafish to different pyriproxyfen (Pestanal®) concentrations (0.125, 0.675, and 1.75 mg/l) for 96 h. We analyzed behavioral parameters, memory, cortisol levels, and gene expression of glucocorticoid receptor (gr) and corticotrophin-releasing factor (crf) after pyriproxyfen exposure. This exposure did not alter locomotion (distance or mean speed), anxiety-like behavior (latency to enter to the top zone of the tank or time in the top zone of the tank), and social or aggressive behavior. However, there was impaired inhibitory avoidance memory at all tested pyriproxyfen concentrations. Cortisol levels were reduced in exposed groups when compared to control or vehicle. However, gr and crf gene expression in pyriproxyfen-treated animals were unaltered when compared to control or vehicle groups. Taken together, these findings indicate that pyriproxyfen may induce cognitive impairment and altered cortisol levels in zebrafish, a non-target species.

Novel object recognition and object location tasks in zebrafish: Influence of habituation and NMDA receptor antagonism.

This study aims to establish a protocol for evaluating the object recognition memory and object location tasks in zebrafish. We evaluated novel the object recognition memory and analyzed the exploration time of the objects during training and testing. Zebrafish explored more the new object in comparison to the familiar object (61% of exploration time during test session). We also tested the object location task and measured the exploration time of each object in the familiar and novel object location. There was a preference to explore the object in the novel location (63% of exploration time during test session). The effect of the non-competitive NMDA receptor antagonist MK-801 was investigated on the object recognition and object location memory. Control (water only) and treated animals (5 µM MK-801) presented a significant preference in exploring the familiar object in comparison to the new object (66 and 68% of exploration time, respectively, during test session); however, 10 µM MK-801-treated animals did not show differences in the exploration time of the objects. In the object location task, the animals treated with the 5 or 10 µM MK-801 did not show a preference for the familiar or novel location whereas the control group had a higher preference in exploring the object in the familiar location (64% of exploration time during test session). Considering the different responses of the control group between original task and in the regimen treatment, we evaluated the impact of habituation on cortisol levels of animals in three different protocols: (1) habituated at the experiment apparatus for 3 days (C1 condition), (2) habituated at the experiment apparatus for 3 days plus treatment tank exposure at fourth day (C2 condition), (3) habituated at the treatment tank and experiment apparatus for 3 days and exposed to treatment tank again at fourth day (C3 condition). The results showed higher levels of cortisol in animals submitted to C2 and C3 conditions compared to animals submitted to C1. When introduced to an acute stressor during C1 condition, we observed an increase in the cortisol levels and an absence of preference for the objects in comparison to control group, which had a preference for novel object and novel location. Fluoxetine treatment induced a decrease in cortisol levels and an absence of preference for the objects in C2 and C3 conditions in comparison to control group, which had a preference for familiar object. However, fluoxetine treatment induced a preference to the novel location in C2 and C3 conditions in comparison to control group, which had a preference for familiar location. These results indicate that treatment tank exposure induced a different performance in object recognition and object location memory due to stress responses. Therefore, these tasks are prone to evaluate memory in physiological and pathological conditions, but its use is limited due to sensitivity to stress caused by manipulation.

Stress responses to conspecific visual cues of predation risk in zebrafish.

Chemical communication relating to predation risk is a trait common among fish species. Prey fish under threat of predation can signal risk to conspecific fish, which then exhibit defensive responses. Fish also assess predation risk by visual cues and change their behavior accordingly. Here, we explored whether these behavioral changes act as visual alarm signals to conspecific fish that are not initially under risk. We show that shoals of zebrafish (Danio rerio) visually exposed to a predator display antipredator behaviors. In addition, these defensive maneuvers trigger antipredator reactions in conspecifics and, concomitantly, stimulate the hypothalamus-pituitary-interrenal axis, leading to cortisol increase. Thus, we conclude that zebrafish defensive behaviors act as visual alarm cues that induce antipredator and stress response in conspecific fish.

Behavioral responses of zebrafish depend on the type of threatening chemical cues.

In fish, defensive reactions are induced by different chemical cues that emanate from sense-related stresses [physical, chemical, and visual (visual contact with predator)] or food stresses (acute fasting and chronic food restriction). Using a shuttle box with a two-chamber unmixed laminar flow that allowed fish to remain or flee from a chemical cue, we showed that the avoidance response depended on the type of the chemical cue. We show that zebrafish (Danio rerio) retreated from water conditioned with chemical cues released by chemically or physically stressed fish and acutely fasted fish, but not from water with cues from fish experiencing visual contact with predatory fish or fish suffering from chronic food restriction. Our data reinforced the hypothesis that fish use a combination of information and the context of the situation to determine their evasion strategy.

Environmental and Pharmacological Manipulations Blunt the Stress Response of Zebrafish in a Similar Manner.

Here we provide evidence that both pharmacological and environmental manipulations similarly blunt the cortisol release in response to an acute stressor in adult zebrafish. Different groups of fish were maintained isolated or group-housed in barren or enriched tanks, and then exposed or not to diazepam or fluoxetine. Acute stress increased cortisol levels in group-housed zebrafish maintained in barren environment. Single-housed zebrafish displayed a blunted cortisol response to stress. Environmental enrichment also blunted the stress response and this was observed in both isolated and group-housed fish. The same blunting effect was observed in zebrafish exposed to diazepam or fluoxetine. We highlighted environmental enrichment as an alternative and/or complimentary therapeutic for reducing stress and as a promoter of animal welfare.

Acute exposure to waterborne psychoactive drugs attract zebrafish.

Psychotropic medications are widely used, and their prescription has increased worldwide, consequently increasing their presence in aquatic environments. Therefore, aquatic organisms can be exposed to psychotropic drugs that may be potentially dangerous, raising the question of whether these drugs are attractive or aversive to fish. To answer this question, adult zebrafish were tested in a chamber that allows the fish to escape or seek a lane of contaminated water. These attraction and aversion paradigms were evaluated by exposing the zebrafish to the presence of acute contamination with these compounds. The zebrafish were attracted by certain concentrations of diazepam, fluoxetine, risperidone and buspirone, which were most likely detected by olfaction, because this behavior was absent in anosmic fish. These findings suggest that despite their deleterious effects, certain psychoactive drugs attract fish.

Waterborne psychoactive drugs impair the initial development of Zebrafish.

The contamination of rivers and other natural water bodies, including underground waters, is a current reality. Human occupation and some economic activities generate a wide range of contaminated effluents that reach these water resources, including psychotropic drug residues. Here we show that fluoxetine, diazepam and risperidone affected the initial development of zebrafish. All drugs increased mortality rate and heart frequency and decreased larvae length. In addition, risperidone and fluoxetine decreased egg hatching. The overall results points to a strong potential of these drugs to cause a negative impact on zebrafish initial development and, since the larvae viability was reduced, promote adverse effects at the population level. We hypothesized that eggs and larvae absorbed the drugs that exert its effects in the central nervous system. These effects on early development may have significant environmental implications.

Waterborne Risperidone Decreases Stress Response in Zebrafish.

The presence of drugs and their metabolites in surface waters and municipal effluents has been reported in several studies, but its impacts on aquatic organisms are not yet well understood. This study investigated the effects of acute exposure to the antipsychotic risperidone on the stress and behavioral responses in zebrafish. It became clear that intermediate concentration of risperidone inhibited the hypothalamic-pituitary-interrenal axis and displayed anxiolytic-like effects in zebrafish. The data presented here suggest that the presence of this antipsychotic in aquatic environments can alter neuroendocrine and behavior profiles in zebrafish.

Effects of waterborne fluoxetine on stress response and osmoregulation in zebrafish.

The presence of fluoxetine in aquatic environments has been reported for decades. Here, we investigate the effects of exposure to fluoxetine on the stress response and osmoregulation in zebrafish. We show that stress response alters osmoregulation and that fluoxetine inhibits these stress-related changes in osmoregulation. The results suggest that the presence of fluoxetine in aquatic ecosystems can cause changes in response to stress and osmoregulation in fish.

My stress, our stress: blunted cortisol response to stress in isolated housed zebrafish.

Here, we show that individually housed zebrafish presented a reduced cortisol response to an acute stressor (persecution with a pen net for 120 s) compared to zebrafish housed in groups of 10. We hypothesized that the cortisol response to stress was reduced in individually housed zebrafish because they depend solely on their own perceptions of the stressor, whereas among grouped zebrafish, the stress response might be augmented by chemical and/or behavioral cues from the other members of the shoal. This hypothesis was based on previous described chemical communication of stress in fish as well on individual variation in stressor perception and potential individual differences in fish personality.

Diazepam and fluoxetine decrease the stress response in zebrafish.

The presence of pharmaceutical products in the aquatic environment has been reported in several studies. However, the impact of these drugs on living organisms is still uncharacterized. Here, we investigated the effects of acute exposure to either diazepam or fluoxetine on the stress response in Danio rerio. We showed that diazepam and fluoxetine inhibited the stress axis in zebrafish. Intermediate concentrations of diazepam suppressed the stress response as measured by cortisol levels, whereas fluoxetine inhibited cortisol increase at concentrations similar to those found in the environment. These data suggest that the presence of psychoactive drugs in aquatic ecosystems could cause neuroendocrine dysfunction in fish.