Novel Tank Test Research Articles

Anxiolytic-like effects of Pseudospondias microcarpa hydroethanolic leaf extract in zebrafish: Possible involvement of GABAergic and serotonergic pathways

Pseudospondias microcarpa is used in ethnomedicine to manage central nervous system diseases. The hydroethanolic extract (PME) from the leaves of the plant has shown anxiolytic-like properties in mice anxiety models. However, its effects in chronic anxiety models and possible mechanism(s) of action were not studied. Therefore, the current study evaluated the anxiolytic-like mechanisms of PME in zebrafish models of anxiety. The zebrafish light dark test (LDT) and novel tank test (NTT) were employed to assess the anxiolytic-like effects of PME (0.1, 0.3, 1.0 mg mL−1), fluoxetine (3 × 10−5 mg mL−1) and diazepam (1.5 × 10−7 mg mL−1). The chronic unpredictable stress (CUS) test was used to further evaluate the extract’s anxiolytic-like properties. The potential mechanisms of anxiolytic action of the extract was evaluated after pre-treated with flumazenil, granisetron, methysergide, or pizotifen, all at 1 × 10−3 mg mL−1. The extract significantly decreased anxiety behaviours in the NT and LD tests. These observed effects of the extract were however counteracted by flumazenil, granisetron, methysergide and pizotifen pre-treatment. In addition, PME treatment significantly reversed CUS-induced anxiety behaviours in zebrafish. Results show that PME possesses anxiolytic-like effects possibly through interaction with serotonergic and gamma-aminobutyric acid mediated pathways.Graphical abstract

Assessing the exploratory profile of two zebrafish populations: Influence of anxiety-like phenotypes and independent trials on homebase-related parameters and exploration

Anxiety is a protective behavior when animals face aversive conditions. The open field test (OFT) is used to assess the spatio-temporal dynamics of exploration, in which both homebase formation and recognition of environmental cues may reflect habituation to unfamiliar conditions. Because emotional- and affective-like states influence exploration patterns and mnemonic aspects, we aimed to verify whether the exploratory behaviors of two zebrafish populations showing distinct baselines of anxiety differ in two OFT sessions. Firstly, we assessed the baseline anxiety-like responses of short fin (SF) and leopard (LEO) populations using the novel tank test (NTT) and light-dark test (LDT) in 6-min trials. Fish were later tested in two consecutive days in the OFT, where the spatial occupancy and exploratory profile were analyzed for 30 min. In general, LEO showed pronounced diving behavior and scototaxis in the NTT and LDT, respectively, in which an “anxiety index” corroborated their exacerbated anxiety-like behavior. In the OFT, the SF population spent less time to establish the homebase in the 1st trial, while only LEO showed a markedly reduction in the latency to homebase formation in the 2nd trial. Both locomotion and homebase-related activities were decreased in the 2nd trial, in which animals also revealed increased occupancy in the center area of the apparatus. Moreover, we verified a significant percentage of homebase conservation for both populations, while only SF showed reduced the number of trips and increased the average length of trips. Principal component analyses revealed that distinct factors accounted for total variances between trials for each population tested. While homebase exploration was reduced in the 2nd trial for SF, an increased occupancy in the center area and hypolocomotion were the main factors that contribute to the effects observed in LEO during re-exposure to the OFT. In conclusion, our novel data support the homebase conservation in zebrafish subjected to independent OFT sessions, as well as corroborate a population-dependent effect on specific behavioral parameters related to exploration.

Cannabis sativa-based oils against aluminum-induced neurotoxicity

The use of terpenoid compounds in different neural-related conditions is becoming useful for several illnesses. Another possible activity of these compounds is the reduction of nervous impairment. Cannabis sativa plants are known for their concentration of two important terpenoids, the delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD). CBD and THC have central peripheral activities already described and their usage in different brain diseases, such as Alzheimer's and multiple sclerosis. Aluminum (Al) is known as an important neurotoxic compound, the physiological action of Al is not known already, and in high concentrations can lead to intoxication and cause neurotoxicity. Here we evaluated the potential effect of two different doses of CBD- and THC-rich based oils against Al-induced toxicity, in the zebrafish model. We evaluated behavioral biomarkers of the novel tank test (NTT) and social preference test (SPT), and biochemical markers: the activity of the enzyme acetylcholinesterase (AChE) and the antioxidant enzymes—catalase, superoxide dismutase, and glutathione-S-transferase. CBD- and THC-based oils were able to increase the AChE activity helping the cholinergic nervous system actuate against Al toxicity which was reflected by the behavioral biomarkers changes. We concluded that the oils have a protective effect and might be used with proposals for neurological and antioxidant impairment avoidance caused by Al intoxications.

Neurotoxicological effects of venlafaxine on Caenorhabditis elegans and Danio rerio

The growing consumption of psychoactive drugs, such as Venlafaxine (VFX), can negatively affect the organisms. Our main hypothesis is to investigate if VFX at human-used doses could exert effects on the behavioral, nervous, and antioxidant systems of two different organisms, zebrafish and C. elegans. We evaluated the effect of acute exposure to VFX at four concentrations (0, 37.5, 75, and 150 mg L−1) using toxicological indicator assessments. We evaluated zebrafish behavior using the novel tank test (NTT), social preference test (SPT), cortisol levels, acetylcholinesterase (AChE) activity, and antioxidant system. In C. elegans, we evaluated body bends, defecation cycles, pharyngeal pumping, AChE activity, and antioxidant system. C. elegans do not show alterations in the behavior analysis of pharyngeal pumping and body bends. Instead, the defecation cycle was increased in the highest dose of VFX. AChE activity also does not have differences compared to the control, the same occurs in lipid peroxidation rates. These results showed that nematodes were more resistant to changes when exposed to VFX. Zebrafish exposed to VFX showed changes in the NTT and SPT test, mainly in the anxiolytic pattern, suggesting that VFX alters this anxiolytic-like behavior. Comparing both organisms, we can observe that zebrafish seems to be more sensitive in this neurotoxicological evaluation.

Novel Chromone-Containing Allylmorpholines Induce Anxiolytic-like and Sedative Effects in Adult Zebrafish.

Chromone-containing allylmorpholines (CCAMs) are a novel class of compounds that have demonstrated acetyl- and butyryl-cholinesterase-inhibiting and N-methyl-D-aspartate (NMDA) receptor-blocking properties in vitro, but their in vivo pharmacological activity remains underexplored. In this work, we evaluated the psychotropic activity of five different CCAMs (1 (9a), 2 (9j), 3 (9l), 4 (33a), and 5 (33b)) using the novel tank test (NTT) and light/dark box (LDB) test in adult zebrafish. The CCAMs were screened in the NTT at a range of concentrations, and they were found to induce a dose-dependent sedative effect. Compound 4 (33a) was also evaluated using the LDB test, and it was found to have anxiolytic-like properties at low concentrations. To assess the potential contribution of the glutamate and cholinergic mechanisms in the effects of the CCAMs, we conducted experiments with pre-exposure to putative antagonists, NMDA and biperiden. Neither biperiden nor NMDA were able to diminish or cancel the effects of the CCAMs, countering the in vitro data obtained in previous studies. The apparent discrepancy could be related to the specifics of CCAM metabolism or to the interspecies differences between the putative target proteins, possibly due to the relatively low identity percentage of their sequences. Although further research in mammals is required in order to establish their pharmacological properties, novel CCAMs may represent an appealing group of psychoactive drug candidates.

Dietary transference of 17α-ethinylestradiol changes the biochemical and behavioral biomarkers in adult zebrafish (Danio rerio)

The endocrine disruptors (ED), even in low concentration, can change the homeostasis of an organism through the biochemical and physiological pathways; and are gaining more relevance due to their well-reported presence in the natural environment. EDs mainly affect non-target animals, which can bioaccumulate, leading to changes in metabolism. Another problem is due to several organisms that compose the aquatic biota serving as a basis of the food chain and transferring it to higher trophic levels. Here we evaluated the dietary transference of 17α-ethinylestradiol (EE2), in adult zebrafish chronically fed by EE2-bioaccumulated brine shrimp (BS). For this, we evaluated behavioral biomarkers such as the novel tank test (NTT), social preference test (SPT), mirror-induced aggressivity (MIA), and biochemical biomarkers such as acetylcholinesterase (AChE), superoxide dismutase (SOD), catalase (CTL), and glutathione-S-transferase (GST) activity, cortisol, and lipid peroxidation levels in adult zebrafish. The behavioral effects can be explained by the changed effects on acetylcholinesterase activity as well as in the antioxidant system mainly affected by the high levels of EE2 identified by HPLC shown that had occurred during a dietary transfer for fish. EE2 has a potential pattern for bioaccumulation and dietary transfer in biological tissue and EE2 can affect the behavior of fish. The observed effects could be dangerous to the environment, affecting, other animals and even human health.

Persistent and transgenerational effects of pesticide residues in zebrafish

Highly toxic chemical compounds are present in rivers and lakes, endangering the survival of non-target species. To evaluate the effects of environmental contamination on non-target species, we used the zebrafish as an animal model. Environmental concentrations of the widely used pesticides, glyphosate (GBH) at 4.8 μg·L−1 and 2,4-dichlorophenoxyacetic acid (DBH) at 3.4 μg·L−1, were used. The animals were exposed during the entire period of organogenesis and evaluated in our previous study regarding initial developmental parameters. In the present study, we evaluate these fish when achieve the adult phase, using the novel tank test (NTT) and the aversivity test. In the second step, the animals were allowed to reproduce, and the initial parameters of development, behavioral parameters in the open field test (OFT) and in the aversivity test (AST), and biochemical biomarkers as acetylcholinesterase (AChE), catalase (CAT), and superoxide dismutase (SOD) in the F1 generation were studied. Fish exposed to GBH showed hypermobility, and their anti-predatory reaction was impaired during adulthood, indicating a persistent effect. We also showed that fish had impaired behavioral and survival changes in the F1 generation as well as effects on AChE activity and antioxidant enzymes, characterizing a transgenerational effect. The fish did not show persistent effects in adulthood due to DBH exposure; however, they were unable to reproduce. Our findings demonstrate the serious impact of pesticides on fish, where the effects of contamination can affect future generations and compromise the species' survival.

Intermittent Fasting Improves Memory, Reduces Anxiety and Enhances Neurotransmitter Levels In Zebrafish (Danio Rerio)

The zebrafish is an excellent model for social, behavioural and neuroscience studies. The brain controls food intake, energy expenditure, insulin secretion, hepatic glucose production, and glucose fatty acid metabolism in adipose tissue and skeletal muscle. Caloric restriction is thought to be the main reason for disease regulation. The sensory system strengthens by altering the union of neurotrophins and synapses. The experimental groups studied in this research included control, uncontrolled diet-(overfed), 12hrs, 24hrs, 48hrs intermittent fasting, respectively. The study groups were subjected to a T-maze test to examine zebrafish memory and learning changes. The novel tank test (NTT) and light and dark (LDT) tests investigated stress-like behaviour. The levels of dopamine, serotonin, epinephrine, and nor-epinephrine were measured using a spectrofluorimetric method. We found behaviour and enhanced neurotransmitter levels in the groups that were subjected to intermittent fasting than the overfed and control groups which may be due to the hippocampus derived neurotrophic factor that in areas memory power and reduce anxiety from this experiment it is evident that intermittent fasting could bring a positive outcome in memory maintenance by lowering the anxiety and thereby keeping a check in neurotransmitter levels.

Differentiating the Neuropharmacological Properties of Nicotinic Acetylcholine Receptor-Activating Alkaloids.

Alkaloids that target nicotinic acetylcholine receptors (nAChR) are of great interest because of the critical role they play in mood and anxiety. However, understanding of the neuropharmacological effects of nicotinic alkaloids, such as cotinine and anatabine, is very limited. In this study, we investigated the neuropharmacological effects of three naturally occurring alkaloids—nicotine, cotinine, and anatabine—in vitro and in vivo. A single injection of nicotine induced anxiolytic-like behavioral features in mice by using the SmartCube® behavioral profiling system, while cotinine and anatabine had no detectable effect. The results were corroborated by using the zebrafish novel tank test (NTT), which showed a profound anxiolytic-like effect induced by multiple doses of nicotine after a single 20-min treatment. When the regulation of dopamine and norepinephrine release—the neurotransmitter systems relevant for anxiety—were examined in vitro, we found that nicotine stimulated the release of both norepinephrine and dopamine, while cotinine and anatabine mainly stimulated the dopamine release. The molecular targets of nicotine were confirmed to be nAChRs with its most potent activities against α4β2 and α6/3β2β3 subtypes in vitro. Anatabine was a weaker agonist for these receptors than nicotine. Cotinine was the least potent nAChR compound, only being able to activate α4β2 and α6/3β2β3 subtypes at high doses and no detectable activities against α3β4 and α7 subtypes at the concentrations tested. The observed effects were unlikely due to the off-target effect, because these alkaloids did not bind or regulate >160 other molecular targets in vitro. Thus, the present results suggest that natural nicotinic alkaloids can induce an anxiolytic-like behavior in nonclinical animal models, potency of which may depend on the activation of various nAChRs and regulation of various neurotransmitter systems. Further investigations would help understand their effects on humans, because non-clinical studies should not be taken as a direct indication for human behavior and nicotine is not risk free.

Statistical Analysis and Machine Learning Used in the Case of Two Behavioral Tests Applied in Zebrafish Exposed to Mycotoxins

Machine learning is a branch of artificial intelligence that allows computer systems to learn directly from examples, data, and experience. Statistical modeling is more about finding connections between variables and consequently the impact of these relationships, while also catering for prediction. It should be clear that these two methodologies are different in terms of their purpose, despite the fact that they use similar means to get there. The evaluation of the machine learning algorithm uses a set of tests to validate its accuracy. Although, for a statistical model, the analysis of regression parameters by confidence intervals, significance tests and other tests can be used to assess the legitimacy of the model. To demonstrate the applications and usefulness of this theory, an experimental study was conducted on zebrafish exposed to mycotoxin. Methods: Patulin (70 µg/L) and kojic acid (100 mg/L, 204 mg/L, and 284 mg/L) were administered by immersion to zebrafish once daily for a period of 7 days before the behavior testing. The following behavioral tests were performed: a novel tank test (NTT) (to assess the explorative behavior and anxiety); and a Y-maze test (which measures the spontaneous explorative behavior). Behavioral tests were performed on separate days. For the behavior tests, the statistical analysis was performed using ANOVA variation analysis (two-way ANOVA). All results are expressed as the mean ± standard error of the mean. The values of the general index F for which p < 0.05 were considered statistically significant. Results: Y-maze—patulin exposure led to an intensification of the locomotor activity and an increased traveled distance and number of arm entries. By increasing the spontaneous alternation between the aquarium’s arms, patulin has shown a stimulating effect on spatial memory. In the case of zebrafish exposed to 100 mg/L kojic acid, the traveled distance was shorter by 27% than the distance attained by those in the control group. The higher doses of kojic acid (204 mg/L and 284 mg/L) led to an increased locomotor activity, distance traveled, number of arm entries, and the spontaneous alternation. The increase in spontaneous alternation demonstrates that 204 mg/L and 284 mg/L kojic acid doses had a stimulating effect on spatial memory. Novel tank test—compared to the control group, the traveled distance of the patulin-exposed fish is slightly reduced. Compared to the control group, the traveled distance of the kojic acid-exposed fish is reduced, due to a shorter mobile time (by 25–27% in the case of fish exposed to 204 mg/L and 284 mg/L kojic acid). Patulin and kojic acid exhibit toxic effects on zebrafish liver, kidney, and myocardium and leads to severe alteration. We continued the analysis by trying some machine learning algorithms on the classification problems in the case of the two behavioral tests MAZE and NTT, after which we concluded that the results were better in the case of the NTT test relative to the MAZE test and that the use of decision tree algorithms leads to amazing results, knowing that their hierarchical structure allows them to learn signals from both classes. Conclusions: The groups exposed to patulin and kojic acid show histological changes in the liver, kidneys, and myocardial muscle tissue. The novel tank test, which assesses exploratory behavior, has been shown to be conclusive in the behavioral analysis of fish that have been given toxins, demonstrating that the intoxicated fish had a decreased explorative behavior and increased anxiety. We were able to detect a machine learning algorithm in the category of decision trees, which can be trained to classify the behavior of fish that were given a toxin in the category of those used in the experiment, only by analyzing the characteristic features of the NTT Behavior Test.

Curcumin micronization by supercritical fluid: In vitro and in vivo biological relevance

Curcumin, a polyphenol extracted from the rhizome of Curcuma longa L. (Zingiberaceae), is shown to have antioxidant, anti-inflammatory, neuroprotective, anxiolytic, and antidepressant properties in both preclinical and clinical studies. However, its low bioavailability is a limitation for its potential adoption as a therapeutic agent. The process of micronization can overcome this barrier by reducing the particle size and increasing the dissolution rate, potentially improving the bioavailability of the compounds of interest. In this study, we compared the in vitro antioxidant effects of curcumin (CUR) and micronized curcumin (MC) and studied their effects on behavioral and neurochemical parameters in zebrafish submitted to unpredictable chronic stress (UCS). MC was processed by the solution enhanced dispersion by supercritical fluids (SEDS) technique obtaining a particle average size of 2.29 µm (8 MPa and 35 °C), which means a reduction of 5.4 times. MC (1 g/L) presented higher antioxidant activity in vitro as compared to CUR, as measured by iron-reducing antioxidant power (FRAP), 1,1-diphenyl-2–2-picyryl-hydrazyl radical removal (DPPH), and deoxyribose tests. UCS increased total distance traveled in the social interaction test (SI), while decreased crossings, time, and entries to the top area in the novel tank test (NTT). No effects of UCS were observed in the open tank test (OTT). The behavioral effects induced by UCS were not blocked by any curcumin preparation. UCS also decreased non-protein thiols (NPSH) levels, while increased glutathione reductase (GR) activity and thiobarbituric acid reactive substances (TBARS) levels on zebrafish brain. MC presented superior antioxidant properties than CUR in vivo, blocking the stress-induced neurochemical effects. Although this study did not measure the concentration of curcumin on the zebrafish brain, our results suggest that micronization increases the bioavailability of curcumin, potentiating its antioxidant activity both in vitro and in vivo. Our study also demonstrates that counteracting the oxidative imbalance induced by UCS is not sufficient to block its behavioral effects.

Positive welfare effects of physical enrichments from the nature-, functions- and feeling- based approaches in farmed rainbow trout (Oncorhynchus mykiss)

The present study aimed to investigate whether environmental enrichment had significant effects on rainbow trout's welfare through nature-, functions- and feeling-based approaches. Fish group behavior was analyzed during the rearing phase, as well as growth performance and health status. We assessed individual's emotional reactivity through a novel-tank test by measuring fear-related behaviors and stress-related physiological responses. Fish boldness and neophobia were then evaluated towards a novel object. We showed that more complex environments decreased aggression levels and improved growth, without impacting fish immune status. Enriched fish were also found less fearful when isolated in a novel tank and bolder when facing a novel object. We concluded that complexifying the environment through the addition of physical structures which stimulate and encourage fish to explore promotes rainbow trout's welfare in farming conditions, according to the three different welfare approaches.

The impact of water changes on stress and subject variation in a zebrafish (Danio rerio) anxiety-related task

BackgroundZebrafish have been widely used to study anxiety-related phenotypes using the novel tank test (NTT). Although the NTT is well-characterized and commonly used by researchers, there is still a lack of information regarding how different experimental variables such as water quality can influence NTT performance. Zebrafish use different chemical cues and olfactory stimuli to communicate in water, so we predicted that water change frequency would affect cortisol, locomotion and anxiety-related parameters in the NTT. New methodsAfter extensive literature research, we found that only about 18% of papers using NTT report partial or complete water changes between subjects. Here, we tested multiple zebrafish in the NTT using the same water up to 9 consecutive times (with no water change) and analyzed cortisol levels, as a stressrelated marker. ResultsWe found that when using the same water for more than 4 trials, data variability is increased and a higher number of extreme values is observed for the time spent in the top zone and immobility. Moreover, after 4 trials with no water change, increased cortisol levels are observed, indicating that animals show increased stressrelated responses with the lack of water changes. ConclusionsThis study shows that lack of water change can significantly influence zebrafish stress-responses in the NTT. Altogether, behavioral experiments should avoid using the same water when testing multiple fish in the task, especially when looking at anxiety in the NTT.

Effects of nicotinic acetylcholine receptor-activating alkaloids on anxiety-like behavior in zebrafish

Alkaloids are a structurally complex group of natural products that have a diverse range of biological activities and significant therapeutic applications. In this study, we examined the acute, anxiolytic-like effects of nicotinic acetylcholine receptor (nAChR)-activating alkaloids with reported neuropharmacological effects but whose effects on anxiety are less well understood. Because α4β2 nAChRs can regulate anxiety, we first demonstrated the functional activities of alkaloids on these receptors in vitro. Their effects on anxiety-like behavior in zebrafish were then examined using the zebrafish novel tank test (NTT). The NTT is a relatively high-throughput behavioral paradigm that takes advantage of the natural tendency of fish to dive down when stressed or anxious. We report for the first time that cotinine, anatabine, and methylanatabine may suppress this anxiety-driven zebrafish behavior after a single 20-min treatment. Effective concentrations of these alkaloids were well above the concentrations naturally found in plants and the concentrations needed to induce anxiolytic-like effect by nicotine. These alkaloids showed good receptor interactions at the α4β2 nAChR agonist site as demonstrated by in vitro binding and in silico docking model, although somewhat weaker than that for nicotine. Minimal or no significant effect of other compounds may have been due to low bioavailability of these compounds in the brain, which is supported by the in silico prediction of blood–brain barrier permeability. Taken together, our findings indicate that nicotine, although not risk-free, is the most potent anxiolytic-like alkaloid tested in this study, and other natural alkaloids may regulate anxiety as well.

Effects of Natural Monoamine Oxidase Inhibitors on Anxiety-Like Behavior in Zebrafish.

Monoamine oxidases (MAO) are a valuable class of mitochondrial enzymes with a critical role in neuromodulation. In this study, we investigated the effect of natural MAO inhibitors on novel environment-induced anxiety by using the zebrafish novel tank test (NTT). Because zebrafish spend more time at the bottom of the tank when they are anxious, anxiolytic compounds increase the time zebrafish spend at the top of the tank and vice versa. Using this paradigm, we found that harmane, norharmane, and 1,2,3,4-tetrahydroisoquinoline (TIQ) induce anxiolytic-like effects in zebrafish, causing them to spend more time at the top of the test tank and less time at the bottom. 2,3,6-trimethyl-1,4-naphtoquinone (TMN) induced an interesting mix of both anxiolytic- and anxiogenic-like effects during the first and second halves of the test, respectively. TIQ was unique in having no observable effect on general movement. Similarly, a reference MAO inhibitor clorgyline—but not pargyline—increased the time spent at the top in a concentration-dependent manner. We also demonstrated that the brain bioavailability of these compounds are high based on the ex vivo bioavailability assay and in silico prediction models, which support the notion that the observed effects on anxiety-like behavior in zebrafish were most likely due to the direct effect of these compounds in the brain. This study is the first investigation to demonstrate the anxiolytic-like effects of MAO inhibitors on novel environment-induced anxiety in zebrafish.

Early chronic hypoxia does not impact rainbow trout behaviour later in life

In trout aquaculture, tight control of water quality is crucial, especially for young life-history stages. This study examined whether chronic exposure to moderate hypoxia during the fry stage affects the rainbow trout’s behavior during later development. One month after yolk-sac resorption, young trout were exposed for 25 days to hypoxia, obtained by decreasing water flow and bubbling nitrogen gas into the tank (Hypoxia group, H: mean O2: 4.85 mg/L; min: 3.83, max: 6.87 mg/L). A control group was maintained in optimal conditions (C: mean O2: 10.41 mg/L; min: 10.12, max: 10.69 mg/L). At 4 days post-hypoxia, basal (resting) whole body cortisol levels were significantly higher in the H group (166.84 ± 20.67 ng/g) as compared to the C group (103.71 ± 8.78 ng/g), but no post-stress differences were evident (values ∼ 175 ng/g). Long-term effects of hypoxia on various behavioural traits were analysed between 3 and 4 months after the return to normoxia and compared to the C group. The exposure of fry to chronic hypoxia did not impact the fish’s behavioural repertoire (activity, stereotypic behaviour, aggression) in their home tanks after re-acclimation to normoxia. Moreover, basal and post-stress plasma cortisol levels, fear-related behaviours (novel-tank test), and risk-taking behaviours (emergence test) did not differ between the C and H groups. However, this early challenge resulted in an enhanced post-hypoxic growth rate in these fish. Our results show that early exposure to moderate hypoxia does not induce any behavioural changes in rainbow trout after several months of return to normoxia.

CHRONIC EXPOSURE TO METHYLPHENIDATE-CONTAMINATED WATER ELICITS SOCIAL IMPAIRMENT TO ZEBRAFISH

Residual contamination of water with MPH represents a severe environmental issue because it can affect non-target animals. Here we describe the behavioral effects in zebrafish after chronic contamination of water containing residues of MPH (0.1875, 1.875 and 3 ug/L). These doses are environmentally relevant since they reflect those found in wastewaters. We evaluated the behavioral effect through the novel tank test (NTT) and social preference test (SPT), and after euthanasia we analyzed oxidative stress parameters. Zebrafish exposed to MPH presented a social impairment, avoiding the conspecifics segment in the social preference test. In addition, MPH in the lowest concentration provoked an anxiolytic effect in the novel tank test. Oxidative stress is not related to these changes. Since the maintenance of an intact behavioral repertoire is crucial for species survival and fitness, our results demonstrate that residual contamination of water by MPH can be a threat to zebrafish, impacting directly to its well-being and survival in the aquatic environment.

Stimulants cocktail: Methylphenidate plus caffeine impairs memory and cognition and alters mitochondrial and oxidative status

Methylphenidate (MPH) is a psychostimulant widely misused to increase wakefulness by drivers and students. Also, MPH can be found in dietary supplements in a clandestine manner aiming to burst performance of physical exercise practitioners. The abusive use of high doses of caffeine (CAF) in these contexts is equally already known. Here, we demonstrate the behavioral, oxidative and mitochondrial effects after acute exposure to high doses of MPH (80 mg/L) and CAF (150 mg/L), alone or associated (80 mg/L + 150 mg/L, respectively). We used zebrafish as animal model due to its high translational relevance. We evaluated the behavioral effects using the Novel Tank Test (NTT), Social Preference Test (SPT) and Y-maze Task and analyzed biomarkers of oxidative stress and activity of mitochondrial respiratory chain complexes. MPH alone induced antisocial behavior. MPH inhibited lipid peroxidation. The association of MPH + CAF presented memory impairment and anxiogenic behavior. In oxidative status, it inhibited lipid peroxidation, increased protein carbonylation and mitochondrial complex II, III and IV activity. Our results demonstrate that MPH and CAF alone negatively impact the typical behavioral of zebrafish. When associated, changes in cognition, memory, oxidative and mitochondrial status are more relevant.

17-α-Ethinylestradiol modulates endocrine and behavioral responses to stress in zebrafish.

The synthetic estrogen, 17-α-ethinylestradiol (EE2), present in contraceptive pills, is an endocrine-disrupting chemical (EDC) that can be found in the aquatic environment. We examined the impacts of EE2 on zebrafish behavioral and physiological responses through the novel tank test (NTT), which measures anxiety-like behavior; the mirror-induced aggression (MIA) test, which measures aggressiveness; and the social preference test (SPT), which measures social cohesion. The steroid hormone levels were also measured. Here, we show that exposure to EE2 impairs stress responses by regulating the levels of specific hormones and eliciting an anxiolytic response, increasing aggression, and reducing social preference in zebrafish. In nature, these changes in behavior compromise reproduction and anti-predator behaviors, which, in turn, affects species survival. The maintenance of an intact behavioral repertoire in zebrafish is essential for their survival. Thus, our results point to the danger of environmental contamination with EE2 as it may alter the dynamics of the prey-predator relationship.

Probiotics reduce anxiety-related behavior in zebrafish

There is increasing evidence that gut microbiome could have effects on neurological processes and on behavior. In this study we used the novel tank test (NTT) to analyze zebrafish exploring behavior after four months’ supplementation with probiotics with probed antioxidant and anti-inflammatory properties. Results showed that prolonged ingestion of Lactobacillus rhamnosus CECT8361 and Bifidobacterium longum CECT7347 significantly alters the swimming pattern and mean swimming speed in the zebrafish model. After treatment, zebrafish strongly reduced their bottom-dwelling geotactic behavior when placed in a new tank, which could be correlated to a lower state of anxiety.