Novel Tank Test Research Articles

Protective mechanism of apigenin in proton pump inhibitor-associated progressive cognitive impairment in adult zebrafish via targeting GSK-3β pathway.

Cognitive impairment is characterized by memory loss and difficulty in focusing, remembering, adhering to directions, and solving problems; commonly seen in an elderly population. Apigenin (APG) (4', 5, 7-trihydroxyflavone) is a flavonoid with several positive health benefits, including chemoprevention, antioxidant and can suppress inflammatory responses by inhibiting TNF-α and IL-1β levels. In this experimental study, we observed the possible neuroprotective effects of APG in the zebrafish model exposed to Lansoprazole (LPZ), a proton pump inhibitor known to induce cognitive impairment through hyperactivation of GSK-3β pathway. This experiment involves 12 adult zebrafish per group, where one group received LPZ (100mg) as a toxin for 7 days and APG (25, 50, and 100mg/kg) as treatment, while DPZ (5mg/kg) served as a standard comparison over the same period. Neurobehavioral tests such as T-Maze, Novel Tank Test (NTT), and Novel Object Recognition (NOR) were performed. Several biochemical assessments were also performed to evaluate the level of lipid peroxidation (LPO), glutathione (GSH), nitrite (NO), acetylcholinesterase activity (AChEs), catalase activity, neurotransmitters (GABA and glutamate), neuroinflammatory markers (IL-1β, TNF-α, and IL-10), and histopathological analysis. The results showed that apigenin enhanced memory function, improved neurotransmitter balance, decreased oxidative stress markers, regulated the production of proinflammatory cytokines, and inhibited GSK-3β activity. Additionally, the co-administration of a GSK-3β inhibitor further promoted neuroprotection and cognitive enhancement facilitated by apigenin, highlighting the importance of the GSK-3β signaling pathway. These findings highlight the potential of apigenin as a natural compound for mitigating cognitive dysfunction. However, this study should also include long-term toxicity assessments and deeper molecular analysis to elucidate Apigenin's mechanism of action fully. Future research should address these gaps to validate its therapeutic potential.

The number of conspecific alarm substance donors notably influences the behavioural responses of zebrafish subjected to a traumatic stress procedure

Zebrafish (Danio rerio) represents a complementary pre-clinical model in stress and anxiety research. Conspecific alarm substance (CAS), an alarm pheromone secreted by injured fish, acts as a warning signal and modulates fear responses. Given their schooling nature and that injury precedes CAS release, varying fresh CAS concentrations extracted from different numbers of CAS-donating zebrafish may uniquely influence trauma-related behaviours. Thus, we investigated the behaviour of juvenile and adult zebrafish exposed to traumatic stress protocols, in the presence of CAS extracted from varying numbers of donating zebrafish. Juveniles were assessed for anxiety and boldness in the light–dark and open field tests (LDT and OFT), while adults were assessed in the novel tank test (NTT) and novel OFT (nOFT). We found that (1) trauma minimally impacted juvenile behaviour regardless of donor-derived CAS concentrations, (2) trauma-exposed adults displayed reduced exploration and heightened risk-taking behaviours in the NTT and nOFT compared to control-exposed fish, (3) NTT and nOFT freezing behaviours were distinctly emulated in adult fish and (4) post-trauma behaviour in adults was influenced by the number of donors. Therefore, CAS concentration as determined by donor number has age-related effects on anxiety- and risk-taking behaviours in trauma-exposed zebrafish, a valuable finding for studies utilising fresh CAS as a stress trigger. While we did not directly investigate CAS concentration through serial dilution, our data are of significant translational and ethological relevance, highlighting the importance of in-house method standardization in stress-related studies utilizing fresh CAS as an alarm cue.

Enhancement of Cognitive Benefits and Anti-Anxiety Effects of Phytolacca americana Fruits in a Zebrafish (Danio rerio) Model of Scopolamine-Induced Memory Impairment.

Phytolacca americana fruits exhibit a wide range of biological activities, including antimicrobial, anti-inflammatory, and anticancer properties. This study aims to investigate the phenolic profile of hydroethanolic extracts from both fresh (PEC) and dried (PEU) fruits of P. americana using high-performance liquid chromatography (HPLC) and to evaluate their impact on anxiety-like behavior, memory, oxidative stress, and cholinergic status in zebrafish (Danio rerio, Tübingen strain) treated with scopolamine (SCO, 100 μM). Acute administration of PEC and PEU (0.1, 0.5, and 1 mg/L) was conducted for one hour per day. In silico analyses were performed to evaluate the pharmacokinetic characteristics of the phenolic compounds discerned in the two extracts, using platforms such as SwissAdme, Molinspiration, ProToX-III, AdmetLab 3.0, PKCSM, and PASS. Anxiety-like behavior and memory performance were assessed through specific behavioral assays, including the novel tank test (NTT), light/dark test (LD), novel approach test (NAT), Y-maze, and novel object recognition (NOR). Subsequently, the activity of acetylcholinesterase (AChE) and the extent of oxidative stress in the zebrafish brain were investigated. Our findings suggest that both PEC and PEU possess anxiolytic effects, alleviating SCO-induced anxiety and enhancing cognitive performance in amnesic zebrafish. Furthermore, these extracts demonstrated the ability to mitigate cholinergic deficits by inhibiting AChE activity and supporting antioxidant defense mechanisms through increased activity of antioxidant enzymes and reduced lipid and protein peroxidation. These results highlight the potential use of P. americana fruit extracts in managing anxiety and cognitive impairments related to dementia conditions.

Posttraumatic anxiety-like behaviour in zebrafish is dose-dependently attenuated by the alpha-2A receptor agonist, guanfacine.

Traumatic stress exposure increases noradrenaline (NA) release, which contributes to anxiety and impaired risk-appraisal. Guanfacine, a selective alpha-2A adrenergic receptor agonist, has been used to treat stress-related disorders characterised by impaired prefrontal cortex function. By acting on both presynaptic inhibitory autoreceptors and postsynaptic heteroreceptors, guanfacine attenuates stress reactivity and enhances cognition. However, its effectiveness in treating trauma-related anxiety and risk-taking behaviour remains unclear. Leveraging the advantages of zebrafish (Danio rerio ) as a sensitive and efficient preclinical model which is ideal for stress research, we explored the impact of traumatic stress exposure combined with varying concentrations of guanfacine in adult zebrafish. Zebrafish were evaluated for trauma-related anxiety using both the novel tank test (NTT) and a novel version of the open-field test (nOFT), the latter which was also used to investigate risk-taking behaviour. We found that (1) traumatic stress exposure led to heightened risk-taking behaviour in the nOFT, and (2) low-to-moderate concentrations of guanfacine (3-20 µg/L) attenuated anxiety-like, but not risk-taking behaviour, with the highest concentration (40 µg/L), showing no effect. These results highlight the complex role of NA in modulating dysregulated behaviours during traumatic events and indicate the potential of guanfacine for improving trauma-related anxiety and risk-taking behaviour.

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

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

Neurotoxicological effects of venlafaxine on Caenorhabditis elegans and Danio rerio

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)

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

Persistent and transgenerational effects of pesticide residues in zebrafish

Persistent and transgenerational effects of pesticide residues in zebrafish

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

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

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

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

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

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.