Impact of ocean warming on a coral reef fish learning and memory.

Tropical ectotherms are highly sensitive to environmental warming, especially coral reef fishes, which are negatively impacted by an increase of a few degrees in ocean temperature. However, much of our understanding on the thermal sensitivity of reef fish is focused on a few traits (e.g., metabolism, reproduction) and we currently lack knowledge on warming effects on cognition, which may endanger decision-making and survival. Here, we investigated the effects of warming on learning and memory in a damselfish species, Acanthochromis polyacanthus. Fish were held at 28-28.5 °C (control group), 30-30.5 °C (moderate warming group) or 31.5-32 °C (high warming group) for 2 weeks, and then trained to associate a blue tag (cue) to the presence of a conspecific (reward). Following 20 training trials (5 days), fish were tested for associative learning (on the following day) and memory storage (after a 5-days interval). The control group A. polyacanthus showed learning of the task and memory retention after five days, but increasing water temperature impaired learning and memory. A thorough understanding of the effects of heat stress, cognition, and fitness is urgently required because cognition may be a key factor determining animals' performance in the predicted scenario of climate changes. Knowing how different species respond to warming can lead to better predictions of future community dynamics, and because it is species specific, it could pinpoint vulnerable/resilience species.

Different housing conditions for zebrafish: What are the effects?

Zebrafish is a popular experimental model in several research areas but little is known about the effects of using different strains or housing conditions. Poor control of genetic background and housing conditions could affect experimental results and data reproducibility. Here we investigated the effects of two possible sources of variation on zebrafish behaviour: fish origin and environmental parameters (light intensity, water temperature and noise). Zebrafish behaviour was then examined using the 'novel tank test', one of the most common paradigms used to assess anxiety-like behaviours in zebrafish. Our results show that an increase in light intensity alters fish behaviour, particularly freezing duration and distance from the bottom of the tank, indicating increased anxiety. Swimming activity increased at the lowest temperature (25 °C). However, different levels of background noise did not cause any significant changes in behaviour. Differences were also found between zebrafish strains and populations: while the AB strain from laboratory 1 was minimally influenced by variation in holding conditions, the AB strain from laboratory 2 was highly affected by changes in temperature, light, and background noise. Our study shows that variation in strains and holding conditions can significantly influence the results of behavioural testing and should be carefully considered in the experimental design and properly reported to improve data interpretation and reproducibility.

Expanding the use of homebase-related parameters to investigate how distinct stressful conditions affect zebrafish behaviors.

Stress is a physiological reaction that allows the organisms to cope with challenging situations daily. Thus, elucidating the behavioral outcomes following different stressors is of great importance in translational research. Here, we aimed to characterize the main factors which explain similarities and differences of two stress protocols on zebrafish exploratory activity. To answer this point, we performed behavioral analyses aiming to simplify the data structure associated with homebase-related measurements in an integrated manner. Adult zebrafish were exposed to conspecific alarm substance for 5 min (acute stress protocol - AS) or submitted to 7 days of unpredictable chronic stress (UCS). Immediately after AS or in the subsequent day following UCS (8th day), fish were individually tested in the open field and the behaviors were recorded for 30 min to posterior identification of homebase locations. For both protocols, behavioral clustering revealed two major clusters, grouping homebase- and locomotor-related parameters, respectively. While AS increased both positive and negative correlations between exploratory and locomotor endpoints, a significant increase in negative correlations was found in UCS-challenged fish. Comparison of the principal component analyses data set revealed a reduced exploratory activity using the homebase in AS group, while decreased locomotion in the periphery and anxiety-like behaviors were evidenced in UCS fish. In conclusion, our findings revealed a different structure of behavior in zebrafish following AS and UCS protocols, supporting the existence of distinct behavioral strategies to cope with acute and chronic stress. Furthermore, we expand the use of homebase-related measurements as a valuable tool to investigate complex behavioral modulations in future translational neuropsychiatry research.

Fish and chips: Using machine learning to estimate the effects of basal cortisol on fish foraging behavior.

Foraging is an essential behavior for animal survival and requires both learning and decision-making skills. However, despite its relevance and ubiquity, there is still no effective mathematical framework to adequately estimate foraging performance that also takes interindividual variability into account. In this work, foraging performance is evaluated in the context of multi-armed bandit (MAB) problems by means of a biological model and a machine learning algorithm. Siamese fighting fish (Betta splendens) were used as a biological model and their ability to forage was assessed in a four-arm cross-maze over 21 trials. It was observed that fish performance varies according to their basal cortisol levels, i.e., a reduced average reward is associated with low and high levels of basal cortisol, while the optimal level maximizes foraging performance. In addition, we suggest the adoption of the epsilon-greedy algorithm to deal with the exploration-exploitation tradeoff and simulate foraging decisions. The algorithm provided results closely related to the biological model and allowed the normalized basal cortisol levels to be correlated with a corresponding tuning parameter. The obtained results indicate that machine learning, by helping to shed light on the intrinsic relationships between physiological parameters and animal behavior, can be a powerful tool for studying animal cognition and behavioral sciences.

Sound stimulus effects on dusky damselfish behavior and cognition.

Anthropogenic noises are widespread and affect marine wildlife. Despite the growing knowledge on noise pollution in the marine environment, its effects on fish cognition are scarce. Here, we investigated the effects of sound exposure on anxiety-like behavior and memory retention on dusky damselfish Stegastes fuscus. The animals were trained in a conditioned place aversion task, and exposed to two daily sessions of music at intensities of 60-70 dBA or 90-100 dBA, while the control group was kept at 42-46 dBA (no music) for five days. After that, fish were tested in the novel tank paradigm and tested for the memory of the aversive task. In the novel tank, animals exposed to sound spent more time still and decreased the distance from the bottom of the tank. Animals also spent more time on the aversive side of the conditioning tank. These results suggest that anthropogenic noise applied through high-intensity music can increase anxiety and decrease memory retention in S. fuscus, suggesting the deleterious potential of noise for reef species.

Alcohol exposure and environmental enrichment effects on contextual fear conditioning in zebrafish.

Environmental enrichment effectively reduces anxiety-like indicators in animals, a potential co-treatment for diseases that generate variations in basal anxiety, such as alcoholism. Here we present an experimental design that allows investigating the effect of enrichment on anxiety-related behaviors using contextual aversive conditioning in zebrafish (Danio rerio). It was first observed whether animals conditioned with an aversive stimulus (electroshock paired with checked background tank) exhibited behavioral variations when previously held in barren (BE) or enriched (EE) environment. Enrichment reduced anxiety-like behaviors. Then, fish was exposed to four alcohol concentrations (0.00%, 0.10%, 0.50%, and 1.00%) after being held in BE or EE, and contextual fear conditioning was tested again. Fish showed dose-dependent and enrichment-related variation in anxiety-like behavior. Together, these results indicate that the experimental design in question is efficient in measuring behavior related to BE and EE, and that enrichment seems to impair the acquisition of dose-dependent effects of alcohol. Our results show that EE can alleviate deleterious effects caused by traumatic events, but it should also consider some effects of enrichment and alcohol exposure interaction.

Memory retention of appetitive and aversive conditioning in the damselfish Stegastes fuscus.

We compared the memory of damselfish Stegastes fuscus in an aversive and appetitive conditioning task. Fish were trained to associate the sides of the tank that corresponded to the presence of a positive (conspecific presence) or negative (electroshock) stimulus. After two conditioning sessions, they were tested for learning. The fish conditioned to the stimulus were then re-tested for memory retention after 5, 10 or 15 days. Both the positive and negative rewards were associated with a specific side of the tank, indicating learning ability. Additionally, in both contexts, S. fuscus stored the information learned and showed similar behavioural patterns after 5, 10 and 15 days, suggesting long-lasting memory. For the ecological context, long lasting memories of social encounters outcomes and negative experiences of threatening situations may confer advantages that ultimately affect fishes' fitness.

Individual differences in response to alcohol exposure in zebrafish (Danio rerio).

Personality traits are related to many aspects of one's life, including risk taking, sociability, and behavioral changes caused by psychoactive substances. This study aimed to investigate individual differences and behavioral changes due to alcohol exposure in zebrafish (Danio rerio). To that end, adult animals were separated into two behavioral profiles: bold and shy, according to their risk taking behavior in an emergence test. Bold and shy fish were allowed to explore a 5-chamber tank and were tested for exploration and sociability (shoaling behavior) following alcohol exposure. The acute drug exposure treatments were 0.0%, 0.1% and 0.5% (v/v%) alcohol. The behavioral parameters evaluated were average and maximum swimming speed, total distance traveled, total time spent immobile, and time spent near a shoal or exploring the tank. For the groups that received no alcohol (0.0% alcohol), shy individuals spent more time near the shoal than bold fish. However, 0.5% alcohol increased bold fish responsiveness to the shoal, while both 0.1% and 0.5% alcohol diminished shoaling in shy fish. Our results show that the behavioral profiles of zebrafish are affected differently by alcohol, with shy animals seemingly more sensitive to behavioral change due to drug exposure. Moreover, we confirm zebrafish as a model for alcohol induced functional (exploration and social behavior) changes that could be useful in high throughput drug screens.

Behavioral Changes Over Time Following Ayahuasca Exposure in Zebrafish.

The combined infusion of Banisteriopsis caapi stem and Psychotria viridis leaves, known as ayahuasca, has been used for centuries by indigenous tribes. The infusion is rich in N, N-dimethyltryptamine (DMT) and monoamine oxidase inhibitors, with properties similar to those of serotonin. Despite substantial progress in the development of new drugs to treat anxiety and depression, current treatments have several limitations. Alternative drugs, such as ayahuasca, may shed light on these disorders. Here, we present time-course behavioral changes induced by ayahuasca in zebrafish, as first step toward establishing an ideal concentration for pre-clinical evaluations. We exposed adult zebrafish to five concentrations of the ayahuasca infusion: 0 (control), 0.1, 0.5, 1, and 3 ml/L (n = 14 each group), and behavior was recorded for 60 min. We evaluated swimming speed, distance traveled, freezing and bottom dwelling every min for 60 min. Swimming speed and distance traveled decreased with an increase in ayahuasca concentration while freezing increased with 1 and 3 ml/L. Bottom dwelling increased with 1 and 3 ml/L, but declined with 0.1 ml/L. Our data suggest that small amounts of ayahuasca do not affect locomotion and reduce anxiety-like behavior in zebrafish, while increased doses of the drug lead to crescent anxiogenic effects. We conclude that the temporal analysis of zebrafish behavior is a sensitive method for the study of ayahuasca-induced functional changes in the vertebrate brain.

Associative learning in the multichamber tank: A new learning paradigm for zebrafish.

The zebrafish has been gaining prominence in the field of behavioural brain research as this species offers a good balance between system complexity and practical simplicity. While the number of studies examining the behaviour of zebrafish has exponentially increased over the past decade, the need is still substantial for paradigms capable of assessing cognitive and mnemonic characteristics of this species. Here we describe and utilize a novel visual discrimination task with which we evaluated acquisition of CS (colour)-US (sight of conspecifics) association in adult zebrafish. We report significant acquisition of CS-US association indicated by the increased time the fish spent in and the increased frequency of visits of the target chamber during a probe trial in the absence of reward. Given the simplicity of the apparatus and procedure, we conclude that the new task may be employed to assay learning and memory in adult zebrafish in an efficient manner.

Irish coffee: Effects of alcohol and caffeine on object discrimination in zebrafish.

Many studies regarding the effects of drugs investigate the acute and chronic use of alcohol, but only a few address the effects of caffeine and alcohol combined to the performance of the zebrafish in cognitive tasks. The zebrafish is an important model for studying the effects of drugs on learning, because it has large genetic similarities to humans and the non-invasive administration of the substances favors translational bias of research. In this study, we observed the effects of alcohol and caffeine on zebrafish cognition through an object discrimination test. We noticed that animals subjected to acute alcohol dose and those under alcohol or caffeine withdrawal did not show discrimination. When fish were treated with associated alcohol and caffeine, those chronically treated with alcohol and subjected to moderate acute dose of caffeine showed learning of the task. Our results reinforce the harmful effects of the alcohol use on cognitive tasks, and suggest that continued use of high doses of caffeine cause cognitive impairment during withdrawal of the substance. However, the acute use of caffeine appears to reverse the harmful effects of alcohol withdrawal, allowing discriminative performance equivalent to control fish. Finally, we reiterate the use of zebrafish as a model for drug effects screening and search for active compounds that modulate the alcohol and caffeine effects.

How Betta splendens finds its way.

This study investigated the Siamese fighting fish Betta splendens performance in associating a stimulus with a specific cue when distractors are present. After trained to associate a specific color cue to a stimulus (conspecific) in a tank containing three colored distractors, the fish was challenged to locate the exact place where the stimulus fish was presented. With only color cues as guides, the Siamese fighting fish spent most of its time close to the color where the stimulus fish was previously presented, regardless of the distractors. However, fish trained to associate an empty place (no cues) to a stimulus fish, and then tested to localize the specific zone where the stimulus was shown, succeeded to locate the place even without any obvious cues/distractors for orientation. This study confirms that Siamese fighting fish show good conditioned learning and cannot be distracted by other stimuli. In addition, an unexpected good performance in the absence of cues may suggest the Betta's ability to orientate by using another sensorial modalities, as magnetic orientation. Collectively, the results of this study confirm Betta as a valid and reliable model for learning and memory tests, and suggest more studies should be developed for the better understanding of the fish's spatial orientation mechanisms.

Acute and chronic alcohol administration: effects on performance of zebrafish in a latent learning task.

Alcohol abuse is a major medical problem. Zebrafish have been proposed to model alcohol related human disorders. Alcohol impairs learning and memory. Here, we analyze the effects of alcohol on performance of zebrafish in a recently developed latent learning paradigm. We employ a 2×3×2 experimental design (chronic×acute alcohol treatment×path blocked). The latent learning task had two phases: one, 30min long exploration trials (16 days, 1 trial/day) with left or right path of a complex maze blocked, and two, a subsequent probe trial with all paths open leading to a goal box that now contained stimulus fish. During the 16 days each fish received one of two chronic treatments: freshwater or 0.50% (v/v%) alcohol. Subsequently, fish were immersed for 1h in one of the following solutions: 0.00 (freshwater), 0.50% or 1.00% alcohol, the acute challenge. Behavior of fish was recorded during the probe trial that commenced immediately after the acute treatment. Path choices, latency to leave the start box and to enter the goal box, time spent in the goal box, distance traveled, and duration of freezing were quantified. We found that acute exposure to 1.00% alcohol after chronic freshwater disrupted learning performance, so did exposure to freshwater after chronic alcohol treatment (withdrawal). We also found exposure to chronic alcohol to diminish the effect of subsequent acute alcohol suggesting development of tolerance. Our results demonstrate that analysis of learning performance of zebrafish allows detection of alcohol-induced functional changes. The simplicity and scalability of the employed task also imply the utility of the zebrafish in high throughput drug screens.

A dose for the wiser is enough: the alcohol benefits for associative learning in zebrafish.

This study aimed to test seeking behavior caused by alcohol and the drug effects on learning in the zebrafish, Danio rerio. Three treatments were conducted: acute, chronic and withdrawal, using 0.10%, 0.25%, and 1.00% alcohol and control (0.00%) (vol/vol.%). For the drug seeking behavior, we used a place preference paradigm (shuttle box tank) before and after alcohol exposure in acute (single exposure) and chronic (7 days) treatments. We observed a change in the basal preference due to the association with alcohol only for 0.25% and 1.00% doses in both acute and chronic offering, indicating an alcohol-seeking behavior after the drug exposure. For the learning task, two treatments were tested: chronic alcohol exposure (26 days including the learning period) and alcohol withdrawal (15 days of alcohol exposure before the learning period). During the learning period, fish received light stimulus followed by food in a pre-defined area of the tank for 8 consecutive days. The low dose group (0.10%) learned the task by the 3rd day both in chronic and withdrawal treatments. The higher doses (0.25% and 1.00%) caused a learning impairment in the chronic treatment group, while fish from the alcohol withdrawal treatment displayed learning on the final testing day. Therefore, we suggest that high alcohol doses impair learning and cause drug seeking behavior, even after drug exposure cessation, while low doses positively affect learning and do not cause seeking behavior. Given our results we propose that the zebrafish is a promising model for identifying active compounds, antibodies or genes which modulate the alcohol dual effects: learning improvement and reinforcing behavior.