Just Keep Swimming
Assessing the Effect of Exercise on Learning in Zebrafish
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ABSTRACT
ABSTRACT
Zebrafish are a beneficial model organism to use in cognitive studies because they have similar physiological attributes and genetic makeup to humans. The first year of research was focused on whether exercised fish could learn an associative task, using a white light stimulus and a food reword, faster than non-exercised fish. This experiment did not demonstrate a benefit of exercise on learning due to the fact that the fish did not eat the food, eliminating the chance of them making a connection. To build off of that research, the second study focused on whether exercised fish, as compared to those unexercised, can more rapidly associate a certain colored light with a section of a decision chamber. Results suggest that exercised fish learned faster than non-exercised fish.
PRESENTATION
PRESENTATION
BACKGROUND
BACKGROUND
Associative learning is the process by which an animal learns to associate a certain response to a particular stimulus (Tan et al., 2022). Common examples would be when a child cleans their room and then receives a positive reaction from their parents (positive reinforcement) or if someone burns their hand from placing it on the stove and learns to associate it with pain (positive punishment). These examples are a type of learning called operant conditioning where rewards and punishment modify behavior (Skiner, 1963). Under operant conditioning there are four methods of associative learning: positive reinforcement, negative reinforcement, positive punishment, and negative punishment. Reinforcement is done with the intention of increasing a behavior, with either providing a reward (positive) or removing an undesirable stimulus (negative). Punishment is done with the intention of decreasing a behavior, with either adding an undesirable stimulus (positive) or taking away a positive stimulus (negative) (OpenStax and Lumen Learning, 2019). Associative learning is a complex type of cognition. Therefore, in order to best understand how learning occurs, it is important to build a firm understanding of cognition and the behavioral processes associated with consciousness.
Cognition is the behavioral process of acquiring knowledge and understanding and is an essential feature of human consciousness. This process involves problem solving, decision making, perception, judgment, memory, and language (OpenStax and Lumen Learning, 2019). However, when these processes malfunction, humans can suffer from cognitive disorders. Some examples are amnesia (the inability to remember events for a period of time), dementia (the impairment of memory and judgment), and delirium (disturbance in mental abilities that result in reduced awareness of surroundings and confused thinking), which are illnesses that include symptoms connected to attention, thought, and perception (Berryhill et al., 2012). New discoveries are essential to learn more about these problems and find treatments to relieve human suffering (Nichols and Newsome, 1999). However, it’s extremely challenging to study these disorders because human behavior is complex and there are ethical limits on the types of experiments that one can perform on people. For these reasons, animal models of human cognition are a desirable approach to studying and understanding cognitive disorders.
Scientists are actively investigating the similarities between human and animal brain function and cognition to discover animal models that could be used to study cognitive disorders in humans. For example, German and Eisch reviewed the literature for papers testing transgenic mice that have been altered to contain mutations known to cause Alzheimer’s disease (2022). They found that transgenic mice with features of Alzheimer’s disease are a successful model for testing potential treatments for humans. For example, these models have been used to test if reduced brain levels of the Aß.42 protein (ß-amyloid) affect the progression of Alzheimer’s disease (Attar et al., 2013) (German and Eisch, 2022). In addition, Laming and McKinney studied normal goldfish, goldfish with telencephalic ablation (destruction of the forebrain), and goldfish with sham operations (surgery with no brain tissue destroyed) to see how their responses would differ to a “light-on” stimulus. Habituation, the diminishing of a physiological response to repeated stimuli, was measured in this experiment by comparing the heart and respiratory rates of the fish before and after the “light-on” stimulus. The time that it took for the fish to achieve habituation (no difference between cardiac and respiratory rates before and after the light stimulation) was compared between the experimental groups. The telencephalic ablation fish had slower habituation rates (impaired learning) as it took them a longer amount of time to not react to the light, demonstrating the importance of the forebrain for learning (Laming and McKinney, 1990). These examples illustrate why animal models are crucial for understanding cognition because certain types of experiments, such as destroying parts of brain tissue, can only be conducted ethically on animals. This research also demonstrates that cognition depends on healthy brain function. Therefore, it is important to understand what interventions, such as exercise, can be used to optimize brain health.
Exercise is the involvement of physical activity in order to improve physical fitness. For humans, exercise improves brain health and increases bone strength (CDC, 2022; Warburton, Nicol, Bredin, 2006; Warburton and Bredin, 2017). Using magnetic resonance imaging (MRI), Steventon et al. collected scans showing that exercise training over seven days increased hippocampal blood flow and microstructural changes (Steventon et al., 2021). Postmenopausal women who regularly exercised had 1.03% less bone loss than those who did not (Howe et al., 2011). Physical activity can also reduce the chances of getting chronic diseases like heart disease, cancer, or type two diabetes (U.S. Department of Health and Human Services, 2018; Colberg et al., 2016). According to Warburton and Bredin, there is a 20-30% decreased risk of developing chronic diseases with regular exercise (Warburton and Bredin, 2017). Aune et al. conducted a meta-analysis, a type of research that pools the results of multiple studies, examining the effects of exercise on the risk of developing diabetes. This research found that, when comparing high physical activity versus low physical activity, the risk of type two diabetes was reduced by 35% (Aune et al., 2015). But, in order for exercise to have a positive effect on one’s body, a certain amount of consistent, physical activity is needed. According to the US federal guidelines on physical activity, for substantial health benefits adults should do at least 150 to 300 minutes of moderate-intensity aerobic exercise - for children sixty minutes - or seventy-five minutes of vigorous-intensity aerobic physical activity each week. Moderate-intensity aerobic activity is defined as anything that gets one’s heart beating faster, whereas vigorous-intensity requires the highest amount of oxygen consumption such as running, swimming, or jumping rope. (U.S. Department of Health and Human Services, 2018). These guidelines were determined by a seventeen-member advisory committee that reviewed the literature of physical activity and health (Piercy and Troiano, 2018). In addition to exercise having an effect on overall health, it also can produce specific health benefits for the brain.
Regular physical activity has a broad range of effects on brain health including an increase in neuron growth, and improvements in sleep patterns and mental health symptoms. Liu and Nusslock reviewed the evidence that brain-derived neurotrophic factor (BDNF) is increased by exercise and proposed that BDNF in turn can increase the supply of blood to the brain and the amount of neurons grown in the hippocampus (Liu and Nusslock, 2018). Kline reviewed the relationship between exercise and sleep and found that exercise can be a treatment for sleep issues (Kline, 2014) which in turn can reduce impairment of cognitive functioning, an effect of sleep deprivation (Luyster et al., 2012). Rebar et al. conducted a meta-meta-analysis (a meta analysis of eight meta analyses) of the effect of physical activity on depression and anxiety and found that exercise decreased these mental health symptoms (Rebar et al., 2015). Ahlskog et al. reviewed the literature to investigate how exercise can help prevent or modify dementia and brain aging. They concluded that physical activity can help decrease the risk of developing dementia, including Alzheimer's, a disease that affects the part of the brain associated with learning and memory (Ahlskog et al., 2011). From these studies, it is clear that exercise has physical benefits and can also improve brain functions like learning and cognition. Thus, one should view exercise as a potential intervention to improve brain function.
Studies utilizing exercise to improve cognition have found that it improves a variety of brain functions. One of the brain functions that exercise can help improve is memory acquisition and recall. Pontifex et al. researched how physical activity affects long-term memory by giving ninety-two adults a list of items, measured physical activity throughout a 12 hour period, and then determined how much of the list they remembered afterward (2016). They found that the time that subjects engaged in physical activity relative to the memory task affected their learning and performance. The researchers found that participants who were physically active one hour prior to the memory task had long term memory retrieval that was significantly better than those who were sedentary. Conversely, the participants who had physical activity immediately after learning the list of items had significantly poorer long term memory retrieval than those who were sedentary during that time (Pontifex et al., 2016). Exercise has also been found to improve motor skills in children. Burns et al. researched the effect exercise had on gross motor skill development (e.g., walking and throwing) by assessing a group of 1,460 school-aged children’s performance in their physical education class. They found that these children improved their gross motor skills by 10% over the course of the 12-weeks (Burns et al., 2017). In addition, exercise can help improve executive functioning (the use of mental skills to help someone plan and achieve goals). Moreau et al. studied if six-weeks of high-intensity training enhanced executive functioning in children, and found statistically significant improvements in measures of cognitive control and working memory as compared to control subjects (Moreau, Kirk, and Waldie, 2017). To determine if exercise improves learning a second language, Liu et al. had a randomized group of forty young adults either ride bikes or be sedentary while being taught. The researchers found that the exercised group had statistically significantly higher performance on the sentence semantic judgment test and word-picture verification task than the sedentary control group. These results show that exercise can help one learn a foreign language vocabulary because the word picture task tested vocabulary meaning and the sentence semantic judgment test evaluated sentence processing (Liu et al., 2017). Exercise not only improves cognitive performance, but it can also change the shape and size of brain regions.
Exercise can increase the volume of the hippocampus, which in turn improves episodic memory and spatial navigation (American Psychological Association, 2020). Aghjayan et al. conducted a study in which they did a meta-analysis of randomized controlled trials to see if aerobic exercise improves episodic memory in adulthood. Their results concluded that episodic memory is positively influenced by exercise for adults aged fifty-five and older (Aghjayan et al., 2022). In terms of improving spatial learning, researchers Herting and Nagel studied if aerobic fitness in adolescents had an influence on hippocampal size, learning, and memory using the virtual Morris Water Task (a water maze task of spatial learning) (Thornberry, Cimadevilla, and Commins, 2021). Their results showed that higher fitness was associated with better learning on the virtual Morris Water Task and larger hippocampal size (Herting and Nagel, 2012). Even though there are many valuable studies collected from human subjects, there are some limitations with this approach. For example, because humans lead complex lives, there are many potential confounding variables such as past learning history, differences in baseline physical health, and variations in environmental conditions (nutrition, stress) that can alter the results. A model organism that is easier to work with than humans, and in turn is potentially better for behavioral studies, is the zebrafish.
Zebrafish, commonly used in experiments regarding translational neuroscience and neurobehavioral studies, are good model organisms because of their similarity to human genetics and neurobehavior (Tan et al., 2022). Classified under the Cyprinidae family, these fish share 70% of the same nucleotide sequence and have analogous brain structures to humans (Kalueff et al., 2014). Zebrafish are also a good model organism because they are easy to maintain and easily bred, which provides many offspring for the researchers (Mrinalini, Tamilanban, and Kumar 2022). Zebrafish’s small size is also an advantage for research, especially when video tracking is used. Zebrafish are increasingly being considered as possible models for studying learning and cognitive processes that are similar to humans.
The genetic, psychological, and neuronal mechanisms that activate behavioral responses in a fish’s brain are similar to other animals and humans (Luchiari et al., 2021). There are also parts of the fish’s brain, specifically the forebrain, called the telencephalon (Cheng, Jesuthasan, and Penney, 2014) that perform cognitive tasks and processes such as executive functioning (Parker et al., 2013) that are similar to those in mammals. Zebrafish can exhibit different forms of learning abilities such as non-associative, associative, locomotor, and social/shoaling learning (Tan et al., 2022). Non-associative learning, such as habituation, is a change in response due to repeated exposure to a stimulus. Locomotor learning is a process where animals learn to move by using environmental feedback (Kawashima et al., 2016). Lastly, social/shoaling learning is the behavior of observing and copying other animals. One possible approach to testing how zebrafish can improve their associative learning and general cognition is by exercising them in a controlled research setting.
Throughout zebrafish research, different methods of exercising fish have been assessed and validated. One group of researchers, DePasquale and Leri, measured the effect exercise has on anxiety-like behavior by using a multi-channel swim tunnel where water was pumped from a sump tank through an inflow pipe to the fish tank (2018). In that tank, there was a honeycomb plastic mesh next to the pipe to establish a uniform water flow/current and to prevent fish from going too close to the pipe. The fish were placed into the tank and forced to swim against the current to be exercised for one hour per day, five days a week for six weeks. After consistent exercise, researchers found that physical activity reduced anxiety-like behavior in zebrafish (DePasquale & Leri, 2018). Another study, conducted by Gilbert, Zerulla, and Tierney researched how aging affected zebrafish swimming performance (2014). To exercise the fish, researchers placed them in a 10 L swimming tube that had a motor creating a consistent current. In order for the fish to stay in place, they had to swim against the current. After doing this exercise routine for at least thirty minutes (or until the fish fatigued) once a week for four weeks, they found that swimming performance improved in young and middle-aged zebrafish but not older zebrafish. However, young and middle-aged zebrafish held onto part of the apparatus (by biting) more often than old zebrafish, which may have allowed them to have a recovery advantage which enabled them to swim longer (Gilbert, Zerulla, and Tierney, 2014). These studies show that exercise procedures are feasible for zebrafish research, but have been limited to swimming in tubes against a current and additional models are needed.
Zebrafish exercise research is a relatively new field and methods for measuring behavioral changes are still being developed (Gerali, 2020). Current methods involving exercise have been limited to forced water currents created by a motor. However, there may be other methods to exercise zebrafish that have not yet been studied. For example, since zebrafish are a shoaling species, there might be a difference in how efficiently they exercise in groups compared to alone. Other important aspects of the exercise procedure that have not yet been optimized are the speed at which the current is set and whether the current can and should be varied. Researchers have not yet determined if it is better to have the speed go up in increments, gradually getting faster, or to keep the current at the same pace throughout the exercise trial. One hypothetical way of exercising fish that does not involve a current would be to scare them with a net to induce them to move back and forth between each side of the tank. This approach could be a more natural experience to trigger exercise, although one that could be too stressful. To optimize this approach, trials would need to be conducted to see how long a simulated predator attack would be necessary for the exercise to improve cognition. However, the best way to house fish for their optimal health and well-being for this type of exercise is not yet known (Lee, Paull, and Tyler, 2022). For example, practical considerations lead researchers to house as many fish in a small tank as possible, but those conditions are not optimal for fish well-being. It is not known whether or not enrichment, like plants, could increase their comfort and, in turn, improve their cognitive performance. Studies could be designed to examine how environmental conditions and exercise methods can be optimized to improve zebrafish exercise research. Ultimately, exercise experiments for zebrafish are a means to investigate other aspects of their physical health and behavior, which may have implications for human research, more specifically the effects on associative learning.
Zebrafish are increasingly being used to study associative learning (Gerlai, 2020). One group of researchers, Sison and Gerlai, studied the relationship between cue and reward by doing two classical learning tasks in a plus-sign shaped maze. In the first task, zebrafish were required to associate a visual cue (a red card placed at the end of one arm of the maze) with a food reward (Sison and Gerlai, 2010). For the paired group (experimental) the red card was placed in the same location where the food would come out, and for the unpaired group (control) the red card was not associated with the food location. The second task was a complex associative learning task (spatial) where the tank was placed on a turntable and food was delivered to the experimental group in the same arm of the plus maze when the tank was oriented a certain way in the room. The fish were required to look for cues outside the maze (eg. fluorescent lighting, equipment in the room) to know when they were going to receive food. The control group received food randomly having no correlation with the tank orientation in the room or place in the maze. For the simple associative task, the difference between the paired and unpaired group was significant (p<0.01) after twenty training trials, where the fish in the experimental group spent more time in the target arm than chance. The zebrafish that received a visual cue food reward pairing learned to associate being in the arm of the plus maze where the food was delivered and the fish in the unpaired group did not. For the complex spatial associative task, the paired group that received food at a constant spatially defined location in the room showed a significant preference (p<0.05) for the target location as compared to the unpaired fish who did not have that same connection. The zebrafish in the paired group learned to associate the exact placement of the tank in the room with receiving food in a certain location in the maze, whereas the unpaired group did not (Sison & Gerlai, 2010). These experiments show that zebrafish are a good model for testing associative learning, which opens the possibility to combine associative learning tasks with other experimental procedures.
Similar to other studies testing the effects of exercise on fish, this year I replicated the research study done by Luchiari and Chacon (2013), to answer the question: Does exercise improve learning in zebrafish? The authors hypothesized that because exercise is expected to enhance brain functions, more specifically associative learning, the fish that were exercised would learn associative tasks faster than fish who were not. In this experiment, the fish were exercised over 20 days in a tube while swimming against a current created by a motor, using similar methods as the study done by Gilbert (2014). Exercised fish (experimental group) were then placed into a testing tank where they performed a light stimulus task daily over the course of a week in which the fish should learn to associate light with being fed. The unexercised fish (control group) were also expected to make this connection but slower than the exercised fish. This study found that the unexercised fish learned this association by the fifth day and the exercised fish made this association by the third day. The extent to which subjects associated the light stimulus with a food reward was measured using an “approach index”. The approach index was calculated by measuring the distance from the feeding area after the light stimulus minus the distance measured before the light stimulus. Using this metric, fish dispersion decreased after the light stimulus over the days for both the control group compared to day one (p=0.01) and the exercise group compared to day one (p=0.03). The control group showed significant approaching of the feeding area on day five, and the exercise group approached the feeding area from day three, which was significantly different between the two groups (p=0.004) (Luchiari and Chacon, 2013). Since the study done by Luchiari and Chacon is the only published study of the effects of exercise on associative learning in zebrafish, and this study only measured the effects of white light as a stimulus, there is a knowledge gap as to the effects of other stimuli on this type of learning under the conditions of exercise.
Herein, I will present a novel research project to fill the aforementioned knowledge gap. I hypothesize that exercised zebrafish, as compared to those unexercised, will more rapidly associate a certain colored light with navigating a plus-shaped maze to find food. I will complete the same exercise process as described by Luchiarai and Chacon; however, instead of having the fish complete the white light stimulus test, I will place them into a decision chamber where they will have to learn the association between a certain colored light and a side of the tank. This study will contribute to the field of zebrafish associative learning, solidify previously published research on the beneficial effects of exercise, and support zebrafish as a good model organism for understanding cognition in general.
Zebrafish offer several advantages over human test subjects as a model for studying associative learning tasks. Research has shown that exercise has measurable benefits for brain function in a variety of ways including improved cognition and, more specifically, associative learning. However, there has been limited research on how exercise can improve learning in zebrafish as well as the best methods to house and exercise zebrafish. Specifically, since limited stimuli have been tested for associated learning tasks, future research could develop these methods further by testing other stimuli. By studying the benefits of exercise on zebrafish cognition, scientists and doctors could potentially apply these findings to help people suffering from cognitive disorders such as dementia, amnesia, and delirium.
METHODS & PROCEDURE
METHODS & PROCEDURE
Overview of Experimental Procedures
The first part of the experiment was based on the results of a study conducted by Luchiarai and Chacon: “Physical exercise improves learning in zebrafish”. The article hypothesized that fish that were exercised would learn to respond to a white light stimulus faster over a course of days than fish that weren't exercised. This is because exercise is supposed to enhance brain functions, more specifically with learning. Based on this study, research was conducted to answer a novel experimental question on whether exercised fish can learn to respond to a colored light stimulus task within a plus maze faster than non-exercised fish. Similar to the replicate study, it was hypothesized that the exercised fish would learn the connection between a color and a certain direction in a plus maze faster than non-exercised fish. In these two experiments the exercised fish were the experimental group, exercise being the independent variable, while non-exercised fish were the negative control.
Safety
While handling the zebrafish and transporting them between tanks, personal protective equipment (lab coats and gloves) and heat resistant gloves (used in order to prevent burns when handling the hot glue gun) were worn. When using the bandsaw it is important to first follow the safety protocol instructions and pass the Bert bandsaw safety test linked here: https://bert.thebetalab.org/getting-started. When using the plexiglass cutter handle with caution as the edge of the cutter is sharp.
2.5 Gallon Tanks and 20 Gallon Tank Set Up
Four home tanks (Aqueon Product #18227) were placed next to an outlet. In order to fill the tanks up with water, a Saedy nine liter bucket (Product #B0C3D1N1S7) was filled up with water to the eight liter line. To ensure that the incoming fish were healthy and in safe water conditions, three conditioners and additives were added to the bucket of water: API Stress Coat Aquarium Water Conditioner (Product #B000255MZG), Seachem Stability Fish Tank Stabilizer (Product #B0002APIJG), and API Tap Water Conditioner (Product #B07BTMK89V). Using a New Canyon micropipette (Product #B08BFHBC7R), 1055 ul of these conditioners were placed into the bucket of water. The water was mixed using a Pawly Aquarium fish net (Product #B07G73V2QH) to ensure that the conditioner and additives were fully diluted and distributed throughout the water. The water was then transferred to the four home tanks using a 1L beaker. The home tanks were filled up so there was an inch from the water line to the top of the tank, requiring multiple buckets of conditioned water to be made and used. After the tanks were filled up with water, the Boxtech filter (Product #4346783286) was set up by following the instructions given in the package, placed on the back left hand side of the tank, and plugged into the outlet. Water was scooped from the tank into the top of the filter using a 1L beaker until water started to continuously pour from the lip of the filter. Next, a Hygger heater (Product #B0CKQNP8F4) was taken out of the packing, suction cupped to the back right hand side of the tank, and plugged into an outlet. The temperature was changed by pressing the “set” button on the cord to 79 degrees F (the temperature of the tank water should always be 79 +/- 1 degrees F). Lastly, thin tubing (PENN-PLAX Product #B0002563MM) was cut so that the air stone (Pawfly Aquarium Product #B076SBR2FB) was able to extend from the inside of the tank to the oxygenator (Active Aqua Product #B07J3GNGZN). One side of the tubing was attached to the oxygenator and the other side of the tubing was pushed over the air stone. The air stone was then dropped into the fish tank. The fish were able to be transferred to the home tanks once the conditioned water and the filter, heater, air stone were placed within them and once they were done acclimating.
A Tetra 20 gallon tank (Product #B08BJ9FQY7) was used as the exercising and acclimation tank for newly arrived zebrafish. The water conditioning done for the four home tanks was repeated in order to fill up the 20 gallon tank. After the tank was filled up with water, the Tetra filter (Product #B01NC2P7BM) was set up by following the instructions given in the package, placed on the back left hand side of the tank, and plugged into the outlet. The Tetra Submersible Aquarium Heater (Product #B000OQRJSG) was suction cupped to the right hand side of the tank and plugged into the outlet. The knob controlling the temperature of the heater was set to 79 degrees F (as explained in the home tank set up, the temperature of the tank water should always be 79 +/- 1 degrees F). Instead of using an oxygenator, the 20 gallon tank used an air pump (Tetra Product #B0009YJ4N6). Similar to the home tank set up, a thin tubing was cut so that the air stone was able to extend from the inside of the tank to the air pump. One side of the tubing was attached to the end of the air pump while the other side of the tubing was pushed over the air stone. The air stone was then dropped into the fish tank. This life support equipment (filters, heaters) differs from those used for the 2.5 gallon tanks because the size of the tank is bigger. New zebrafish can be placed inside the 20 gallon tank for acclimation after the tank is set up and has been cycling for 24 hours.
Care and Handling of Zebrafish
A group of 24 zebrafish were purchased from The Wet Spot, which were shipped in a bag with oxygenated water (Product: Danio Rerio). When they arrived, the bags were placed in the 20 gallon testing tank water for fifteen minutes so that the temperature of the water inside the bag matched the tanks’ temperature. Then, the top of the bag was cut and the fish were poured into a 1 L beaker. In order for the fish to acclimate, some water from the beaker was poured into the sink, a net covered the top to ensure that no fish escaped, until only 500 mL of water was left in the beaker. 500 mL of conditioned tank water was put into the beaker using another 1 L beaker. This process was repeated three times so that almost the entire beaker was conditioned water. The zebrafish were then placed into the four home tanks (6 in each).
The fish’s health was maintained by feeding them carefully and having frequent water changes. The fish were fed every other day with a pinch of the TetraMin tropical flakes (Product #16106). The tank water was tested every other week, using test strips, to ensure that the fish were swimming in clean water (FUNSWTM Product #B08MZDLC15). If the water appeared unhealthy (when the Ph levels on the test strips do not read between 6.8-7.8 or the ammonia was a number higher than zero) then a water change was conducted: a quarter of the tank was emptied and was refilled with conditioned water, as done when setting up the home tanks. In an article titled “Maintenance of Fish Health in Aquaculture”, Assfer and Abunna explain that monitoring and maintaining the water quality of the tank is an important aspect of taking care of fish (Assfera and Abunna, 2018). In addition, when the water level became low in the tanks (evaporation from the heaters) they were also filled back up with conditioned water. When filling the water back up, the temperature of the fish tank should be 79 +/- 1 degrees Fahrenheit.
Exercising Fish
To manipulate the independent variable for the experiment, zebrafish were exercised five days a week for four weeks. A workout schedule was created to accommodate for three day weekends or days off from school. The workout schedule required the fish to be exercised three times a week (Monday, Wednesday, Friday during a five day week and Tuesday, Wednesday, Friday after a three day weekend). This workout schedule was different from last year; last year the fish were exercised every day, but to eliminate any risk of over-exercising the fish the schedule changed to three times a week (giving the fish a day off in between trials). To exercise the zebrafish, they were placed within a 20 gallon tank and inside an exercise tube. The 20 gallon tank was set up similar to that of the home tanks with a different filter, heater, and air pump. The exercise tube was created from a 4 to 5 inch diameter and 2 to 3 feet long tube (McMaster-Carr Product #8585K55). The tube was cut, using a bandsaw, to 22 centimeters long (Grizzly Industrial Product #G0948). Before using the bandsaw, ensure that the safety protocols are completed (as listed above). An elbow piece (McMaster-Carr Product #4880K28) was attached to one end of the tube and a mesh grate (FSWCCK Product #B0B7J3RY46) was hot glued (Gorilla Product #B07K798MK9) to the other. A plastic flexible sheet was wrapped around the edge of the tube, closest to the mesh grate, where there was four inches of plastic sheet above the tube (KTRIO Product #B07X7WYKP9). This was added to the tube halfway through the exercising period due to fish being sucked into the motor after leaving the exercise tube. This plastic sheet created a barrier so that all of the current was being pushed into the tube. The air pump (JEREPET Product #B09NNHMQN4) was placed on the bottom left hand side of the tank and was secured by a magnet (that accompanied the pump) on the outside of the tank. The pump was set to level 2.
From a beaker, six fish were poured inside the opening of the elbow of the tube and swam into the longer part (22 centimeters) of the exercise tube. The fish swam against a current made by air pumps facing inside the exercise tube. Many researchers, such as DePasquale and Leri and Pettinau et al., have efficiently used this exercise method (swimming against a pump) in their own research (DePasquale & Leri, 2018 and Pettinaue et al., 2022). The fish swam against the pump for four minutes at level two. This differs from last year’s exercise regime, where the motor was turned off when all of the fish left the tube. This part of the procedure changed because the new pump created a stronger current than last year's model. In order to not over-exercise the fish the current was turned off after four minutes. The pump was updated from last year because that motor only had one setting (Sears Product #A024929319) whereas the new pump has twenty different flow rate levels. After exercising, fish were placed inside one of the four home tanks to separate them from the other group of fish. The amount of fish that lasted the entire four minutes was recorded in a data table. This exercise process was repeated that same day with another group of six fish in order to have a total of twelve exercised zebrafish. After both groups of six had exercised, the number of fish from each group that lasted the full four minutes was averaged and that number was recorded in the data table. At first, the fish were immediately pushed out of the tube but as the days went on the zebrafish were able to withstand the current and swim against it for the full duration of the four minutes. Using the method described by Luchiari and Chacon (2013), the fish achieved a high level of respiratory fitness after many training sessions. This is similar to what these researchers reported from their experiment (Luchiari and Chacon, 2013).
Setting up the 5.5 Gallon Tank for Replicate Study
A Tetra 5.5 gallon tank (Product #B08BJBYB4S) was used for the replicate study. This tank was set up the same way and used the same equipment as the home tanks (conditioned water, filter, heater) except it used an air pump for 5.5 gallons (Tetra Product #B0009YJ4N6) as done in the 20 gallon set up. In order to set up the tank for the light stimulus task a clear plexiglass (DYCacrlic Product #B0BQGGKYXS) was cut using a plexiglass cutter (BAISALJI Product #B08Y7RZ1WD) to a height of 3 inches and a width of 10.5 inches. The plexi glass divider was glued using the Aqueon Silicone Sealant (Product #B0002ASD2K) an inch away from the left side of the tank where the top of the glass was right above the waterline. Sheets of white 8.5x11 inch paper were placed around the outside walls, covering the whole tank except for the long side of the tank that was directly facing the wall. This coverage made it so that the fish completing the task could not see the researcher about to feed them. A desk lamp (BOHON Product #B08SK4DMHR) was placed next to the tank positioned in which the head of the lamp was facing down in the middle of the tank. This tank set up was done by Luchiari and Chacon when they completed this same experiment (Luchiari and Chacon, 2013).
Replicate Study
To measure the effects of exercise on associated learning in zebrafish, fish completed a white light stimulus test. Non-exercised fish served as a negative control for this experiment. The non-exercised fish when exposed to the light stimulus task would show the baseline rate of learning. The exercised fish served as the experimental group and when their learning rate was compared to the non-exercised fish, the effect of exercise could be determined. The non-exercised fish were conditioned over five days to a white light stimulus task, a test done to test other aspects of fish learning (habituation) as seen by Lamming and McKinny’s research (Lamming and McKinny, 1990). Nine non-exercised fish, one at a time, were placed in a 5.5 gallon tank. The fish spent one minute acclimating to the new environment and then after that one minute, a white light (a desk lamp placed above the tank) was turned on for 10 seconds. As soon as the white light was turned off, a pinch of TetraMin tropical flakes were placed within the feeding area (diagram shown in the “Setting up the 5.5 Gallon Tank” section). The fish were recorded by using an iPhone throughout this process to track the amount of time it took for them to find and eat the food after the light stimulus. The dependent variable was the time that it took for the fish to enter the feeding area and eat the food and this time was recorded in the data table. The same process was repeated with the nine exercised fish. The data was collected by watching the videos and calculating how long (in seconds) it took for the fish to eat the food after the light stimulus. A two tailed correlated t-test was done to compare the amount of time it took the exercised fish and the non-exercised fish to enter the feeding area and eat the food. The t-test resulted in a p value greater than 0.05. These results suggest that there was no significant difference between the negative control and experimental arm. This data differs from the results Luchiari and Chacon found (Luchiari and Chacon, 2013). This means that the exercised fish did not learn faster than the non-exercised fish during the associative learning task, so this data did not support the article’s hypothesis.
Setting up the Testing Tank: Decision Chamber
Before beginning, Personal Protective Equipment (PPE), googles, was put on. A saw was used by a professional in order to cut the 24 x 48 inch piece of white acrylic plexiglass into one 24 x 24 inch piece (KLiHDSM Product #B00IW9691K). One of the 24 x 24 in pieces of white acrylic plexiglass was placed into the blade platform after ensuring the Laser Cutter (Product #B0CLRKPZJ1) was free of dust and place. GlowForge Pro was then used to create one 23.5 x 32 cm rectangle. In this rectangle, a 8 x 18 cm rectangle was created 14 cm from the top of the sheet and 2.5 cm from the leftmost side. A second 8 x 18 cm rectangle was created 2.5 cm to the right of the first, such that it was also 14 cm from the top and 2.5 cm from the rightmost side of the first 23.5 x 32 cm rectangle. These 8 x 18 cm rectangles were created to be the entrances to the decision chamber.
The file was saved as “[First and Last Initials] - Testing Aquarium Dividers Draft 1 and was exported to the Laser Cutter. The circular “start” button was pressed on the laser cutter. Once the laser cutter finished, it took two minutes before the plexiglass was ready, allowing the machine to clear out any fumes. Next, two sheets of plexiglass (Lesnlok Product #B097JQ3F1N) were cut, using a plexiglass cutter (BAISALJI Product #B08Y7RZ1WD), into two 4 inch x 10 inch rectangles. Using the same saw that was used to create the entrances, a 10 x 10 inch square was cut from the white acrylic sheet (KLiHDSM Product #B00IW9691K).
A Tetra ten gallon tank (Prodict #B09Y7M25BT) was set up in the same way as the home tanks. Once the tanks were set up, two LED light strips (MingDak Product #B07W2YG3C4) were plugged into an outlet nearby the tank. One LED light strip was suction cupped on the outside of the back side of the tank. The suction cups were dipped in water in order for them to stick to the tank better. In order to set up the LED lights for the blue light stimulus, the LED light strip was turned on and set it to the blue color. It was then turned off until needed (during the trials). The second LED light strip was suction cupped to the front side of the tank. The suction cups were dipped in water in order for them to stick to the tank better. In order to set up the LED lights for the yellow light stimulus, the LED light strip was turned on and set it to the yellow color. It was then turned off until needed (during the trials). The white acrylic cut out was placed vertically ten inches from the side of the left side of the tank. Positioned in which the openings were facing downward. The white acrylic divider was placed horizontally in between the white acrylic cut out and the left side of the tank, creating two chambers. The two plexiglass rectangles were placed right in front of each opening facing the acclimating section of the tank.
Current Study Negative Control
The negative control is an experiment with a known outcome that shows no correlation between two variables. The control usually does not have a manipulated independent variable. This is helpful because it provides baseline data that the experimental arm can be compared to. In this case, the fish that were not exercised before they performed the associative learning maze test were the negative control. The control fish were tested Monday through Thursday in a decision chamber. Similar to that of the exercise regime, other researchers such as Sison and Gerali, Tierny et al., and Benvenutti et al. used a maze to test cognition and learning (Sison & Gerali, 2010, and Tierny et al., 2019, and Benvenutti et al., 2021).
Five fish, one at a time, were placed into the testing tank. Fish were poured from a 1L beaker into the bigger section of the tank (acclimating section). In this experiment, TetraMin tropical flakes (Product #16106) were placed in the left or right part of the decision chamber when a certain color was turned on; when the yellow light went on the food was placed in the left chamber and when the blue light was turned on the food was placed in the right chamber. These colors were chosen for the associative task based on the results of Nguyen and a team of researcher’s experiment where they conducted a light-based color preference study to see which colors zebrafish preferred (Nguyen et al., 2021). Each day the fish were trained twice, once with the blue light stimulus and once with the yellow light. Fish acclimated for three minutes and thirty seconds. After the acclimation period the blue light was turned on and the plexiglass blocking the entrance to the right chamber was removed. As soon as the fish entered the right chamber they were rewarded with food. Similarly, after the acclimation period, when the yellow light was turned on, the plexiglass blocking the left chamber was removed. Once the fish entered the left chamber they were rewarded with food. A stopwatch (Lavatools Product #B07G84DT5D) was used to time the trials to collect data on the amount of time it took the fish to go to the chamber. This test was done to see if fish were able to associate a color with a section of the tank. This research, using a colored light stimulus with a food reinforcement was also done by Buatois et al. (Buatois et al., 2023).
Current Study Experimental Arm
To measure any potential effects that exercise has on learning in zebrafish, the exercised zebrafish performed the colored light task as described above. For the experimental arm, zebrafish performed the same decision chamber light test that non-exercised fish completed. Comparing data with the negative control will help determine if the exercised fish can learn a connection between light and direction faster than non-exercised fish.
Data Collection
In order to collect data, the amount of time it took for the fish to enter the chamber was taken by using a stopwatch. The amount of seconds it took each fish a day was averaged and recorded in a Google Sheet. The mean time it took the non-exercised fish to eat the food, associated with the colored light, after the colored light stimulus was compared to the data from the exercised fish by a two tailed correlated t-test. The t-test results, a p value, show whether or not the two sets of data have a statistically significant difference. In order for data to be significantly different the p value needs to be less than 0.05. In this case, after completing a two tailed correlated t-test, the p value should be less than 0.05 to conclude that there was a statistically significant difference between the time in which the exercised fish learned the associative learning task than the non-exercised fish.
Materials List and Procedure (Click to View)
Materials List and Procedure (Click to View)
Materials:
One 16 oz bottle of Aqueon Water Conditioner (ASIN: B0010729SS).
One box of Tubing (for oxygenator) (Item #ST8).
2 bags of Zebrafish - unsexed (Item #145562).
Two 1 L Beakers.
One Marineland Penguin PRO Power Filter for up to 75 Gallons (Item # AQ-78181).
One Seachem Stability.
One black sharpie.
Scissors.
Pencil.
One sheet of Mesh (Item #B0B7J3RY46).
Two packs of TetraMin Tropical Fish Food (Item #16106).
4" - 5" diameter 2-3 feet long Tube.
One Elbow Piece.
iPhone.
Stopwatch.
One bottle of pH strips (Item #B097R5CSJS).
One pack of Plexiglass (Item #B097JQ3F1N).
Setting up the Home Tanks (2.5 gallon fish tank)
Get a 2.5 gallon fish tank (exercise home tank) and place it on an even counter nearby an outlet.
If the tank is on an uneven surface, it can lead to it tipping or falling.
Rinse it out with warm water to eliminate any dust in the tank.
Check the tanks for any cracks or anywhere that looks broken that will lead to a leak.
Fill up a 9 liter bucket with water to the 8 liter mark.
The temperature of the water does not matter.
Fill up the 10 mL graduated cylinder with 5 mL of conditioner. This will be used for 1.055 mL of conditioner.
Note: 1.055mL was found using the unit conversion from L to gallons and gallons to mL from the instructions on the bottle.
Grab the 100-1000 uL Volumetric pipette, use the knob at the end of the pipette and turn it so that the number displaced on the pipette reads 1000.
Grab the 20-200 uL Volumetric pipette, use the knob at the end of the pipette and turn it so that the number displaced on the pipette reads 55.
Using the pipette from step 6, press the plunger half way down and then dip the tip into the conditioner solution in the 10 mL graduated cylinder. Release the plunger to collect the conditioner.
Bring the pipette over to the bucket of water, dip the tip into the water and then press fully down on the plunger to release the conditioner.
Repeat steps 8-9 with the pipette from step 7.
Mix the water with the fish net and then pour it into the fish tank.
Repeat steps 4-11 to create more condition water if necessary and fill the tank until the tank water is 1 inch from the top.
Open the Boxtech Aquarium hang filter packaging and follow the instructions to put it together.
Put on the filter in the back left hand side of the fish tank.
See image below (circled in red).
Plug the filter in.
Use a 1000 mL sized beaker to scoop water from the tank into the top of the filter.
Repeat step 16 until the water starts continuously pouring from the lip of the filter.
Unpack the box containing the Freesa Aquarium Heater. Inside, there should be a heater, a thermometer, and a suction cup.
Attach the black suction cups to the Freesa Heater.
Using the knob on the top, set the heater to 79 +/- 1 degrees fahrenheit.
Using the suction cup, stick the heater to the side of the tank.
View image below.
Circled in red is the nob used to change the temperature.
Circled in blue is the suction cup.
22. Using suction cups, attach the device that displays/shows the temperature on the other side of the tank from the heater (in this case the left side), and keep the actual device outside of the tank.
23. Plug the heater into an outlet.
24. Repeat steps 4-5, but instead fill the bucket up to 4 liters and use .530 mL of conditioner.
25. Use the 100-1000 pipette and turn the knob on the top to 530.
26. Repeat steps 8-9 with the pipette from step 25.
27. Place an airstone into the bucket with the conditioned water and let it soak for 30 minutes.
28. Plug the oxygenator into an outlet.
29. Grab the tubing and cut it so that it will stretch from the oxygenator to the inside of the tank.
30. Attach one side of the tubing into the oxygenator.
31. Push the other side of the tube over the air stone.
32. Drop the airstone into the fish tank.
33. Take one of the clear breeder boxes and suction cup it to the bottom right hand side of the inside of the tank.
34. Put the top of the fish tank onto the fish tank.
35. Repeat steps 1-34 with another 2.5 gallon tank for the control home tank.
36. Do not place fish into the tank until it has been 24-48 hours since the fish tank has been cycling with the filters.
Once the time is up, proceed to the “Placing Fish into Home Tank” section.
Setting up two 20 Gallon Tanks (That will be used for the exercise tank and the testing tank)
Repeat steps 1-12 from the “setting up home tank” procedure but with a 20 gallon tank.
Set up the Marineland Power filter.
“Unbox the contents of the package containing the Marineland Power Filter.
Open the hinged lid of the filter. Inside, there should be one biowheel.
Get two Masters filter cartridges.
Place the cartridges into the bucket to soak.
While the cartridges are soaking, place the biowheel into their correct position and construct the extension tube.
Attach the extension tube to the bottom of the filter.
Take the filter cartridges out of the bucket and place them into the cartridge slots.
Close the hinged lid of the filter” (Swetow, 2022).
Put on the filter in the back left hand side of the fish tank.
Plug the filter in.
Use a 1000 mL sized beaker to scoop water from the tank into the top of the filter.
Repeat step 13 until the water starts continuously pouring from the lip of the filter.
Unpack the box containing the Tetra Submersible Aquarium heater. Inside, there should be a heater and suction cups.
Attach the two black suction cups to the heater.
Using the suction cups, stick the heater to the side of the tank using the suction cups attached to the heater.
Plug the heater into an outlet.
Repeat steps 24-26 from the “setting up home tanks” section.
Get the Penn Plax airline tubing and the Tetra Whisper air pump.
Use scissors to cut a 100 cm long piece of the Penn Plax tubing.
Note: Use caution when handling scissors.
Push the side of the tube described in step 21 over the outlet of the air pump (the knob sticking out of the air pump).
Push the other side of the tube over the air stone.
Drop the airstone into the fish tank and turn on the air pump by plugging it into an outlet.
Repeat steps 1-24 for the other 20 gallon tank.
Setting Up the Exercise Tank (made in steps 1-24 of the “Setting up the Two 20 Gallon Tank” section)
Take the clear long tube and use a bandsaw to cut it so it is 22 centimeters long.
Have a professional cut it.
Wear safety gear (safety goggles when standing near the cutting).
Take the elbow piece and connect/secure it to the end of a clear pipe.
Grab the mesh cap and use the hot glue gun to attach it to the other end of the pipe.
This will prevent the fish from swimming out of the pipe.
View image below.
Get the EcoPlus 75 GPH (284 LPH, 5.75W) pump.
Facing the 20 gallon tank use the suction cups to secure the pump on the bottom of the left side of the tank 3 inches from the front glass.
View image below.
Suction cup the second pump directly above the first pump.
Place the tube on the bottom of the tank where the mesh is directly touching the pumps (the part where the water flow exits).
Note: before each trial of exercising make sure the mesh cap is touching the pumps.
After the first 2 weeks of using this level pump, repeat steps 5-8 for the second level pumps.
Placing Fish into the Home Tank
The zebrafish will be shipped in a plastic bag. When the zebrafish arrived, immediately place the bag into the water of the home tank for 20 minutes to allow for the water in the bag to adjust to the temperature of the tank.
Get a 1 L beaker.
After waiting for 20 minutes, use a pair of scissors to cut open the top of the bag.
Gently pour the water (and zebrafish) from the bag into the 1 L beaker.
Over the sink pour out the water until it reaches the 500 mL line.
Using a net cover the top of the beaker so the fish do not fall into the sink.
Using a different beaker, scoop out water from the fish tank and fill up the 1 L beaker, from step 4, to the 1000 mL line.
Let the beaker sit for 5 minutes.
Repeat steps 5-7 for 3 more times.
This is changing the water from what they came in into the water made for them previously.
If there is more than one bag repeat steps 3-8 for as many bags there are.
Once the water “combination” has been completed, the fish need to be transferred into the 20 gallon tank.
Use a fish net to transfer all of the fish into the 20 gallon tank.
After the fish acclimated to the tank for three days (no deaths) fill up a 2L beaker of water from the fish tank and use a fish net to transfer 12 of the fish into the beaker.
Use a fish net to transfer the fish from the beaker into the control group home tank (set up in step 35 of the “setting up home tanks” section). Once all the fish have been transferred, put the lid of the fish tank back on.
“Note: Do not keep the fish out of water for more than 7 seconds when transferring”. (Swetow, 2022).
Note: Use a fish net on top of the beaker to prevent the zebrafish from jumping out.
Repeat steps 12-13 but move the other 12 of the fish into the exercise home tank (set up in steps 1-34 of the “setting up home tanks” section).
When all the fish are in the tank, feed them a pinch of the TetraMin Tropical Flakes food. If the food is consumed in under 30 seconds add another pinch.
Feed the fish this same amount once a day.
Check the temperature of the water by looking at the number displayed on the thermometer (set up in step 21 in the “Setting up the Home Tanks” section).
Do this every time the fish are fed.
If the temperature is not what it is supposed to be, adjust the heater to target temperature (79+/- 1 degrees F).
Once a week, check the pH levels by sticking the strip into the tank water and follow the directions on the bottle to compare the color on the strip to the key.
The pH level should be 6.8-7.8 but if it is not that, do a water change (partially empty out the water using a 1000 mL beaker and then repeat steps 1-12 from the “setting up home tanks” section.)
Once a week test the water for ammonia by using the “FreshWater Aquarium Test Kit”.
When using the test there is a small glass graduated cylinder with a top provided/ Collect 5 mL of the water from the fish tank into the cylinder.
Use the bottle labeled “Ammonia bottle #1”, and use 8 drops of that into the cylinder.
Use the bottle labeled “Ammonia bottle #2”, and use 8 drops of that into the cylinder.
Shake vigorously and wait 5 minutes to compare the color with the key from the kit.
If the water has high ammonia levels, do a water change (partially empty out the water using a 1000 mL beaker and then repeat steps 1-12 from the “setting up home tanks” section.)
Exercising the Fish
Check the “Workout Schedule for Zebrafish” table for what days to exercise the fish and what pump setting to use.
Hold the exercise tube so that the elbow of the tube is above the water line and the rest of the tube is submerged in the water.
Transfer a fish from the home exercised fish tank into the exercise tank’s exercise tube using a fish net.
Repeat steps 2-3 more times until 11 fish are safely in the exercise tube.
Only 9 will be used for the experiment but exercise two more in case there are deaths.
After placing the fish into the long tube, place the cap over the elbow so that the fish do not leave the tube.
Place the mesh of the long tube right up against the pumps.
Do this each trial.
Open the clock app on the iPhone.
Go to the stopwatch setting.
Once they are all in the long part of the tube, take off the cap and plug in the air pumps to turn it on.
Press start on the stopwatch.
Stop the timer when all the fish have exited the tube and into the open tank.
Write this data into the data table below.
Transfer the fish one at a time into the breeding boxes using the fish net.
Repeat steps 1-13 daily according to the Workout Schedule for Zebrafish table.
After all the exercise days are complete, proceed to the experimental arm procedure.
Creating the Dividers for the Testing Tank
“Put on Personal Protective Equipment (PPE).
Because this procedure involves glassware, PPE is goggles.
Note: Do not remove PPE at any time during the procedure.
Obtain the white acrylic plexiglass sheet, aquarium glue, a ruler, and a sharpie.
Use a saw to cut the 24 x 48 inch piece of white acrylic plexiglass into one 24 x 24 inch piece.
Note: This is extremely dangerous and should only be done by a professional.
Ensure the Laser Cutter is free of dust and place one of the 24 x 24 in pieces of white acrylic plexiglass into the blade platform.
Using GlowForge Pro on a desktop, create one 23.5 x 32 cm rectangles.
In the first rectangle, create a 8 x 18 cm rectangle, 14 cm from the top of the sheet and 2.5 cm from the leftmost side.
Create a second 8 x 18 cm rectangle, 2.5 cm to the right of the first, such that it is also 14 cm from the top and 2.5 cm from the rightmost side of the first 23.5 x 32 cm rectangle.
Note: These 8 x 18 cm rectangles are the entrances to the decision chamber.
Refer to the diagram below for a visual representation of steps 6-7.
Save the file as “[First and Last Initials] - Testing Aquarium Dividers Draft 1.”
Export the file to the Laser Cutter.
Press the circular “start” button on the laser cutter.
Once the laser cutter has finished, wait two minutes before retrieving the plexiglass to allow the machine to clear out any fumes.” (Ava MM, 2023-2024).
Use a plexiglass cutter to cut two 4 inch x 10 inch rectangles from a sheet of plexiglass.
This will be used to cover up the openings.
Use the same saw from step 2 and cut a 10 x 10 inch square from the white acrylic sheet.
This will be used to divide the two chambers.
Setting up the Testing Tank (10 gallon tank)
Complete steps 1-32 from the “setting up home tank” to fill up the 10 gallon tank.
Plug two LED light strips into an outlet nearby the tank.
Suction cup one LED light strip on the outside of the back side of the tank.
The suction cups might need to be placed in water first in order to stick better.
Turn on the LED light and set it to the blue color.
Turn off the LED light strip until needed (during the trials).
Suction cup the second LED light strip to the front side of the tank.
The suction cups might need to be placed in water first in order to stick better.
Turn on the LED light and set it to the yellow color.
Turn off the LED light strip until needed (during the trials).
Place the white acrylic cut out from steps 2-11 of the “Creating the Dividers for the Testing Tank” section vertically 10 inches from the side of the left side of the tank.
Make sure the openings are facing downward.
Place the diver from step 13 of the “Creating the Dividers for the Testing Tank” section horizontally in between the white acrylic cut out from step 9 and the left side of the tank.
This will create two chambers.
Refer to the diagram below.
Place the two plexiglass rectangles made in step 12 of the “Creating the Dividers for the Testing Tank” section right in front of each opening facing the acclimating section of the tank.
Refer to the diagram below.
Creating Google Drive to Store Data (Videos)
Create a Google Drive folder that will contain all the videos collected throughout this procedure.
Open up Google Drive on Google.
Sign in using a preferred email and password.
When logged into Google Drive, press “new” (which should be in the upper left hand corner if working on a laptop).
Press “new folder”.
Title the folder “Videos for Zebrafish Research (Negative Control)”.
Repeat step 5 and title the folder “Videos for Zebrafish Research (Experimental Arm).”
Negative Control (non-exercised zebrafish)
Open the clock app on the iPhone.
From the fishtank-home base, use a fish net to scoop and transfer one non-exercised fish to the test arena/tank.
Place the fish into the acclimating section of the testing tank.
Start the stopwatch in the clock app by pressing the “start” button.
Let the fish acclimate for 3 minutes and 30 seconds.
Turn the LED light strip on to blue.
Remove the divider blocking the right chamber.
Once the fish enters the right chamber, place the divider back on.
Place the food into the chamber.
This allows them to associate the blue light with the right side of the tank using food as a reward.
Once the fish enters the chamber, stop the timer.
Subtract the acclimating time (3 minutes and thirty seconds) from the total time.
This is the amount of time it took the fish to enter the chamber.
Record the time in the table below.
Take the fish out using the net and transfer it into the breeding boxes in the home control group fish tank.
If the fish did not eat all the food, use the fish net to remove the remaining food.
Repeat steps 1-14 4 more times using a different fish each time.
Repeat steps 1-6 but instead turn the LED light on to yellow.
Remove the divider blocking the left chamber.
Repeat steps 8-15.
Repeat steps 1-18 for another three more days.
Once the experimenting is complete, upload the videos to Google Drive folder titled “Videos for Zebrafish Research (Negative Control)” and name them according to the replicate.
Example: “Day 1 Fish 1 Trial 1”.
Clean up by transferring the fish using the fish net and place them in the clear breeding boxes in the negative control group home tank.
If running out of time, this would be a good place to stop for the day.
Experimental Arm (exercised zebrafish)
Repeat steps 1-16 of the negative control arm but use the exercised fish to complete this.
Clean up by placing the most recent test fish in the clear breeding boxes, using the fish net, in the exercised group home tank.
Place videos in the “Videos for Zebrafish Research (Experimental Arm)” Google Drive folder.
Final Test
Before testing make sure that the LED light strip is set to the blue color.
Place one exercised fish in the acclimating section of the tank.
Let the fish acclimate for 3 minutes and 30 seconds.
Turn on the LED light strip.
Remove both dividers to the chambers.
This gives the fish two options to swim to.
Once the fish enters either chamber, record which side the fish went to in the table below.
Place fish in a breeder box in the home tanks.
Before placing the second zebrafish in the testing tank, change the LED light strip and set it to the yellow color.
Place the second exercised fish in the acclimating section of the tank.
Repeat steps 3-7.
Before placing the third exercised fish in the testing tank set the LED light strip to the blue color.
Place the third exercised fish in the acclimating section of the tank.
Repeat steps 3-7.
Before placing the fourth exercised fish in the testing tank set the LED light strip to the yellow color.
Place the fourth exercised fish in the acclimating section of the tank.
Repeat steps 3-7.
Before placing the fifth exercised fish in the testing tanks set the LED light strip to the blue color.
Place the fifth exercised fish in the acclimating section of the tank.
Repeat steps 3-7.
Repeat steps 1-19 with non-exercised fish.
Data and Collection
Collect a computer.
Open Google.
It should be connected to the account made in the “Creating Google Drive to Store Data (Videos)” section of the procedure.
On the top right hand corner click on the 9 dots in the square.
Scroll until the app “Sheets” is visible.
Click on that app.
Under the label “start a new spreadsheet” press the blank option.
Title this google sheet “Data for Zebrafish Colored Light Decision Chamber Experiment”.
On the bottom where it says “Sheet 1” double click it and rename it “Data for Control Group Zebrafish Colored Light Decision Chamber Experiment”.
Press the plus button on the bottom left to create a new sheet.
On the bottom where it says “Sheet 2” double click it and rename it “Data for Exercised Zebrafish Colored Light Decision Chamber Experiment”.
Using the “Data for Control Group Zebrafish Colored Light Decision Chamber Experiment” google sheet, label the google sheet columns according to the example below.
Reference image.
Repeat step 11 for the “Data for Exercised Zebrafish Colored Light Decision Chamber Experiment” Google Sheet.
Open the Google Drive where all of the negative control videos are stored (the Google Drive folder called “Videos for Zebrafish Research (Negative Control)”.
Go in order in which the videos are labeled and click on the video “Day 1 Fish 1 Trial 1” (Then “Day 1 Fish 1 Trial 2, etc) to watch.
Record the data of how long it took for the fish to go to eat the food into the sheet according to its labels.
For example: for the first video, put the date “Date”, the number 1 under “Fish Number”, the number 1 under “Trial Number”, and the amount of time under the “Time Stamp at the Beginning”.
Repeat steps 13-15 for the remainder of the videos in this google drive folder.
Repeat steps 13-16 for the Google Drive folder named “Videos for Zebrafish Research (Experimental Arm)”.
PROCEDURE FIGURES AND CHARTS
PROCEDURE FIGURES AND CHARTS
Step 14
Step 5
Step 11
Step 21
Step 7
Workout Schedule
Step 3
Step 11
Data Collection
RESULTS AND DISCUSSION
RESULTS AND DISCUSSION
Results
Junior year research was replicated based on the study conducted by Luchiari and Chacon where they exercised fish and had them complete an associative task in which they associated white light with a food reward. Their results supported their hypothesis that exercised fish would learn the associative task faster than non-exercised fish (2013). During junior year research, it was difficult to collect sufficient data due to the fact that the fish did not eat the food reward during light conditioning trials. This prevented them from forming a connection between the white light and food reward. Thus, the data collected (see supplemental materials) did not support the hypothesis that exercised fish can learn an associative task faster than non-exercised fish. To fill any further knowledge gap, during senior year, data was collected to compare the time it took for non-exercised and exercised fish to complete a colored light associative task using a food reward. It was hypothesized that the exercised fish would learn the task faster than non-exercised fish. Data collection occurred over the course of two weeks (testing only occurred during days two to five and days nine to twelve, four days for the negative control and four days for the experimental) when four non-exercised and five exercised fish completed the associative task. Each fish completed the blue and yellow stimulus task once a day, and they were tested across four days. Herein data presented may suggest that exercise helps zebrafish learn an associated task earlier in a training regiment. Not only do they learn the task earlier, but they also demonstrate improved learning completing the task with higher efficiency and fidelity at the end of a four day testing period.
Figure 1. Average Time (Seconds) for Non-Exercised Fish to Eat the Food After Colored Light Cue. Trial times for food consumption (seconds) after either the yellow or blue light cues were averaged together to get an average time to eat the food reward each day of training. Day 1= 128 +/- 47 seconds, Day 2 = 60 +/- 41 seconds, Day 3 = 118 +/- 71 seconds, Day 4 = 76 +/- 25 seconds. The error bars represent standard deviation of the mean. Two tailed correlated t-tests were conducted to compare the time it took to consume the food between day 1 and days 2-4. P value from day 1 and day 4 was < 0.05 and all the other t-tests had a p value > than 0.05.
There were four zebrafish that completed the negative control. The non-exercised fish were the negative control and this data is used for comparison to the experimental group (exercised fish). Day one of the training was used as a baseline since it was the first time the fish completed the task, therefore the most naive. Analyzing data was completed using a statistical t-test, comparing day one to the preceding days, to assess which day they had improved learning on. The time it takes to eat the food after the light stimulus is indicative of strong learning, meaning that shorter times are better. A two-tailed correlated t-test was performed comparing the time it took to consume the food on days one (128 +/- 47 seconds) and four (76 +/- 25 seconds), which showed a significant difference (p = .011, p < .05). Another two-tailed correlated t-test compared data from day one (128 +/- 47 seconds) and day three (118 +/- 71 seconds) and did not show significance (p = 0.67, p > 0.05). Lastly, a two-tailed independent t-test was conducted comparing the data from day one (128 +/- 47 seconds) and day two (60 +/- 41 seconds) and did not show a significant difference (p = .035, p> .05). These results suggest that the non-exercised fish did not learn the associative task until day four.
Figure 2. The average time (seconds) it took for the exercised fish to consume the food after the colored light cue. Trials for both the yellow light and the blue light are averaged together. Day 1 = 151 +/- 83 seconds, Day 2 = 122 +/- 80 seconds, Day 3 = 67 +/- 37 seconds, Day 4 = 33 +/- 31 seconds. The error bars represent standard deviation. Two tailed correlated t-tests were conducted to compare the time it took to consume the food on day 1 with days 2-4. P value from day 1 and day 4 was < 0.05 and p value from day 1 and day 4 was = 0.05, all other days were > than 0.05.
A two-tailed correlated t-test was performed comparing the time it took to consume the food on days one (151 +/- 83 seconds) and four (33 +/- 31 seconds) which showed a significant difference (p = 0.0016, p < .05). The results from this t-test suggest that the exercised fish learned the associative task by day four. To better understand how fast it took for the exercised fish to learn the associative task, another two-tailed independent t-test was done comparing the data from day one (151 +/- 83 seconds) and day three (67 +/- 37 seconds). The p-value was equal to 0.05 suggesting a trend toward a significant difference but not a clear indication that the fish learned by day three. One of the main sources of error is the limited amount of fish used per trial. If there had been more fish (greater than five) it is possible that there would have been a significant difference (p < 0.05). Lastly, a two-tailed correlated t-test showed that there was no significant difference (p = 0.47, p > .05) between day one and day two. By looking at the bar graph visually, the apparent pattern is that the time to eat the food gradually decreased throughout the four days from 151 to 33 seconds. The decrease in the time it took for the exercised fish to eat the food after the colored light stimulus suggests that exercise improves learning abilities in zebrafish.
Figure 3. A bar graph comparing the average time (seconds) it took for the non-exercised and exercised fish to consume the food after the colored light cue. The purple bars represent the experimental group (exercised fish) and the blue bars represent the negative control (non-exercised fish). The error bars represent standard deviation.
Figure 3 compares the data from the non-exercised and exercised fish side-by-side in a bar graph in order to visually compare the trends. A two tailed independent t-test was performed comparing exercised and unexercised fish each day of the training for days one to four. Significant differences in the time it took to get to the chamber occurred on day four (p = 0.0058, p < 0.05), suggesting that the exercised fish learned the associative task faster.
Figure 4. Percentage of the non-exercised and exercised fish that swam to the correct side after the colored light cue in the final test. Percentage of negative control = 25%, percentage of experimental arm = 80%.
Figure 4 shows the data collected from the final test after the learning training on day four. The final test was conducted to see if a fish could go into the correct section of the tank based on the colored light when both sections were unblocked. By looking at the graph it is clear that the exercised fish retained a better memory of the color associative task because the percent of exercised fish that went to the correct side of the tank was higher than the percent of non-exercised fish.
Discussion
Overall, the results of the study did not irrefutably show that exercised fish were able to learn faster than the non-exercised fish. Regardless if the fish were exercised or not, they all learned by day four, validating that the colored light associative training regiment is successful in general. The negative control, non-exercised fish, showed an expected outcome. When completing the training, it was crucial that the non-exercised fish showed significant learning during the four days and were successfully taught to associate a colored light with a food in a specific location. It was necessary in order to make the claim that fish in general, exercised or not, are able to learn this task within the time frame provided. Not only did the fish show that they are able to learn the task they, more importantly, only learned it by the last day. This is optimal data for a negative control because it showed that it took them the duration of the entire training period and provided a benchmark for the exercised fish to learn between days one and three. While the p-value from the t-test (Figure 2) comparing day one and three in the exercised fish was just barely significant (p = .05), this value implies a low confidence that the correlation is due to the exercise rather than random chance. However, these initial results are promising and are approaching significance, which suggests that the exercised fish are trending in the direction of learning faster than the non-exercised fish.
There is such uncertainty due to the small sample size. During the exercise and training process the amount of fish used in this experiment decreased to five, due to deaths. This is further supported by Figure 3 where the overall trend shows the exercised fish had a clear pattern of learning improvement compared to the non-exercised fish. However, across all subjects, there is still variability in the time it took them to learn as evidenced by the large standard deviation (error bars) in the graphs. The results from the final test also support this claim. By the end of the four days, the percentage of fish that were able to go to the correct side based on the colored light cue was higher in the exercised group (80% versus 25%). This shows that the exercised fish retained a better memory of the association of colored light and food. In addition to that piece of data, the exercised fish learned the task faster (p = 0.0058) on day four (Figure 3) than the non-exercised fish, further supporting my hypothesis.
The main source of potential error (that led to the large error bars on the graphs) was due to the limited number of fish used in the research. At the beginning of the study, the plan was to use nine fish for testing. However due to unexpected deaths, the amount of fish dropped from nine to seven at the beginning of the two week training period. In addition to this, there were fish that showed anxiety-like behavior (hiding in the bottom corner of the tank for the duration of the trial) and, therefore, were unable to complete the research which led to a decrease in the amount of fish used, and with that data points. If this research was to be done again, allowing the fish to acclimate to their home tanks for at least a week would eliminate that behavior. By the end of the two weeks, there were only five fish that produced sufficient data points. Having only a few fish tested limited the ability to show significant differences between the groups. With additional trials it's possible that the p-value would have been less than 0.05 for days one and three during the exercised fish trials. If this study were to be replicated, it would be valuable and crucial to use more data points. By adding data points, the effect of how fast the fish learned would be more prominent and the p-value from the t-tests would be more beneficial.
By looking at the results, bar graph trends indicate that exercise improves learning in zebrafish. This is significant due to the fact that zebrafish are a great model organism to continue to use in research studying human behavior because the nervous system health of zebrafish can give us insight to human health. This study could give scientists an understanding of how to increase learning ability in humans. Even though there was not a significant difference (due to the p-value being right on the cut off of 0.05) this study was able to further understand how exercise affects cognition, more specifically associative learning. It would be interesting to take this study a step further and add more chambers to the tank. By doing so, it would be even clearer if the fish can make an association due to less random chance. However, when doing this next experiment it would be beneficial to use more data points in order to truly see if there is a significant difference in the speed of which exercised fish learn the associative task versus non-exercised fish. To fill any further knowledge gaps, it would be useful to see how exercise could affect cognition in other forms of learning: non-associative tasks and memory.
To the knowledge of the researchers, this study was the first known use of a colored light associative task to investigate if exercise has an improvement on learning in zebrafish. Since this was a more challenging task than white light, a light usually used to test cognition, it showed that fish, especially those that are exercised, can even learn complex tasks in a short amount of time. This task was challenging due to the fact that there were two colors involved and with that, the fish had to be trained to go to a specific chamber out of two options, whereas during junior year research, using the white light, the fish only had one option to go to. Due to the limited amount of research done on exercise and zebrafish cognition, this was a great study to add to that field of research.
Ruby K.-M. '2024
Ruby K.-M. '2024
Science has always been my favorite subject in school, but I particularly am interested in psychology and neuroscience. How the brain functions, what parts of the brain are involved with cognition, and how we improve learning abilities are aspects of psychology and neuroscience that pushed me to work on associative learning in zebrafish. When I go to Skidmore for college I am going to be on the pre-med track and will major in either both psychology and neuroscience or just one.
SUPPLEMENTAL INFO AND FIGURES
SUPPLEMENTAL INFO AND FIGURES
Replicate Study Figures
Replicate Study Figures
Junior Year Data: Negative control (non-exercised fish) data from the replicate study.
Junior Year Data: Experimental arm (exercised fish) data from the replicate study.
Negative control (non-exercised) and experimental arm (exercised fish) data placed side by side.
Link to Lab Notebook
Link to Lab Notebook
WORKS CITED
WORKS CITED
Aghjayan, S. L., Bournias, T., Kang, C., Zhou, X., Stillman, C. M., Donofry, S. D., Kamarck, T.
W., Marsland, A. L., Voss, M. W., Fraundorf, S. H., & Erickson, K. I. (2022). Aerobic exercise improves episodic memory in late adulthood: a systematic review and meta-analysis. Communications medicine, 2, 15. https://doi.org/10.1038/s43856-022-00079-7
Ahlskog, J. E., Geda, Y. E., Graff-Radford, N. R., & Petersen, R. C. (2011). Physical
exercise as a preventive or disease-modifying treatment of dementia and brain aging. Mayo Clinic proceedings, 86(9), 876–884. https://doi.org/10.4065/mcp.2011.0252
American Psychological Association. (2020, March 4). Working out Boosts Brain Health. American Psychological Association. https://www.apa.org/topics/exercisefitness/stress#:~:text=Exercise%20can%20build%20the%20mind%27s%20muscles&text=In%20resea rch%20with%20rodents%20
Assefa, A., & Abunna, F. (2018). Maintenance of Fish Health in Aquaculture: Review of Epidemiological Approaches for Prevention and Control of Infectious Disease of Fish. Veterinary medicine international, 2018, 5432497. https://doi.org/10.1155/2018/5432497
Attar, A., Meral, D., Urbanc, B., & Bita, G. (2013, November 22). Assembly of amyloid β-protein variants containing familial alzheimer’s disease-linked amino acid substitutions. Bio-nanoimaging. https://www.sciencedirect.com/science/article/abs/pii/B9780123944313000389
Aune, D., Norat, T., Leitzmann, M., Tonstad, S., & Vatten, L. J. (2015). Physical activity and the risk of type 2 diabetes: a systematic review and dose-response meta-analysis. European journal of epidemiology, 30(7), 529–542. https://doi.org/10.1007/s10654-015-0056-z
Benvenutti, R., Marcon, M., Gallas-Lopes, M., de Mello, A. J., Herrmann, A. P., & Piato, A.
(2021). Swimming in the maze: An overview of maze apparatuses and protocols to assess zebrafish behavior. Neuroscience and biobehavioral reviews, 127, 761–778. https://doi.org/10.1016/j.neubiorev.2021.05.027
Berryhill, M.E. , Peterson, D., Jones, K., Tanoue, R., Cognitive Disorders, Editor(s): V.S.
Ramachandran, Encyclopedia of Human Behavior (Second Edition), Academic Press, 2012, Pages 536-542, ISBN 9780080961804, https://doi.org/10.1016/B978-0-12-375000-6.00096-3.
Buatois, A., Nguyen, S., Bailleul, C., & Gerlai, R. (2021). Colored-Light Preference in Zebrafish
(Danio rerio). Zebrafish, 18(4), 243–251. https://doi.org/10.1089/zeb.2020.1977
Buatois, A., Siddiqi, Z., Naim, S., Marawi, T., & Gerlai, R. (2023). A simple semi-automated home-tank method and procedure to explore classical associative learning in adult zebrafish - behavior research methods. Retrieved from https://link.springer.com/article/10.3758/s13428-023-02076-7
Burns, R. D., Fu, Y., Fang, Y., Hannon, J. C., & Brusseau, T. A. (2017). Effect of a 12-Week
Physical Activity Program on Gross Motor Skills in Children. Perceptual and motor skills, 124(6), 1121–1133. https://doi.org/10.1177/0031512517720566
Centers for Disease Control and Prevention. (2022, June 16). Benefits of physical activity. Centers for Disease Control and Prevention. https://www.cdc.gov/physicalactivity/basics/pahealth/index.htm#:~:text=Being%20physically%20active%20can%20improve,activity%20 gain%20some%20health%20benefits
Cheng, R. K., Jesuthasan, S. J., & Penney, T. B. (2014). Zebrafish forebrain and temporal
conditioning. Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 369(1637), 20120462. https://doi.org/10.1098/rstb.2012.0462
Colberg, S. R., Sigal, R. J., Yardley, J. E., Riddell, M. C., Dunstan, D. W., Dempsey, P. C.,
Horton, E. S., Castorino, K., & Tate, D. F. (2016). Physical Activity/Exercise and Diabetes: A Position Statement of the American Diabetes Association. Diabetes care, 39(11), 2065–2079. https://doi.org/10.2337/dc16-1728
DePasquale, C., & Leri, J. (2018). The influence of exercise on anxiety-like behavior in
zebrafish (Danio rerio). Behavioural processes, 157, 638–644. https://doi.org/10.1016/j.beproc.2018.04.006
Gerlai R. (2020). Evolutionary conservation, translational relevance and cognitive
function: The future of zebrafish in behavioral neuroscience. Neuroscience and biobehavioral reviews, 116, 426–435. https://doi.org/10.1016/j.neubiorev.2020.07.009
German, D. C., & Eisch, A. J. (2004). Mouse models of Alzheimer's disease: insight into
treatment. Reviews in the neurosciences, 15(5), 353–369. https://doi.org/10.1515/revneuro.2004.15.5.353
Gilbert, M. J., Zerulla, T. C., & Tierney, K. B. (2014). Zebrafish (Danio rerio) as a model for
the study of aging and exercise: physical ability and trainability decrease with age. Experimental gerontology, 50, 106–113. https://doi.org/10.1016/j.exger.2013.11.013
Herting, M. M., & Nagel, B. J. (2012). Aerobic fitness relates to learning on a
virtual Morris Water Task and hippocampal volume in adolescents. Behavioural brain research, 233(2), 517–525. https://doi.org/10.1016/j.bbr.2012.05.012
Howe, T. E., Shea, B., Dawson, L. J., Downie, F., Murray, A., Ross, C., Harbour, R. T.,
Caldwell, L. M., & Creed, G. (2011). Exercise for preventing and treating osteoporosis in postmenopausal women. The Cochrane database of systematic reviews, (7), CD000333. https://doi.org/10.1002/14651858.CD000333.pub2
Kalueff, A. V., Stewart, A. M., & Gerlai, R. (2014). Zebrafish as an emerging model for
studying complex brain disorders. Trends in pharmacological sciences, 35(2), 63–75. https://doi.org/10.1016/j.tips.2013.12.002
Kawashima, T., Zwart, M. F., Yang, C. T., Mensh, B. D., & Ahrens, M. B. (2016). The
Serotonergic System Tracks the Outcomes of Actions to Mediate Short-Term Motor Learning. Cell, 167(4), 933–946.e20. https://doi.org/10.1016/j.cell.2016.09.055
Kline C. E. (2014). The bidirectional relationship between exercise and sleep:
Implications for exercise adherence and sleep improvement. American journal of lifestyle medicine, 8(6), 375–379. https://doi.org/10.1177/1559827614544437
Laming, P. R., & McKinney, S. J. (1990). Habituation in goldfish (Carassius auratus) is
impaired by increased interstimulus interval, interval variability, and telencephalic ablation. Behavioral neuroscience, 104(6), 869–875. https://doi.org/10.1037//0735-7044.104.6.869
Lee, C. J., Paull, G. C., & Tyler, C. R. (2022). Improving zebrafish laboratory welfare and
scientific research through understanding their natural history. Biological reviews of the Cambridge Philosophical Society, 97(3), 1038–1056. https://doi.org/10.1111/brv.12831
Liu, F., Sulpizio, S., Kornpetpanee, S., & Job, R. (2017). It takes biking to learn:
Physical activity improves learning a second language. PloS one, 12(5), e0177624. https://doi.org/10.1371/journal.pone.0177624
Liu, P. Z., & Nusslock, R. (2018). Exercise-Mediated Neurogenesis in the Hippocampus
via BDNF. Frontiers in neuroscience, 12, 52. https://doi.org/10.3389/fnins.2018.00052
Luchiari, A. C., & Chacon, D. M. (2013). Physical exercise improves learning in zebrafish,
Danio rerio. Behavioural processes, 100, 44–47. https://doi.org/10.1016/j.beproc.2013.07.020
Luchiari, A. C., Málaga-Trillo, E., Tran, S., & Gerlai, R. (2021). Editorial: Zebrafish
Cognition and Behavior. Frontiers in behavioral neuroscience, 15, 659501. https://doi.org/10.3389/fnbeh.2021.659501
Luyster, F. S., Strollo, P. J., Jr, Zee, P. C., Walsh, J. K., & Boards of Directors of the
American Academy of Sleep Medicine and the Sleep Research Society (2012). Sleep: a health imperative. Sleep, 35(6), 727–734. https://doi.org/10.5665/sleep.1846
Moreau, D., Kirk, I. J., & Waldie, K. E. (2017). High-intensity training enhances executive
function in children in a randomized, placebo-controlled trial. eLife, 6, e25062. https://doi.org/10.7554/eLife.25062
Mrinalini, R., Tamilanban, T., Naveen Kumar, V., & Manasa, K. (2023). Zebrafish - The
Neurobehavioural Model in Trend. Neuroscience, 520, 95–118. https://doi.org/10.1016/j.neuroscience.2022.12.016
Nichols, M., Newsome, W. The neurobiology of cognition. Nature 402 (Suppl 6761),
C35–C38 (1999). https://doi.org/10.1038/35011531
OpenStax and Lumen Learning. (2019) Introduction to Psychology.
https://pressbooks.online.ucf.edu/lumenpsychology/
Parker, M. O., Brock, A. J., Walton, R. T., & Brennan, C. H. (2013). The role of zebrafish
(Danio rerio) in dissecting the genetics and neural circuits of executive function. Frontiers in neural circuits, 7, 63. https://doi.org/10.3389/fncir.2013.00063
Pettinau, L., Seppänen, E., Sikanen, A., & Anttila, K. (2022b). Aerobic exercise training with optimal intensity increases cardiac thermal tolerance in juvenile rainbow trout. Retrieved from https://www.frontiersin.org/articles/10.3389/fmars.2022.912720/full
Piercy, K. L., Troiano, R. P., Ballard, R. M., Carlson, S. A., Fulton, J. E., Galuska, D. A.,
George, S. M., & Olson, R. D. (2018). The Physical Activity Guidelines for Americans. JAMA, 320(19), 2020–2028.
https://doi.org/10.1001/jama.2018.14854
Pontifex, M. B., Gwizdala, K. L., Parks, A. C., Pfeiffer, K. A., & Fenn, K. M. (2016). The
Association between Physical Activity During the Day and Long-Term Memory Stability. Scientific reports, 6, 38148. https://doi.org/10.1038/srep38148
Rebar, A. L., Stanton, R., Geard, D., Short, C., Duncan, M. J., & Vandelanotte, C. (2015). A
meta-meta-analysis of the effect of physical activity on depression and anxiety in non-clinical adult populations. Health psychology review, 9(3), 366–378. https://doi.org/10.1080/17437199.2015.1022901
Sison, M., & Gerlai, R. (2010). Associative learning in zebrafish (Danio rerio) in the plus
maze. Behavioural brain research, 207(1), 99–104. https://doi.org/10.1016/j.bbr.2009.09.043
Skinner, B. F. (1963). Operant behavior. American Psychologist, 18(8), 503–515.
https://doi.org/10.1037/h0045185
Steventon, J. J., Chandler, H. L., Foster, C., Dingsdale, H., Germuska, M., Massey,
T., Parker, G., Wise, R. G., & Murphy, K. (2021). Changes in white matter microstructure and MRI-derived cerebral blood flow after 1-week of exercise training. Scientific reports, 11(1), 22061. https://doi.org/10.1038/s41598-021-01630-7
Tan, J. K., Nazar, F. H., Makpol, S., & Teoh, S. L. (2022). Zebrafish: A pharmacological model for learning and memory research. Molecules, 27(21), 7374. https://doi.org/10.3390/molecules27217374
Tierney, A.J., Baker, A., Forward, J. et al. (2019). Response and place learning in crayfish spatial
behavior. Learn Behav 47, 80–90 https://doi.org/10.3758/s13420-018-0345-y
Thornberry, C., Cimadevilla, J. M., & Commins, S. (2021). Virtual Morris water maze: opportunities and challenges. Reviews in the neurosciences, 32(8), 887–903. https://doi.org/10.1515/revneuro-2020-0149
US Department of Health and Human Services (2108) Physical Activity Guidelines for
Americans, 2nd Edition https://health.gov/sites/default/files/2019-09/Physical_Activity_Guidelines_2nd_edition.pdf
Warburton, D. E. R., & Bredin, S. S. D. (2017). Health benefits of physical activity: a
systematic review of current systematic reviews. Current opinion in cardiology, 32(5), 541–556. https://doi.org/10.1097/HCO.0000000000000437
Warburton, D. E., Nicol, C. W., & Bredin, S. S. (2006). Health benefits of physical activity:
the evidence. CMAJ : Canadian Medical Association journal = journal de l'Association medicale canadienne, 174(6), 801–809. https://doi.org/10.1503/cmaj.051351
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