Using live vertebrate animals to demonstrate learning and memory is typically not done in high school biology classes. We designed an apparatus and protocol by which students observe learning in fish. Students generate questions and discover answers (e.g., does age, sex, species, or chemical exposure impact learning and memory outcomes)?

Introduction

Getting students to be scientifically literate and possess analytical skills to critically assess what they hear and read can be a challenge. Since zebrafish are increasingly used to model biological processes (Wilk et al., 2018) and behavior (Templeton, 2017) in high school biology classes, we designed a method so that students can ask questions specifically about learning and memory that are relevant to them. Students can develop experiments that lead to a data-determined realization that even small amounts of chemicals can substantially affect learning and memory. Our approach uses research-based teaching strategies to engage learners by fostering questions that lead to outcomes promoted by the Next Generation Science Standards (NGSS).

The questions for teachers are (1) how are students provided with hands-on experiences that effectively model learning and (2) how do teachers provide a foundation for students to examine simultaneous variables (e.g., chemical exposure at different ages)? Finding answers to such questions in most high school settings is difficult because using animals, especially vertebrates, in classrooms to test hypotheses assessing learning (acquiring knowledge and/or skills) and memory (storing and recalling information) is generally not attempted. The difficulty of maintaining vertebrate animals in classrooms, short class periods in relation to the time it normally takes to conduct behavior experiments, the ability to use simple protocols that parallel those used in scientific laboratories, and the cost of apparatuses are significant barriers to conducting learning tests. It becomes unlikely, therefore, that students will have opportunities to collect meaningful data and analyze outcomes from classroom experiments investigating learning.

High schools and colleges use experiments examining simple learning in invertebrate species (Abramson, 1990; Phelps et al., 2004) but avoid experiments in the more complex processes of vertebrate cognition (College Board, 2012). Advance Placement (AP) Psychology courses do not use animal experimentation and limit lab exercises to examinations of human behavior (May & Einstein, 2013; College Board, 2014). In short, there is a significant gap in high school curricula using vertebrate models to study learning and memory in terms of the range of variables (e.g., age, sex, species, and chemical exposures) that affect learning outcomes. We have met this challenge by designing and field testing an apparatus that is portable, inexpensive ($20–30/unit), and simple to build, requires no electronics to operate, is easy for students to use, and allows students to design experiments that match their own personal interests using small fish from pet stores.

To ensure that the use of animals is ethical and scientifically justified, and that the animals are treated humanely and receive good-quality care, there is careful oversight by the Animal Care Program at UW-Milwaukee (UWM). In addition, teachers in the program undergo training in the ethical use of animals in research, which is taught by the UWM research animal veterinarian. Also, teachers must sign an agreement that they will care for the fish according to the regulations of the National Research Council (2011).

Why Use Fish?

Space is limited in most classrooms, and resources to keep animals are scarce. Small fish (e.g., minnows, zebrafish, comet goldfish, and guppies) serve as excellent models of complex learning and are easy to obtain and maintain, requiring minimal space to allow large numbers of animals to be held for statistically relevant sample sizes. These species are especially useful because they can be bred in classrooms in order to examine intergenerational learning abilities affected by specific environmental variables. While students assume that fish cannot learn complex tasks, fish do develop a decision hierarchy, show goal-oriented behaviors, and display both spatial and nonspatial discrimination abilities (Wyers, 1985; Arthur & Levin, 2001). In fact, fish can be trained quickly to do even complex tasks (view a goldfish “dribbling” a ball and “shooting” it through a hoop: http://psheplus.blogspot.com/). While fish brain anatomy has significant differences from the mammalian brain, the areas involved in learning and memory are both analogous (functionally similar) and homologous (developmentally similar) to it (Salas et al., 2003). This means that fish learning and learning deficits have parallels to mammalian behavior that are important for the students' understanding of themselves.

Goals & Objectives

Our apparatus (see Supplemental Material with the online version of this article) enables direct observations of cognition by using inquiry-approach methods to investigate student-driven hypotheses. Teachers who worked with us as we developed this module have identified numerous standards within the NGSS for which this learning and memory exercise applies (Table 1).

Table 1.
Comparing high school student performance expectations of the Next Generation Science Standards (NGSS Lead States, 2013) to learning and memory module outcomes.
NGSS Performance ExpectationsStudent Outcomes
HS-LS1-2: Develop and use a model to illustrate the hierarchical organization of interaction systems that provide specific functions at the organism system level such as nutrient uptake, water delivery and organism movement in response to neural stimuli. 
  • Students compare brain development in fish vs. human and relate that to behavioral controls.

  • Students assess interactions between sensory neurons, brain function, and behavioral outcomes in fish and humans.

 
HS-LS2-7: Design, evaluate and refine a solution for reducing the impacts of human activities on the environment and biodiversity. 
  • Students compare learning outcomes under different chemical exposure regimens (e.g., no exposure vs. exposure vs. exposure followed by no exposure vs. exposure followed by treatment with detoxifying agents).

  • Students compare learning outcomes using environmental enrichment techniques of their design to overcome effects of toxic exposures on learning.

 
HS-LS2-8: Evaluate the evidence for the role of group behavior on individual and species' chances to survive and reproduce. Emphasis is on: (1) distinguishing between group and individual behavior, (2) identifying evidence supporting the outcomes of group behavior, (3) developing logical and reasonable arguments based on evidence. 
  • Students can design experiments to examine effect of group vs. solitary housing on learning and memory.

  • Students can design experiments in which fish are tested individually or in groups to assess rate of learning and ability to remember learned tasks.

 
HS-LS3-3: Apply concepts of statistics and probability to support explanations that organisms with an advantageous heritable trait tend to increase in proportion to organisms lacking this trait. Emphasis is on analyzing shifts in numerical distribution of traits and using these shifts as evidence to support explanations. 
  • Students learn to breed fish (zebrafish or fathead minnows are easiest for classroom settings).

  • Students do crossbreeding to assess heritability of learning behavior.

 
HS-LS4-6: Create or revise a simulation to test a solution to mitigate adverse impacts of human activity on biodiversity. Emphasis is on designing solutions for a proposed problem related to threatened or endangered species or to genetic variation of organisms for multiple species. 
  • Students use techniques described above as a means to assess interspecific comparisons.

  • Students observe that mitigation techniques for one species may have different results for other species.

  • Students gain firsthand knowledge of the difficulty in designing solutions to mitigate adverse impacts of human activity on biodiversity.

 
NGSS Performance ExpectationsStudent Outcomes
HS-LS1-2: Develop and use a model to illustrate the hierarchical organization of interaction systems that provide specific functions at the organism system level such as nutrient uptake, water delivery and organism movement in response to neural stimuli. 
  • Students compare brain development in fish vs. human and relate that to behavioral controls.

  • Students assess interactions between sensory neurons, brain function, and behavioral outcomes in fish and humans.

 
HS-LS2-7: Design, evaluate and refine a solution for reducing the impacts of human activities on the environment and biodiversity. 
  • Students compare learning outcomes under different chemical exposure regimens (e.g., no exposure vs. exposure vs. exposure followed by no exposure vs. exposure followed by treatment with detoxifying agents).

  • Students compare learning outcomes using environmental enrichment techniques of their design to overcome effects of toxic exposures on learning.

 
HS-LS2-8: Evaluate the evidence for the role of group behavior on individual and species' chances to survive and reproduce. Emphasis is on: (1) distinguishing between group and individual behavior, (2) identifying evidence supporting the outcomes of group behavior, (3) developing logical and reasonable arguments based on evidence. 
  • Students can design experiments to examine effect of group vs. solitary housing on learning and memory.

  • Students can design experiments in which fish are tested individually or in groups to assess rate of learning and ability to remember learned tasks.

 
HS-LS3-3: Apply concepts of statistics and probability to support explanations that organisms with an advantageous heritable trait tend to increase in proportion to organisms lacking this trait. Emphasis is on analyzing shifts in numerical distribution of traits and using these shifts as evidence to support explanations. 
  • Students learn to breed fish (zebrafish or fathead minnows are easiest for classroom settings).

  • Students do crossbreeding to assess heritability of learning behavior.

 
HS-LS4-6: Create or revise a simulation to test a solution to mitigate adverse impacts of human activity on biodiversity. Emphasis is on designing solutions for a proposed problem related to threatened or endangered species or to genetic variation of organisms for multiple species. 
  • Students use techniques described above as a means to assess interspecific comparisons.

  • Students observe that mitigation techniques for one species may have different results for other species.

  • Students gain firsthand knowledge of the difficulty in designing solutions to mitigate adverse impacts of human activity on biodiversity.

 

Students are enabled to articulate and refine their own broad questions about cognition and environmental variables that may affect these processes. They become acquainted with appropriate tools and techniques to conduct a controlled learning experiment using live animals. They propose hypotheses that link their personal health to environmental hazards that will ultimately help in personal and social decision making. Students gather and analyze data regarding cognitive behaviors of control and experimental organisms. Finally, students interpret these data to draw conclusions, generate explanations, and predict trends in the effects of environmental agents on vertebrate cognition as compared to non-exposed animal models. Under teacher supervision, students will learn how to handle chemicals (including wearing of appropriate lab clothing), use varying chemical exposure regimens, and properly dispose of chemicals.

Using positive punishment (agitating water near fish for wrong choice, a method that avoids physical injury to the fish) and positive reinforcement (being left alone for correct choice), students test cognitive flexibility, the mental ability to restructure knowledge in multiple ways depending on changing situational demands. The ability to switch between thinking styles or to simultaneously think about several concepts is a key aspect of higher-level learning. It is used by students when taking tests requiring the application of knowledge.

Protocol for Left-Right Learning & Memory Test

Materials

  • One T-maze unit for each group (for building instructions, see Supplemental Material)

  • Clean containers to hold 4 L of dechlorinated water (to prevent adding any additional chemicals to the water, simply allow tap water to stand for one to two days to allow chlorine to volatilize) for each T-maze session

  • Holding tank (1 L beaker with aeration) for each fish being tested (renew water once per day)

  • Marking tape/pens to label tanks

  • One fish net for each T-Maze

  • Optional: One or two floor mats for each T-maze (to reduce danger of slipping on wet floor)

Building a T-Maze

T-mazes are simple apparatuses that provide fish with a straightforward choice – left or right. The T-maze (Figure 1) is easy to build, affordable, easy to store, easy to use, and allows complex behavior to be studied within a single class period. Two or three students and even a single, highly motivated student can conduct all the tasks.

Figure 1.

Completed T-maze and data collection roles (numbers) for each student in the group.

Figure 1.

Completed T-maze and data collection roles (numbers) for each student in the group.

Methods

Students should practice testing protocols until they feel comfortable conducting actual experiments. Students achieve competency within 15–20 minutes and are well prepared to start collecting data the following day. While our lab provides teachers with adult zebrafish that have been exposed to 0 or 10 μM Pb2+ during embryonic development to model human fetal exposures, students have used our approved chemicals list to identify items that have particular interest to them. They research that chemical and identify appropriate concentrations and exposure regimens for this module that will match their specific research question. Examples of the flexibility of the module for student-generated background research and hypotheses are embryonic exposures to 0 or 0.2 mg nicotine/L (see Figure 5B) and embryonic or lifetime exposures to either 0 or 22 μM tartrazine (yellow dye 5; see Figure 5C, D) before testing for changes in adult zebrafish learning.

Student Roles

Within each group there are specific tasks (Figure 1 – numbers in figure are associated with each step below). Each group member should practice each role so that everyone is competent in fish handling and T-maze operation.

  1. Start Box: Student opens and closes gate to release fish into raceway.

  2. Netting: When using positive punishment, student agitates water with handle of fish net, then nets fish out of maze and returns it to start box.

  3. Arm Gate: Student lowers gate of incorrect arm once fish has entered correct side.

  4. Data and Time: Student records data and keeps track of time fish is in start box.

The protocol (Figure 2) is done each day, where Day 1 = Training (i.e., shaping skill set), Days 2 and 3 = Testing (i.e., developing improvement in function), and Day 8 = Memory (i.e., stability of learned behavior).

Figure 2.

Testing protocol for learning and memory experiment involving acclimation to environment, training with positive reinforcement and punishment, and memory.

Figure 2.

Testing protocol for learning and memory experiment involving acclimation to environment, training with positive reinforcement and punishment, and memory.

Fish Acclimation

Fish are acclimated to the T-maze for 5 minutes only on Day 1 to assist them in learning about and feeling comfortable with their new surroundings. During this time there are no gates (except at starting block) in the T-maze. As an option, during the acclimation period, count the number of times a fish goes into each arm to identify a preference. Start actual testing with either the preferred or non-preferred arm as the correct choice. To be consistent and ensure that all data are comparable, the class should decide which arm initially will be “correct.” The directions below and Figure 3 assume that the RIGHT arm is the correct side in the initial phase of the reversal sequence.

Figure 3.

Student photographs of various steps in experiments using T-maze. Photographs provided by Sheridan Schaffer, Greendale (WI) High School (parental permission provided). (A) Place individual fish into starting block. (B) Allow fish to swim down raceway. (C) Fish chooses LEFT side, lower gate on RIGHT arm. (D) Vigorously stir for positive punishment.

Figure 3.

Student photographs of various steps in experiments using T-maze. Photographs provided by Sheridan Schaffer, Greendale (WI) High School (parental permission provided). (A) Place individual fish into starting block. (B) Allow fish to swim down raceway. (C) Fish chooses LEFT side, lower gate on RIGHT arm. (D) Vigorously stir for positive punishment.

Left-Right Discrimination (30–40 minutes)

  1. Place individual fish into starting block (Figure 3A).

  2. After 15 seconds, open starting block gate to allow fish to enter the raceway. Close gate.

  3. Allow fish to swim down raceway. If it enters the RIGHT arm lower the gate on the LEFT arm and record arm choice on your data sheet. Be sure fish is actually in T-maze arm before closing gate. Allow fish to remain undisturbed for 45 seconds. Then gently net it and place it back into the starting block for 15 seconds (Figure 3B). Repeat.

  4. If fish chooses the LEFT side, lower gate on the RIGHT arm (Figure 3C), agitate the LEFT side vigorously for 20 seconds (Figure 3D; student photograph shows right side but positive punishment procedure is same for either side), net fish, and place back into starting block for 15 seconds. Record choice on data sheet (Figure 4).

  5. Conduct correction routine. This step is included only when fish chooses the wrong side. No data are recorded during correction trials. This is a positive reinforcement procedure to help fish learn correct pattern.

    • Lower gate on the LEFT arm (i.e., wrong side).

    • Open starting box gate and let fish swim to correct arm. If necessary, use gentle touches with blunt probe to encourage fish to enter correct arm.

    • Allow fish to remain in correct arm for 45 seconds. Then gently place back in starting box.

    • Remove gate on incorrect arm and continue trials.

  6. Continue conducting trials until a maximum of 20 trials or five correct choices within a string of six trials occurs – whichever comes first.

    • Fish is considered to have learned task of correctly choosing the RIGHT arm if it has done so for five out of six trials (e.g., sequence of R R L R R R is successful but L R L R R R is not because over string of 6 trials only 4 were correct; Figure 4).

    • In Figure 4, fish learned the task at trial 15. We call this reaching criterion, the standard by which something is judged or decided. In other words, fish has successfully accomplished task as defined. A 5/6 criterion is common in learning studies and, in terms relevant to the students' experience, is a grade of “B” (83% correct).

    Upon reaching criterion, place the fish back into starting block. Repeat above procedure until fish reaches criterion, except now correct side is LEFT arm. This is termed a reversal task and measures cognitive flexibility. In reversal learning, the individual first learns to choose right in a spatial discrimination problem and then must choose left. Such reversals can be difficult and directly measure cognitive deficits, especially when testing for sensitivity to environmental chemicals (Evans et al., 1994).

  7. Entire procedure is done on Day 1 as a training method, on Days 2 and 3 for learning, and on Day 8 for memory.

  8. When all trials are completed, drain the water, rinse T-maze and all gates with distilled water, and dry overnight. If students find that they can test more than one fish per day, fresh dechlorinated water must be used for the second fish. During the trials, fish may excrete alarm substances that affect behaviors of other fish.

Figure 4.

Sample group data sheet.

Figure 4.

Sample group data sheet.

Optional Test – Double Reversal Task

To fully test cognitive flexibility, scientists may use the more challenging double reversal task (e.g., Saili et al., 2012). In this test, the animal first learns the task, which is followed by a reversal. However, the animal is then asked to reverse its choice back to the original correct side. Such double reversals usually require more time (30 trials/session) than a single class period allows, so such a test would be limited to those students who are conducting the experiment on their own time.

Data Analysis

It is best to display results comparing the control to the experimental variable using a graph. Many students have difficulty constructing and interpreting this presentation of data (Berg & Phillips, 1994; Berg & Smith, 1994). Importantly, with this skill students discover that different graphs can explain different aspects of the learning and memory process.

  1. Number of trials to reach criterion for first and second task: How long does it take for fish to learn (Figure 5A, B, D)?

  2. First correct trial: How long does it take for fish to discover the correct side?

  3. Longest string of correct trials (maximum = 5; Figure 5C): How consistent is fish in choosing correct side?

  4. Longest string of incorrect trials, the Perseveration Index (maximum = 20), where perseveration is the tendency to continue the same sequence of behaviors even though conditions have changed and require new or modified behaviors (i.e., get “stuck” on a wrong choice; Cañas et al., 2006). Perseveration is often associated with brain damage.

  5. Number of trials needed to successfully reverse task (trials between end of successful completion of first task and beginning of string of trials ending in successful completion of second task): How easy is it for fish to display cognitive flexibility?

  6. Number of fish successfully completing tasks 1 and 2: What percentage of sample population shows cognitive flexibility?

It is important for students to understand that because testing is done at discreet time points and data are not continuous, they should use a bar graph. Line graphs incorrectly imply behavioral changes during the day. Using the bar graph, the x-axis represents Time (days) or Specific Task (Training, Testing, Reversal) and the y-axis represents the dependent variable. Bars of both the control and experimental variable should be placed on the same graph for easier comparisons.

The correct statistical test to analyze data within and between test days is a repeated-measures analysis of variance (ANOVA) because the same fish is tested repeatedly (Tamura & Buelke-Sam, 1992). Most statistical software will have this test. If not, one-way ANOVA tests are sufficient. If students are not familiar with this, then use means and standard errors with graphs to show the data. If the standard error bars overlap, the means are not statistically different.

Figure 5.

Sample student data. Sheridan Schaffer (Greendale High School) compared two fish species (A). Brianna Karweick (Seymour Community High School) studied the effect of developmental nicotine exposure on adult learning (B). Amanda Linskins (Seymour Community High School) examined the effect of only embryonic (Y-C) vs. lifelong (Y) exposure to tartrazine/yellow dye 5 (C, D). Parental permission to publish these graphs was provided. (A) Number of trials for fish of different species to reach criterion for first task. (B) Number of trials to successfully complete task 1 after developmental exposure to nicotine. (C) Longest string of incorrect trials for task 1 (data for task 2 not shown) in fish exposed to developmental or lifetime concentrations of tartrazine. (D) Number of fish exposed to developmental or lifetime concentrations of tartrazine that successfully completed task 2.

Figure 5.

Sample student data. Sheridan Schaffer (Greendale High School) compared two fish species (A). Brianna Karweick (Seymour Community High School) studied the effect of developmental nicotine exposure on adult learning (B). Amanda Linskins (Seymour Community High School) examined the effect of only embryonic (Y-C) vs. lifelong (Y) exposure to tartrazine/yellow dye 5 (C, D). Parental permission to publish these graphs was provided. (A) Number of trials for fish of different species to reach criterion for first task. (B) Number of trials to successfully complete task 1 after developmental exposure to nicotine. (C) Longest string of incorrect trials for task 1 (data for task 2 not shown) in fish exposed to developmental or lifetime concentrations of tartrazine. (D) Number of fish exposed to developmental or lifetime concentrations of tartrazine that successfully completed task 2.

Conclusion

Observations of live animal behavior provide an exciting context for understanding physiological mechanisms and anatomical structures involved in learning and memory. Students may have questions about the process of learning and the animal's ability to learn under new conditions. In answering these student-generated questions, the experimental procedure described meets four basic goals of psychology research (Plotnik & Kouyoumdjian, 2014): description (what is happening), explanation (why is it happening), prediction (will it happen again), and control (how can it be changed).

Numerous online sources on environmental chemical exposures, especially in students, can compare how chemicals in air, water, and food may impact their own lives. The message learned through this inquiry-based approach is that environmental chemicals have profound, real-world effects on animals and, by extension, people. It is our experience that some students develop research questions that have not been addressed previously and, therefore, make a real contribution to science.

Learning and memory experiments can be applied to ecology. For example, how is spatial learning important in migration, foraging, or predator–prey relationships? Through comparison of multiple fish species or fish vs. human, students learn basic concepts and principles of living organisms as they relate to learning and memory (e.g., brain structure and function, impact of sensory motor inputs to regulating behavior, and the new field of cognition ecology; Healy & Jones, 2002). A unit in genetics may find use for experiments in learning; for example, do different strains of zebrafish learn differently? What happens to the offspring if the smart fish are bred together and those that are more challenged in learning the reversal task are bred together? A similar experiment can be conducted to see if fish that are exposed to an environmental chemical pass on their learning difficulties to the next generation as observed in lead-exposed zebrafish (Xu et al., 2016). Life-stage learning abilities can be investigated to model age-related cognitive decline in humans. We have developed a protocol for classroom breeding of zebrafish (see Tomasiewicz et al., 2014).

Using fish in T-mazes to study learning and memory has many advantages for high schools. From generating enthusiasm in laboratory experiences because students are using a live animal to creatively meeting NGSS for Life Sciences, this easy-to-use, inexpensive apparatus has the flexibility to meet many of the needs teachers have in developing challenging examinations on learning and memory.

This work was supported by a Science Education Partnership Award (SEPA) grant from the Office of the Director, National Institutes of Health (award no. GM129191). Content is solely the authors' responsibility and does not necessarily represent official views of the National Institutes of Health. We thank teachers and students for valuable feedback. Student-generated data from Brianna Karweick and Amanda Linskins (Seymour Community High School, Seymour, WI) and Sheridan Schaffer (Greendale High School, Greendale, WI) were provided with parental permission. The following Wisconsin high schools field tested this module: Greendale (Amy Zientek, teacher), Seymour (Cassandra Cobb and Carrie Schmidt, teachers), Hamilton (Milwaukee; Kevin Schiebenes, teacher), Germantown (Stacey Bast and Mark McClellan, teachers), and Wauwatosa East (Mary Haasch, Science Club coordinator). Use of live animals in this module was approved by the University of Wisconsin-Milwaukee Institutional Animal Care and Use Committee. For more information about our SEPA program, visit http://people.uwm.edu/winstep/.

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Supplementary data