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 &
• One T-maze unit for each group (for building instructions, see
• 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.
Students should practice testing protocols until they feel comfort-
able 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, stu-
dents 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 life-
time exposures to either 0 or 22 µM tartrazine (yellow dye 5;
see Figure 5C, D) before testing for changes in adult zebrafish
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
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
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.
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).
Figure 1. Completed T-maze and data collection roles
(numbers) for each student in the group.