Documentation

Introduction

The latent learning apparatus is used to analyze the learning and memory function in zebrafish. The apparatus analyzes the learning ability by using the spatial exploration process in which the fish explores the maze by following its natural tendency to explore novelty, without any reward or punishment reinforcement. The latent learning apparatus provides an environment to perform a vast range of investigations that span from evaluating the basis of learning mechanisms to the causes and treatment of learning deficits.

Jensen (2006) explains latent learning as a process in which the subject learns about the spatial layout of the complicated maze while wandering in it without any reinforcer. After training, when a reinforcer is introduced in the maze, the individuals use spatial learning to find out the reinforcer more quickly than those individuals that were not trained for unreinforced exploration task.

The latent learning apparatus can be used to assess disruption in the learning abilities of the zebrafish after acute and chronic drug exposure. It can also be applied to study the effects of neurotransmitters on learning acquisition. The apparatus is a powerful tool for evaluating underlying biological and neurological mechanisms along with molecular and cellular structures involved in learning. Various investigations inquiring genetic basis of learning and memory trait in zebrafish also make use of the latent learning apparatus.

The latent learning apparatus contains a start box and a goal box connected via tunnels. The goal box is provided with stimuli to attract the subjects. The movement of the fish in and out of the goal box can be controlled by guillotine doors. The positions of these guillotine doors can be modified according to the experimental design. The walls of the maze are made of acrylic to ensure that subject can have clear visual access to the goal box from all locations.  Other learning based apparatus used for zebrafish are Zebrafish Associative Learning and Zebrafish Bifurcating T Maze.

Apparatus and Equipment

The latent learning maze contains a 15 x 15 cm start box and a 15 x 15 cm goal box that are linked through a set of tunnels. The height of the apparatus is 10 cm and is constructed of clear acrylic. A 5 cm wide straight tunnel extends from the start box, and after 10 cm it divides to form a 4-way intersection creating an upward, a backward, a right and a left path, all leading to the goal box. The upward channel measures 15 cm from the intersection point to the goal box.  In addition to the straight channel, the start box is also equipped with tunnels on its right and left side leading to the goal box. Both tunnels measure 5 cm wide and are 35 cm long. The start box and the goal box are provided with floor drains. The goal box contains three entrance points equipped with guillotine doors to control fish movement. The goal box is surrounded by a 25 cm long and 5 cm wide path from all sides, connecting it to the right and left tunnels.

Training Protocol

Clean the apparatus and equipment thoroughly before use. Use similar quality, chemical composition and temperature of water used in the habituation period for training and probe trial.  An external tracking and recording systems such as Noldus Ethovision XT can be used with the latent learning apparatus for recording the fish behavior.

Exploration Training

Fill the Latent Learning Maze with 10 cm of water. Confine the experimental group in the start box for 30 seconds to acclimate to the environment. Close the passage towards the straight path and either the left or the right tunnel leading to the goal box. Perform a 30-minute exploration session. Repeat sessions as required.

Latent Learning Probe Trial

Fill the maze with water up to 10 cm height. Place a shoal of zebrafish as a reinforcer in the goal box. Place one fish at a time in the start box. After 30 seconds of acclimation, allow the fish to explore the maze without any restrictions of the paths. Conduct a 10 min trial for each fish.

Evaluation of the Effects of Alcohol on Zebrafish behavior in a Latent Learning Task

Luchiari, Salajan, and Gerlai (2015) investigated the effects of alcohol on zebrafish performance by using the latent learning apparatus. The subjects were treated in six treatment groups using different alcohol concentration C0.00A0.00, C0.00A0.50, C0.00A1.00, C0.50A0.00, C0.50A0.50, C0.50A1.00, where ‘C’ represents alcohol percentage in chronic treatment and ‘A’ represents alcohol percentage in 1-hour acute treatment. The fish behavior towards zebrafish conspecifics in the goal box was observed in the probe trial. The results showed that the control group (C0.00A0.00) made maximum correct choices and represented a spatial bias in tunnel preference. C0.00A0.50 and C0.00A1.00 groups showed significant impairment of memory recall displayed as a wrong tunnel choice and less time spent in the correct tunnel. The chronic alcohol treatment groups C0.50A0.50 and C0.50A1.00 showed significant tolerance behavior towards the acute challenge. The withdrawal group C0.50A0.00 showed a marked disturbance in behavior represented as an incorrect tunnel choice, increased time in the wrong tunnel, less time in the goal box, increase time in the start box, less distance traveled and, elevated freezing time.

Evaluation of Latent Learning Behavior in Zebrafish

Gómez-Laplaza and Gerlai  (2010) studied the learning and memory function of zebrafish in a latent learning task. The subjects were reared in five groups. Each group was trained for different tunnel preferences in the latent learning apparatus without any reinforcer present in the goal box. Each fish from all five groups was tested in a probe test for the display of latent learning acquired during the training phase. A group of five zebrafish conspecifics was placed in the goal box during probe trial to attract the subjects. It was observed that the subjects trained on right tunnel route preferred right tunnel and those trained on left tunnel preferred left tunnels in a probe trial. Subjects trained on both tunnels made random choices. Thus, it was concluded that the zebrafish successfully acquired latent learning in the absence of a reinforcer and showed learning manifestation in the probe trial.

Investigation of the role of Dopamine Receptors in Latent Learning

Naderi, Jamwal, Ferrari, Niyogi, & Chivers (2016) investigated the role of dopamine receptors in the acquisition and consolidation of latent learning. The subjects were classified into three categories, 10 treatment groups, 2 trained control groups, and 1 untrained control group. The treatment groups were treated with dopaminergic agonists (Apomorphine, SKF-38393, and Quinpirole) and dopamine antagonists (SCH23390, and Eticlopride) using the solution immersion method. After a 16 days training period, the learning and memory performance of each subject was analyzed individually in the latent learning apparatus containing 6 zebrafish stimuli in the goal box. Results showed impaired cognitive function in subjects treated with dopamine agonists. It was concluded that D1 and D2 receptors mainly govern the acquisition and consolidation of memory in zebrafish. D1 receptor agonists showed no effect on learning when administered before training. On the contrary, D1 antagonists improved memory on post-training exposure. D2 receptor antagonists showed significantly enhanced learning performance in both pre and post training exposure.

Data Analysis

Following parameters are analyzed by using Latent Learning Apparatus

• Time spent in the right tunnel
• Time spent in the left tunnel
• Number of fish present in the right tunnel
• Number of fish present in the left tunnel
• Latency to leave the start box
• Latency for entering the goal box
• Time spent in the goal box
• Duration of immobility or freezing time
• Total distance traveled

Strengths and Limitations

Strengths

The latent learning apparatus is a simple yet efficient tool requiring minimum handling. The apparatus can be used for high throughput screenings due to its feature to operate under minimal monitoring in parallel with other similar apparatus. The duration of the probe test conducted in the latent learning apparatus is short, that makes it eligible for larger sample population analysis. The apparatus does not require the presence of an experimenter to conduct an experiment. It has high scalability due to minimum experimenter intervention during the trial.

Limitations

The training of zebrafish in a latent learning maze can be time-consuming. Cleaning of the apparatus is important to prevent chances of lingering olfactory cues affecting the fish behavior. The temperature and quality of water used in the apparatus should comply with the habituation tank’s water specifications. Mishandling of the subjects may induce fear and stress that may disrupt task performance.

Summary

• The latent learning apparatus analyzes the memory and learning behavior of zebrafish based on spatial exploration process.
• In a latent learning apparatus, the learning is acquired during a training phase in the absence of a reward and the manifestation of learned information is observed in the presence of a reinforcer during probe trial.
• The experiment in a latent learning maze can be performed without any intervention.
• A single latent learning maze needs prolonged time for the training of a large number of subjects; however, multiple similar latent learning apparatus can be used in parallel to analyze a larger sample population.
• The apparatus can be used for high throughput drug screening and genetic mutation investigations.
• The latent learning apparatus can be used to study the effects of a drug on learning and memory behavior in zebrafish.
• The apparatus also helps in studying brain structures and neurotransmitters regulating cognitive functions.

References

1. Gómez-Laplaza, L. M., & Gerlai, R. (2010). Latent learning in zebrafish (Danio rerio). Behavioural Brain Research, 208(2), 509-515. doi:10.1016/j.bbr.2009.12.031
2. Jensen, R. (2006). Behaviorism, latent learning, and cognitive maps: needed revisions in introductory psychology textbooks. Behavioral Analysis, 29(2), 187–209.
3. Luchiari, A. C., Salajan, D. C., & Gerlai, R. (2015). Acute and Chronic Alcohol Administration: Effects on Performance of Zebrafish in a Latent Learning Task. Behavioural Brain Research, 282(1), 76–83. doi:10.1016/j.bbr.2014.12.013.
4. Naderi, M., Jamwal, A., Ferrari, M. C., Niyogi, S., & Chivers, D. P. (2016). Dopamine receptors participate in the acquisition and consolidation of latent learning of spatial information in zebrafish (Danio rerio). Progress in Neuropsychopharmacology & Biological Psychiatry, 67(1), 21-30.