The Water Escape T-maze apparatus is used to evaluate spatial learning and memory in rodents when placed in a stressful environment. The apparatus combines the design features of the Morris Water Maze and the T-maze, thereby allowing a range of possible investigations.

The Water Escape T-maze relies on water-escape motivated navigation to investigate behaviors and processes involved in learning and memory. In general, the task involves the subject to learn the position of the escape platform placed in one of the goal arms. Since rodents tend to have a strong aversion to water, the subjects are highly motivated to escape. This learning behavior is then assessed by reversing the position or removing the escape platform. Subjects with impaired cognitive abilities tend to display poorer performance in reaching the correct goal arm. In addition to investigating escape-behaviors and spatial learning, the Water Escape T maze can also be effectively used in the assessment of repetitive behaviors (Guariglia & Chadman, 2013), cognitive abilities in neuropsychiatric and neurodegenerative models, drug testing and effects of behavioral manipulations.

The Water Escape T-maze consists of a long start alley that bifurcates into two goal arms to create a T-shaped apparatus. The goal arms are equipped with sliding doors where they meet the start alley. The apparatus comes with escape platforms that can be placed in the arms submerged below the water surface. Other apparatus used in spatial learning and memory include the Y maze, Barnes Maze, Morris Water Star Maze, and Radial Arm Maze.

The water escape T-maze consists of a 1 cm thick, grey Plexiglas T-maze pool. The dimensions of the main alley of the maze are 100 cm x 20 cm x 40 cm. The alley bifurcates into two side arms of dimensions 45 cm x 20 cm x 40 cm. The main alley and the two side arms are connected by two sliding doors which are manually operated to close either arm. At the end of each arm is a Plexiglas platform (15 cm x 18 cm). The platform is submerged 2 cm below the surface of the water.

Clean the water escape T-maze apparatus before and after usage. Observation and recording of the movements of the subjects can be done using a tracking device, such as the  Noldus EthoVision XT. Train the subjects during the dark cycle under red light. Between intra-trials and intertrial runs, place the subject inside a cage.

Habituation and Pre-training

Prior to the actual trials, train the subjects on the Water T-maze over the course of 4 days.

Day 1
Do not place the escape platform in the apparatus. Gently immerse the subject into the Water Escape T-maze apparatus for 1 minute.

Day 2 and 3
Perform 10 trial pairs of forced-forced alternations. In the first trial, allow the subject to enter the goal arm of its choice. Follow this trial by blocking the previously visited arm, thus, forcing it to visit the previously unvisited arm.

Day 4
Perform 10 trial pairs of forced-choice runs. In the first trial, place the escape platform in only one arm (left or right) and block the entry to the other arm. Following this trial, place the escape platform in the previously blocked arm. Allow subject to access both arms. Consider the subject trained once it meets the learning criterion.

Delayed Alternation Task

Place the escape platform in the predetermined goal arm and perform forced-choice runs (forced trial and choice trial pairs) in a pseudorandomized manner. Following the forced-choice trial, place the platform in the previously non-accessible arm and allow the subject to access both arms. If the subject fails to choose the arm with the platform, close its sliding door. Let the subject remain in the water for 10 seconds and then open the sliding door to allow access to the arm with the submerged platform. In order to evaluate retention memory, introduce an intra-trial delay (10 seconds or more) between the forced trial and choice trial. Further, maintain an appropriate inter-trial interval between trial pairs.

Evaluation of the effect of environmental enrichment on acute stress and working memory performances

Del Arco, Segovia, Garrido, De Blas, and Mora (2006) assessed whether environmental enrichment affected the release of dopamine and acetylcholine in the prefrontal cortex (PFC) following acute stress. Three months old, male Wister rats were divided into an enriched environment group or standard cage environment group. The enriched environment group had access to running wheels, rearrangeable plastic tunnels, an elevated platform, and toys that were changed every 5 to 6 days. Animals were first evaluated for their spontaneous motor activity using an Open-Field test, following which working memory was evaluated in a Water Escape T-Maze. The enriched group displayed faster habituation to the Open-Field as observed from the significantly lower total activity as opposed to the control group. Prior to assessing working memory, the animals were placed in a brightly lit Open-Field for 10 minutes to induce stress. Immediately following stress induction, animals were tasked with performing a delayed alternation task in the Water Escape T-Maze. Though not statistically significant, stress resulted in a decrease of task performance at 10-second retention interval in both groups. Additionally, no significant effect of housing conditions was observed to affect performance. Further analysis using microdialysis, following 40 minutes of handling stress, revealed that enriched conditions resulted in stress increased the release of dopamine only. On the other hand, in the control group, stress resulted in a significant increase in dopamine and acetylcholine concentrations.

Investigation of the effects of MK-801, scopolamine, and MPEP on working memory performance

Locchi, Dall’Olio, Gandolfi, and Rimondini (2007) evaluated the performances of male Sprague-Dawley rats in delayed alternation task using the Water Escape T-maze following pharmacological manipulations. Animals that met the learning criterion for the Water Escape T-maze task were treated with either MK-801 (0.125 – 0.25 mg/kg), scopolamine (1 mg/kg), MPEP (1 mg/kg) or a combination of MK-801 and MPEP 35 to 40 minutes prior to testing. Drug administration was every 7 days for a maximum of 3 times. During the 7-day interval, the animals were evaluated for their working memory in the maze. The results of these trials showed that Scopolamine and MK-801 significantly decreased the performance in the delayed alternation tasks. While MPEP did not significantly affect the subject’s performance when injected alone, it resulted in enhancement of MK-801 induced working memory impairments.

Investigation of the effect of 7NI treatments on 6-OHDA-induced spatial learning deficits

Haik et al. (2008) investigated the potential of 7-nitroindazole (7NI) treatment as an early, presymptomatic pharmacotherapy for Parkinson’s disease. Male Sprague-Dawley rats were divided into 3 treatment groups; 7NI/6-OHDA, vehicle/6-OHDA, and vehicle/saline. Simulation of presymptomatic Parkinson’s disease was achieved via bilateral injections of 6-OHDA into the neostriatum of the rats. Animals spatial learning abilities were evaluated 21 days after the surgery on the Water Escape T-maze. Rats were evaluated using spatial reversal task that incorporated two sets of reversals of escape platform location (SR1 and SR2) following every 10 consecutive correct choices. It was observed that, in comparison to the controls and 7NI groups, the 6-OHDA group made more errors during the second round of spatial reversal. Subjects were also evaluated for their sensorimotor abilities using the Open-Field Test, Balance Beam, Grip Strength, and Paw-Placement. Dopaminergic immunohistochemistry analysis further revealed that 6-OHDA lesions resulted in an average loss of 35% of the dopamine neurons in comparison to the control group.

Investigation of effects of cannabinoid discriminative stimulus using Water Escape T-maze

Wiley et al., 2017 investigated cannabinoid discrimination using FAAH ^(-/-) and FAAH ^(+/+) mice (C57BL/6 background) using a Water Escape T-maze. The water maze was chosen to eliminate the influence of food rewards on the observations that often plague traditional appetitive reinforcer-based tasks. Subjects were injected with JZL184 two hours prior to the sessions while rimonabant was injected immediately before THC or AEA that was administered 30 minutes prior to the session. The vehicle-treated group served as the controls for the experiment. The acquisition trials involved placement of the escape platform in one of the goal arms based on the treatment (drug versus vehicle/no-drug).  Subjects that chose the injection-appropriate choice on the first trial with the overall correct choice of 80% or more, in addition to completing 8 of the 10 trials within 60 seconds, were selected for the test sessions. The test sessions were carried out with both goal arms equipped with the escape platform, and performances were measured using the same parameters as the acquisition trials. It was observed that acquisition of the discrimination task was faster in FAAH ^(-/-) mice (18.9 ± 4.45 and 14.6 ± 1.61 choice sessions for THC and AEA discrimination, respectively) compared to the FAAH ^(+/+) mice. However, across procedures and genotype, THC and AEA had relatively minor potency differences. Additionally, it was observed that JZL184 only partially substituted in both THC-trained and AEA-trained FAAH ^(-/-) mice.

The following data can be analyzed by using the water escape T-maze apparatus:

• Ratio of correct and incorrect choices
• Number of times the correct choice arm was chosen
• Number of times the incorrect choice arm was chosen
• Total number of incorrect choices
• Number of times an arm was chosen first.

Strengths

The Water Escape T-maze apparatus effectively assesses spatial learning and working memory of subjects. The maze allows assessment of both reference and working memory simultaneously. The maze relies on using rodent’s aversiveness to water to motivate them to find the escape platform. By combining the T-maze principle, the maze simplifies the assessment of choice behaviors and reduces the number of navigational paths that can be seen with a Morris Water Maze. The addition of guillotine doors allows the introduction of delays or adaptation of the maze for different protocols and experiments.

Limitations

Factors such as the species, their motor functions, and the presence of visual cues can affect task performance. Contamination such as the release of pheromones could give faulty result in the experiment. Fear of water can result in the subject becoming distraught and not participating in the experiment. Subjects may have their olfactory senses damaged if submerged in water for too long.

• The Water Escape T-maze apparatus is used in spatial learning and memory retention tasks.
• The apparatus combines the aversive factors of the Morris Water Maze with the two-choice design of the conventional T-maze apparatus.
• The choice arms are equipped with sliding doors that allow controlling the movement of the subject into the arms.

1. Delarco, A., Segovia, G., Garrido, P., Deblas, M., & Mora, F. (2007). Stress, prefrontal cortex, and environmental enrichment: Studies on dopamine and acetylcholine release and working memory performance in rats.Behavioral Brain Research, 176(2), 267-273. doi:10.1016/j.bbr.2006.10.006
2. Guariglia, S. R., & Chadman, K. K. (2013). Water T-maze: A useful assay for determination of repetitive behaviors in mice. Journal of Neuroscience Methods, 220(1), 24-29. doi:10.1016/j.jneumeth.2013.08.019
3. Haik, K. L., Shear, D. A., Hargrove, C., Patton, J., Mazei-Robison, M., Sandstrom, M. I., & Dunbar, G. L. (2008). 7-nitroindazole attenuates 6-hydroxydopamine-induced spatial learning deficits and dopamine neuron loss in a presymptomatic animal model of Parkinson’s disease.Experimental and Clinical Psychopharmacology, 16(2), 178-189. doi:10.1037/1064-1297.16.2.178
4. Locchi, F., Dall’Olio, R., Gandolfi, O., & Rimondini, R. (2007). Water T-maze, an improved method to assess spatial working memory in rats: Pharmacological validation.Neuroscience Letters, 422(3), 213-216. doi:10.1016/j.neulet.2007.06.023
5. Wiley, J. L., Lefever, T. W., Pulley, N. S., Marusich, J. A., Cravatt, B. F., & Lichtman, A. H. (2017). Just Add Water: Cannabinoid Discrimination in a Water T-Maze with FAAH(−/−) and FAAH(+/+) Mice. Behavioral Pharmacology,  27(5), 479-484. doi:10.1097/FBP.0000000000000228