The Cincinnati Water Maze (Multiple T-Maze or Labyrinthine Maze) is composed of nine intersecting “T’s”.  The Water Maze serves as an excellent tool in behavioral studies, enabling scientists to investigate the cognitive effects of various factors, such as drugs, aging, or neurological conditions. This complex maze assesses egocentric navigational learning and memory in rodents. As a result, the test can be performed in darkness to test an animal’s ability to navigate.

How Does Cincinnati Water Maze Work?

At its core, the Cincinnati Water Maze consists of a large circular pool filled with water, made opaque with the addition of non-toxic white paint. A hidden escape platform serves as the goal for the rodent subject.

Assessing Learning & Navigation Using Cincinnati Water Maze

Allocentric Navigation

The CWM enables researchers to examine allocentric navigation, which involves spatial learning and memory based on external cues. Through meticulous experimentation, scientists can study how rodents develop strategies for locating the hidden platform by relying on visual cues, such as distal landmarks.

Egocentric Learning

In addition to allocentric navigation, researchers can also explore egocentric learning, a form of spatial cognition centered on the organism’s body. This perspective considers the position of the escape platform in relation to the subject itself.

Techniques for Cincinnati Water Maze Analysis

The data generated by Cincinnati Water Maze experiments can be complex, requiring specialized techniques for analysis. Researchers typically measure several performance parameters, including latency to find the platform, distance traveled, and time spent in target quadrant.

Customizable Features of the Cincinnati Water Maze (CWM)

The following are a list of customizable features that can be completed by the Anilocus Animal Behavior team to create a research environment tailored to your research needs:

Environment Features:

• Size and shape of the chamber
• Pool Dimensions (size and depth)
• Lighting conditions
• Soundproofing
• Platform Placement
• Maze Configurations
• Environmental Landmarks
• Temperature Control
• Accessories

Stimulus, Control, and Test Article Administration for CWM Test:

• Experimental Groups (e.g., saline, treated, vehicle, etc.)
• Method of drug administration (e.g., injection, oral, or vapor)
• Stimulus Type (Cues, Darkness, Alcohol, Drugs, Food, etc.)

Trial Conditions for CWM Test:

• Type of conditioning stimulus (e.g., light, sound, or odor)
• Number of conditioning sessions
• Duration of conditioning sessions
• Time between conditioning sessions
• Trial Duration

Protocol for Cincinnati Water Maze (CWM) Experiment

The Cincinnati Water Maze (CWM) is an innovative tool that has has revolutionized experimental protocols for assessing cognitive function developed by Dr. Charles V. Vorhees at the University of Cincinnati. It offers researchers invaluable insights into egocentric and allocentric learning, memory, and navigational strategies. The Cincinnati Water Maze, often referred to simply as the Water Maze, is a laboratory apparatus designed for behavioral studies in rodents. It is specifically tailored to investigate cognitive functions related to spatial learning and memory.

Materials

Item	Purpose	Manufacturer	Catalog
Cincinnati Water Maze	Experimental	Anilocus	SKU: 16767
Animals	Test subjects	Anilocus	Contact Us
4K Webcam: Studio-Quality Webcam	Behavior Recording	Lumina	B09T749G1S
Panlab SMART video tracking system	Automated Video Behavior Tracking Software	Harvard Apparatus	V3.0
Fisherbrand™ Timer	Timekeeping	Fisher Scientific	02-261-840
Near Infrared Combo Red Light	Lighting Conditions	Varies	Varies
Towels and Bedding	Drying/Warming Animal	Varies	Varies

Cincinnati Water Maze (CWM) for Rats: Experimental Protocol

Step 1: Preparations

Day 1

• Morning: Prepare the CWM pool by filling it with water and adding non-toxic dye for opacity. Ensure the water temperature is within the desired range.
• Afternoon: Set up any required visual cues around the maze. Ensure the escape platform is submerged and hidden.

Step 2: Habituation

Days 2-6

• Morning (2-6 hours/day): Place rats in the CWM without the escape platform. Allow them to swim freely for gradually increasing durations each day (e.g., start with 10 minutes and progress to 30 minutes).
• Afternoon (1-2 hours/day): After the morning habituation session, dry and warm the rats, ensuring their comfort.

Step 3: Training

Days 7-11

• Morning (3 hours/day): Introduce the escape platform at a fixed location in the maze. Each day, perform multiple training trials, with a maximum duration of 60 seconds per trial. Record the latency to find the platform.
• Afternoon (1-2 hours/day): Dry and warm the rats after each morning training session.

Step 4: Probe Trials

Days 12-13

• Morning (3 hours/day): Conduct probe trials where the escape platform is either removed or placed at a novel location. Record latencies and errors.
• Afternoon (1-2 hours/day): Dry and warm the rats after each morning probe trial session.

Step 5: Data Analysis

Days 14-16

• Morning (4-6 hours/day): Analyze the collected data, including latency times, swimming paths, and errors.
• Afternoon (2-3 hours/day): Prepare reports and summaries of the findings.

Step 6: Post-Experiment

Day 17

• Morning: Conclude the experiment by removing the rats from the CWM. Perform any required post-experiment assessments.
• Afternoon: Begin cleaning and maintaining the CWM pool and equipment.

Step 7: Data Interpretation and Reporting

Days 18-20

• Morning (4-6 hours/day): Interpret the results, conduct statistical analyses, and prepare a detailed report of the experiment’s findings.
• Afternoon (2-3 hours/day): Finalize the report, including tables, graphs, and figures.

Step 8: Documentation and Ethical Considerations

Day 21

• Morning: Ensure all experimental documentation is complete, including ethical considerations, animal welfare records, and data transparency.
• Afternoon: Submit any necessary reports or documentation to relevant authorities or institutions.

Data Analysis

1. Latency Analysis: Evaluate the time taken by subjects to find the platform, assessing learning and memory.
2. Path Analysis: Analyze swimming paths to identify spatial strategies employed by subjects.
3. Error Analysis: Record errors such as platform crossings and revisitations as measures of cognitive performance
4. Software Utilization: Process and visualize data using dedicated CWM analysis software.
5. Analyze the data using appropriate statistical tests (e.g., t-test, ANOVA) to determine the significance of the scores.
6. Interpret the results and draw conclusions based on the experimental objectives.

Ethical Considerations

1. Minimize stress and discomfort to the animals during the experiment.
2. Obtain necessary approvals and permissions from relevant ethics committees.
3. Ensure that the experiment adheres to ethical guidelines and regulations for animal research. If your team needs guidance with this study for an outlined IACUC-approved protocol, please contact us for more information.

References

1. Vorhees, C. V., & Williams, M. T. (2016). Cincinnati water maze: A review of the development, methods, and evidence as a test of egocentric learning and memory. Neurotoxicology and teratology, 57, 1–19. doi:10.1016/j.ntt.2016.08.002.
2. Vorhees, C. V., Herring, N. R., Schaefer, T. L., Grace, C. E., Skelton, M. R., Johnson, H. L., & Williams, M. T. (2008). Effects of neonatal (+)-methamphetamine on path integration and spatial learning in rats: effects of dose and rearing conditions. International journal of developmental neuroscience : the official journal of the International Society for Developmental Neuroscience, 26(6), 599–610. doi:10.1016/j.ijdevneu.2008.04.002.
3. Williams MT, Sugimoto C, Regan SL, et al. Cognitive and behavioral effects of whole brain conventional or high dose rate (FLASH) proton irradiation in a neonatal Sprague Dawley rat model. PLoS One. 2022;17(9):e0274007. Published 2022 Sep 16. doi:10.1371/journal.pone.0274007.