A Method for Assessing Working Memory in Rats Using Controlled Virtual Environment

Abstract

The aim of the study is to develop an experimental method to effectively assess the working memory in rats. The method uses a state-of-the-art controlled virtual environment with a virtual maze. The setup includes a treadmill for rodents, a fixation system, a dome for displaying virtual environment, and a control unit.

Materials and methods: Biological part of the investigation: In our study, young healthy Wistar rats aged 6-7 months were used. The initial stage involved habituating the experimental animals to the experimenter over a period of two weeks. The habituation process was conducted in several successive steps. First, the rats were acclimated to wearing a jacket, which is part of the apparatus that holds the animal in the experimental setup. Next, they were familiarized with the fixation system. Following this, the rats were introduced to the treadmill (a sphere), and finally, they were acclimated to the entire setup. Subsequently, the rats were gradually habituated to the virtual maze and the associated reward system through positive reinforcement. This approach helped minimize stress and facilitated their adaptation to the new conditions. The second stage involved exploring the virtual space and learning the features of the virtual maze, including walls, turns, and the end goal. During the learning phase, the animals received positive reinforcement in the form of sugared water from the automatic water dispenser for correctly performed tasks. To navigate the T-maze, the rats used visual cues such as wall color and figures on the wall. At this stage, the rats learned to use virtual space to achieve their goals. Once the rats showed evident progress in learning the virtual environment, we implemented a protocol to assess their working memory. This assessment was based on the time it took for the rats to find the maze arm that provided positive reinforcement.Engineering part of the investigation: The animal is positioned on a foam plastic sphere with a 30 cm radius, using a custom device that allows its head and paws to remain mobile. Bearing fix the sphere in place, enabling the rat to rotate freely around its vertical axis. The rat's forward and backward movements cause the sphere to rotate, simulating a treadmill. The sphere's movements are detected by two infrared sensors (adapted from optical LED mice with USB interfaces) and transmitted to a computer, which generates an image of the virtual environment - a maze with landmarks on its walls. The virtual environment, created using the Unity Real-Time 3D Development Platform, is projected onto a custom-designed dome display containing the sphere and the lab rat. The setup provided the rat with a 360° field of view.

Conclusion: In our study, we present a setup that includes a projector, a dome display, a sphere (treadmill), a virtual T-maze, motion capture sensors, systems for securing animals to the sphere, and positive reinforcement delivery systems. We have developed an optimal protocol for immersing laboratory animals into a virtual environment and evaluating their cognitive functions, particularly working memory. The application of virtual reality in biological experiments enables more precise control over study conditions and allows for the creation of highly accurate and realistic behavioral protocols to assess cognitive functions in animals. This approach enhances our understanding of the mechanisms underlying working memory and their relationship with behavioral processes in rats and other animals.

Keywords: T-maze; virtual environment; working memory in rats.

Figure 1.. Variations of the setup for creation of virtual environment:

(a) the maze is demonstrated to the rats using a display screen; (b) the maze is projected on a cone display; (c) the maze is projected on the spherical display

Figure 2.. Variations of animal fixtures:

(a) rigid head fixation; (b) rigid body fixation; (c) flexible body fixation

Figure 3.. Schematic representation of the jacket for holding the experimental animal in the virtual reality system:

(a) a general view of the jacket inside and outside; the central part consists of the fabric rectangles; fabric ribbons serve as lateral fixtures for securing the animal; (b) a view of the jacket on the experimental animal

Figure 4.. Schematic view of the developed fixture for the tested rat on the sphere and positive reinforcement presentation of sugared water from the drinker

Figure 5.. General view of the setup with the tested rat in the virtual maze

The treadmill (sphere) is attached to the aluminum profiles. The rat motion sensors are mounted on the hoop on the bottom of the sphere. The tested rat in the fixing jacket is placed on the upper central part of the sphere with a fixture, which is connected with the jacket and half of the hoop on top directly with the entire setup for free movement. The virtual T-maze is projected to the cone display using a projector with a pattern of grey circles on the walls. If the rat turns correctly, it receives sugared water from the tube via the water supply system attached to half of the hoop from above

Figure 6.. Virtual reality image for the tested rats inside the 360° cone display with a green tower as an endpoint landmark

Figure 7.. User interface of the first page of the virtual maze program

Figure 8.. Virtual maze image (left) and display of the second monitor screen necessary for program control (right)

Figure 9.. Illustration of the changed shape of the virtual maze (left) and display of the second monitor screen necessary for program control (right)

Figure 10.. Variants of different element shapes on the virtual maze walls:

(a) white walls with grey triangles; (b) black walls with white circles; (c) white walls with black circles

Figure 11.. Illustration of the developed virtual T-maze with a central track, left and right turns and two towers at the end of the 2D projection

Figure 12.. Schematic view of the developed protocol for evaluation of the working memory in the tested rats based on remembering the desired arm of the T-maze in accordance with the wall color