AMBITUS system for investigation of several parameters related to motor activity and cognitive functions in rodents


Keywords: behavioral investigations, memory, motivation, motor activity, sensors for movements’ detection, rat, rodents, software, learning



The normal cognition require the cooperation of several brain structures (Vorhees and Williams, 2014;Buzsaki, 2013), and their impairments may lead to several neuropsychiatric diseases. The preclinical investigations essential for the determination of action mechanisms and testing of new drugs. Based on PubMed data the number of rodents studies related to the learning functions continuously increasing at the last 20 years (Fig. 1).



Figure 1. A: Number of studies investigated the learning functions in rodents. B: Number of studies investigated different mazes in rats.


A number of behavioral paradigms have been developed to assess motor activity, learning and memory performance in rodents. The rodent tasks with food rewards in different mazes and hole-boards are useful methods to evaluate memory functions (Bevins and Besheer, 2006; Casarrubea et al., 2010; Chambon et al., 2011; Chrobal et al., 1987; Fitzgerald et al., 1985; Gordan et al., 2012; Hauser et al., 2012; Kekesi et al., 2015; Locklear and Kritzer, 2014; McLean et al., 2009; Petrovszki et al., 2013; Roof and Stein, 2001; Schroeder et al., 2000; van der Staay et al., 2012; Vorhees and Williams, 2014; Xu et al., 2013). It is very important that the experimental setups need to be designed within the normal range of behavior, and the experimenter has to be sure that the animals are given the opportunity to solve the problems with which the test procedure confronts them (Heim et al., 2000). Searching for food is a naturally motivated behavior; thus, rodents are able to learn to retrieve food rewards located in different setups. It is well-known that in contrast to a hole-board with an open field, where the rats can show significant anxiety, rats prefer the narrow tunnels (Kimchi and Terkel, 2004). Based on this assumption, a circular corridor with or without a few side-boxes containing food rewards are applied to evaluate the exploratory activity and/or learning abilities of rodents (Monaco et al., 2014; Nadal et al., 2005; Newman et al., 2014; Wikenheiser et al., 2013; Wikenheiser and Redish, 2011, 2015). Unfortunately, both methods may require time-consuming data collection and/or they need long training sessions, or the commercial video-tracking systems (e.g., EthoVision XT) require special software which can analyze precisely the behavior of the animals. If cognitive function and behavioral activity of a large number of animals have to be investigated routinely, simple and fast tests are required.

A new instrument named the AMBITUS system was developed (AMBITUS is a Latin term literally meaning “to go round”), which is a square corridor with side-boxes along the inner and outer sides; thus, it is a combination of hole-board and corridor tests (with several food rewards but without open field). It offers the animals the opportunity of exploring a new environment and finding several food rewards. The AMBITUS system can be applied for the investigation of a variety of parameters associated with locomotor, exploratory and rearing activities and cognitive performance (task acquisition and learning flexibility) of rodents.


The AMBITUS apparatus

A rectangular corridor was constructed of a clear Plexiglas on black floor with an outer diameter of 80 cm, width of 8 cm and height of 50 cm (Fig. 2). The rats can move around the track between the walls in forward and backward directions (Fig 2A), but, depending on the task, one or two corridor can be closed (Fig 2B).

Each of the four corridors has four side-boxes (2 on the internal wall and 2 on the external wall) of equal size (side-boxes: 5x5x5 cm) with one food reward (puffed rice: 20 mg). Altogether, 16 side-boxes extending from the corridor are numbered consecutively (1–16; Fig. 2). Each box is equipped with an infrared LED (light emitting diode) at one side and a photocell at the other side to measure the exploratory activity (visits) into the boxes by the disruption of light beams. The locomotor and rearing activity was detected similarly by infrared beams located midway in each corridor and along the corridors, respectively. All of the photocell units are interfaced to a microcontroller unit. The apparatus automatically records individual beam breaks associated with each side-box visit with 1 ms time resolution, and stores the information about the location, the duration and the number of visits by custom-made software. Based on our preliminary experiments, all emitters and receivers were located 1 cm above the floor and from the entrance of the side-box to detect the visits into the side-boxes to the food rewards. This location of the sensors ensures that whenever a rat interrupts the infrared beam, its nose should necessarily be located within the side-box. The movement sensors can automatically detect spontaneous visits into the side-boxes giving objective information about the exploratory activity. Thus, the system permits the detailed and automatic recording of the visits of the animal into the side-boxes (exploratory behavior) and in concert with the video records, eating behavior can also be determined. In concert with the detection of the eating activity by the analysis of the video records, several parameters related to learning and memory functions could also be determined.

After insertion the food-rewards into the planned side-boxes, trials commenced by placing the rats into the same starting point within the corridor (Fig. 1); thereafter, the experimenter immediately left the room. The animals are allowed to explore the corridor and collect food rewards. The number of food rewards eaten is detected at the end of each trial by the experimenter. Experiments are recorded using an infrared video device equipped above the apparatus. When the animals had eaten all the available food rewards, the video recordings are analyzed offline to determine the time required to complete task (eating time).

Manifold parameters are either recorded automatically by photocells or manually by the experimenter (only the eating time) or are calculated from the baseline data by a custom-made software. Thus, the exploratory activity is evaluated as the latency, number, and duration of visits into the side boxes revealing the motivation to search. Furthermore, latencies to complete the task, the number of the eaten food rewards may provide data for the motivation and/or cognitive functions. However, some other parameters can also be considered, e.g., the number of different side-boxes inspected, revisited boxes or repetitions can reveal the perseverative behavior of the animals (Horvath et al. 2019). Regarding the locomotor activity, both the number of visits into different corridors, the whole circle number, and the direction of movements can be detected. The rearing activity can be characterized by the number of rearing during a test. All of these parameters can be detected for the whole phase and up to the collection of all of the food.

The AMBITUS system records multiple behavioral items simultaneously, which can be displayed immediately after the completion of the experiment. While the video-records should be checked by an investigator for detection of the eating time, if the rat ate all of them. However, the analysis of video-records is a fast process, requires only a few minutes/records, and together with the automatic registration of exploratory behavior, it significantly decreased the time required for data collection, and it could also prevent some mistakes originated through an oversight in case of manual data collection.

It has repeatedly been hypothesized that anxiety related behavior and cognitive processes may interact in a fundamental manner. Most of the behavioral tests are accompanied by different levels of stress, which might be due to exposure to a novel environment, open field and/or food restriction.

Since the narrow corridor in the AMBITUS system can simulate the natural environment of the animals; thigmotaxis has no influence on the behavior of the rats.

Most rodents show high levels of learning in the AMBITUS system, suggesting that the task has not been overly stressful.

Thus, the rats’ activity can be a confounding factor in spatial learning tasks, which may lead to the difficulty of selection bias that is a common problem in appetitive tasks, where there can be significant dropout rates. However, this device might be appropriate for the investigation of restricted interest of animals, too.

Very important errors might have been the tail or foot entering into the side-boxes and scored as visits. Fortunately, since the foot is below the body, it never happened that the feet were entering the side-boxes accidentally, which might occur in the hole-board test. Furthermore, since the position of the photocell is 1 cm above the floor and the distal part of the rat’s tail is thinner, this error has not been observed, too. 

The AMBITUS may be adapted to many other experimental conditions, e.g., more and/or longer learning trials, habituation periods without food rewards, providing more palatable food or various configurations of stimuli. The structural analysis of the exploratory activity can give further data about exploratory strategy of the animals, thus the separate analysis of each side-box or clustering of the inner and outer ones may give several possibilities for the investigation of spatial learning.



Figure 2. Ground plan of the corridor with 16 side-boxes equipped with photo beams with a rat at the starting point with opened (A) or closed (B) corridor. The height of the apparatus is 50 cm. Photographs of the device (C,D).



Test procedures

Different paradigms (number and locations of rewards) and training sessions can be applied to characterize the behaviors of the animals in different aspects. The Figure 3. describes one paradigms with different task.

The recent results showed that healthy animals can perform the simple tasks after a few repetitions (Horvath et al. 2017, 2019). The detailed analyses of the individual parameters related to task performance justified that most of the healthy rats learned the pattern and showed cognitive flexibility using this system.  It was found that normal rats were able to learn the task to a high level of performance during a short period of time, and they exhibited some improved performance at the second trial, suggesting that this simple task could be acquired easily. Schizophrenia-like rats showed decreased exploratory activity and learning ability in the AMBITUS system, suggesting that this system can be applied to reveal the abnormalities in these parameters of rats with different impairments. Preliminary data also proved that 8 trials for two days in two blocks each day seemed to be enough for the animals to acquire the Task 1 with gradually decreased time required to complete the task. Thus, healthy rats showed clear improvement in the learning capacity during the investigated period. Two recent articles applied the Ambitus system and characterized healthy and schizophrenia-like (Wisket) rats based on different task (Horvath et al., 2017,2019). The evaluation of these animals in this system proved the suitability of the AMBITUS system for the analysis of exploratory, locomotor and rearing activity and cognitive functions of rats.

Regarding the correlation analysis between manual and computer-based data acquisition on exploratory behavior, significant correlation was shown between these values, proving the reliability of the device (Horvath et al. 2017,2019).



Figure 3. Example for a complex paradigm (Green: 16 rewards; Red: 8 rewards in the inner boxes; Blue: 8 rewards in the outer boxes; Grey: 5 reward in the 16th box).


Important features are:

   1, This system based on reward, there is no any punishment.

   2, Several paradigms can be applied.

   3, The AMBITUS system is also appropriate to detect cognitive flexibility after different manipulations of the tasks.

   4, In contrast to Water maze test no stress effect of cold water.

   5, In contrast to Holeboard test, no stress effects of open field.

   6, Since the experiments were performed in dim light without external cues, the learning in the corridor could essentially be considered a form of an egocentric task solved by learning the association between its egocentric body response and the reward. The square shape provides important egocentric information due to the four corners and the inner and outer side-boxes containing the food rewards.

   7, The large number of reward increases the possibility to the faster learning.

   8, The simple corridor system provides modest difficulty for the rodents, therefore the training phase might be short.

   9, The system is unaffected by insufficient contrast between the rat and the background, as well as by lighting conditions, as it could be the case with some video-based methods.

  10, All the above mentioned data are recorded automatically and saved for later data processing.

  11, The data files are analyzed with a software developed for it, and the results are exported to a spreadsheet file.

  12, It provides simple assessment of the behavior of a large number of animals within a short period of time.



In conclusion, the AMBITUS system, containing photocells for automated recording of exploratory behavior and video-tracking for the detection of the eating activity, is a fast, efficient, objective and reliable way to assess the activity and learning capacity and memory functions of rats after minimal training sessions. The advantages of this new system include the shape (square corridor without open field), the large number of food rewards with different locations, and the possibility for separate analysis of each box or in the chain of inner and outer ones that ensures the investigation of spatial learning functions and learning flexibility applying different protocols. The software working with data obtained by photocells gives immediate data acquisition, the assurance of experimenter-independent measurements, and the reproducibility of results. The repeated use of the AMBITUS system can also be applied in the same animals to assess time-dependent effects such as in neurodegenerative and aging models and to evaluate the effects of different treatments. In summary, the AMBITUS system may be a valuable tool to screen for the effects of compounds or diseases which are believed to act on rat exploratory activity and memory capacity during a short time period, but additional studies are required to validate the AMBITUS system with other protocols. Altogether, the corridor combined with the advantages of the hole-board could quantify the activities of rats and reflect the degree of learning ability and memory as well.



Szeged, 2020. January 23.




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