RotaRod is a simple test to evaluate motor function and coordination in rodents. Motor coordination or fatigue can be assessed by measuring the time the animal remains on a rotating rod before falling off. It is a widely used device described in animal experimentation that has seen improvements since its invention. In the latest models, the rod can spin constantly, accelerate, sway (forward and backward), or move through complex ramps of acceleration/deceleration [9, 10].

$\bullet$ Purpose: Assessment of Motor coordination, balance, ability to maintain posture.

$\bullet$ Materials: Motorized rotating cylinder device with lane separators [11] (Fig. 1).

$\bullet$ Procedure: Each mouse is placed in a lane in the rotating cylinder. The cylinder is set at height of 40–50 cm to discourage the mice from jumping off. A padded surface beneath the cylinder prevents injuries from falls. The rotational speed is increased in a specific time interval, usually from 4 to 40 rpm in 300 s [12]. Some protocols have a duration test of 600 s [13].

$\bullet$ Variables recorded:

- Latency to Fall: Time in seconds until the mouse falls off the rotating rod at a set speed. This measures initial motor coordination and balance. The faster the cylinder rotates, the faster the mice will fall. Mice can be tested at a set speed for different trials or at a gradually accelerated speed.

- Fall Speed: Time in seconds for the mouse to reach the padded base once it starts falling. Impaired mice exhibit slower fall speeds compared to controls.

- Number of Rotations: Count of rotations the mouse completes with the cylinder before falling off. Increased rotations suggest compensatory motor strategies or decreased ability to disengage from the rotating surface [14].

$\bullet$ Advantages:

- Ease of Use: The rotarod test is straightforward to perform and analyze, making it accessible for assessing motor deficits in MS models [15].

- High Throughput: Multiple mice can undergo testing simultaneously, increasing efficiency in data collection.

$\bullet$ Limitations:

- Not reliable and not sensitive to detect subtle motor alterations.

- Mouse behaviour: Mice may refuse to balance on the cylinder by choosing to fall or cling to the cylinder and rotate with it.

- Results can be influenced by the mouse weight, heavier mice tend to fall easier.

Offers the ability to detect subtle deficits in motor skills and balance than other motor tests. The test evaluates an animal’s ability to walk on a long, narrow beam.

$\bullet$ Purpose: Assessment of fine coordination and balance [16, 17].

$\bullet$ Materials:

- A 6- or 12-mm wide beam that is 100 or 120 cm in length.

- A 60-watts lamp at the starting point as a stimulator to encourage the mice to start walking across the beam.

- A black box with nesting materials at the endpoint to encourage completion.

- A padded nylon hammock is placed beneath the beam to ensure gentle mice falls.

- A camera for recording trials and analysing performance [18] (Fig. 2).

$\bullet$ Procedure: The beam is positioned 50 cm above the ground. The mouse is positioned at the starting point and should traverse the beam. If the mouse falls it will be gently placed again on the beam. In case the mouse doesn’t start walking, the experimenter may poke, prod or push gently the mouse from behind with his fingers during training. The test is performed in 3 consecutive days: Day 1 and 2 are for training and day 3 is for testing. During training sessions, the mouse walks 3 times across the 12 mm beam and afterwards another 3 times using the 6 mm beam. There is a 10-minute break after each training session with every type of beam. The test is carried out twice a week and the cut-off trial time is 60 seconds [18].

The beam can be inclined to increase complexity and sensitivity of the test to facilitate detecting subtle motor deficits. It can be inclined downwards to encourage the mice to move forward [19, 20].

$\bullet$ Variables recorded:

- Walking Time: The time taken for the mouse to traverse an 80 cm section of the beam. Motor-impaired mice typically require more time [19, 21].

- Number of footslips: Count of footslips, with notes on whether fore- or hindlimbs were involved [19, 21].

$\bullet$ Advantages:

- Reliable and more sensitive to detect motor coordination deficits than other motor tests (e.g., rotarod test).

$\bullet$ Limitations:

- Confounding factors includes motivation of the mice to cross the beam. Overtraining may lead to boredom, while anxiety can affect their ability to complete the task [16].

Motor behavior is mediated by a complex neural network that originates in the cortex and ends in the skeletal muscles, involving neuronal tracts and connection pathways of the first and second motor neuron. Various methods exist to determine gait abnormalities in small rodents, including the rotarod test paradigm. The Motor Skill Sequence (MOSS) is designed to detect latent deficits in motor performance. In a first step, we habituate mice to training wheels composed of regularly spaced crossbars until achieving maximum performance functionality.

$\bullet$ Purpose: The MOSS test evaluates bimanual motor coordination, fatigue, and endurance in rodent models [22, 23].

$\bullet$ Materials:

- MOSS cage equipped with a 38-rung training wheel to allow initial spontaneous running behaviour assessment.

- MOSS cage equipped with a 16-rung complex running wheel for advanced motor skill evaluation.

- Activity Monitoring System Software for tracking and recording motor activity (Fig. 3).

$\bullet$ Procedure: In a first step the mouse is placed in the MOSS cage with a training wheel with regularly spaced crossbars for two weeks. This phase allows the mice to become accustomed to voluntary running. In the second step, the previous wheel is replaced by a complex wheel with irregularly spaced crossbars for a week. Voluntary running in both wheels are continuously recorded [24].

$\bullet$ Variables recorded:

- Cumulative Distance Travelled: Measured in minutes, calculated based on the number of wheel revolutions and the distance covered per revolution.

- Maximum Running Velocity: Recorded in revolutions per minute (rpm).

- Number of Individual Runs: The total count of distinct running bouts.

- Maximum Run Duration: The longest continuous running period.

$\bullet$ Advantages:

- The test is highly sensitive and reliable for detecting latent motor deficits, since it requires complex bilateral motor coordination.

- Voluntary wheel running can test fatigue at the same time, which is a frequent symptom in MS patients.

$\bullet$ Limitations: The MOSS test is complex to perform and analyze, requiring specialized equipment and software [23].

This test is based on the fact that rodents ability to grip is inversely proportional to the bar diameter.

While the Horizontal Bar Test and the Rotarod Test both assess motor coordination, they do so in different ways. The Rotarod Test adds an additional dimension of difficulty by incorporating speed and acceleration, which challenge the animal’s ability to balance dynamically as the rod rotates. This is a critical difference because dynamic balance and motor coordination are crucial for evaluating more subtle motor impairments, particularly in early-stage disease models. In contrast, the Horizontal Bar Test focuses solely on the animal’s static grip strength and does not assess the animal’s ability to respond to changing conditions like increasing speed, as seen in the Rotarod Test [25].

$\bullet$ Purpose: The Horizontal Bar Test is used to assess motor coordination, grip strength, and endurance in rodent models [21].

$\bullet$ Materials:

- 38 cm long Bar: Typically 3 bars are used of a 2 mm, 4 mm and 6 mm width.

- Supporting Frame: A sturdy frame to securely hold the bar at a fixed height, usually around 50 cm [25, 26, 27] (Fig. 4).

$\bullet$ Procedure: Mice are tested in horizontal bars of increasing thickness and the time to remain in the bar is recorded. The bar is placed above a padded surface to prevent gentle falls. Maximum test time (cut-off time) is 30 sec.

$\bullet$ Variables recorded:

- Hang Time: The duration (in seconds) that the rodent can hang from the bar before falling. The scoring system is as follows: Falling between 1–5 sec = 1, Falling between 6–10 sec = 2, Falling between 11–20 sec = 3, Falling between 21–30 sec = 4, Falling after 30 sec = 5, Placing one forepaw on a bar support without falling = 5 [25].

$\bullet$ Advantages:

- The test is sensitive to detect neuromuscular deficits and changes in motor coordination.

- The test is reproducible and easily standardized. It yields reliable, consistent results across different trials.

- Easy to set and conduct.

- Cost-Effective. The test requires inexpensive materials and is straightforward to perform in most laboratory settings [26, 27].

$\bullet$ Limitations:

- Lack of Dynamic Components: The Horizontal Bar Test primarily measures static motor abilities and does not challenge the animal’s ability to maintain balance under changing conditions. This absence of a dynamic component limits its ability to mimic more complex motor tasks.

- Results can be influenced by the mouse weight, heavier mice tend to fall easier.

The Forelimb Grip Strength Test measures the muscle strength of rodents by having them grip a bar while an experimenter gently pulls their tail. This test, which can be conducted horizontally or vertically, is praised for its ease of use and reliability, despite variability due to motivational factors and body mass differences [28].

$\bullet$ Purpose: To measure the forelimb muscle strength of animals [7, 23].

$\bullet$ Materials:

- Grip Strength Meter or Monitoring Devise (Fig. 5).

$\bullet$ Procedure: The mouse grips a bar connected to the monitoring apparatus while the experimenter gently pulls the tail of the rodent in a horizontal plane until the device registers the strength exerted by the mouse.

This test can also be conducted vertically. This modification increases validity, reliability and lowers variability. It is theorized that mice are more motivated to grip the bar in the vertical position due to a greater fear of falling. Additionally, the vertical test better utilizes the force of gravity and the pull of the mouse’s tail by the experimenter, resulting in more accurate and less variable recorded results. In this position, the total forces involved are more effectively transmitted to the transducer [29].

$\bullet$ Variables recorded:

- Forelimb strength as recorded by the apparatus.

$\bullet$ Advantages:

- Easy and quick to perform.

- No need of animal training.

$\bullet$ Limitations:

- Mouse motivational factors to grip the bar may increase variability in results.

- Differences in animal body mass can alter the results. This can be predicted if animal muscle force is proportional to body mass. However, a recent study has shown no correlation between body mass and the strength values recorded by the device [29]. Alterations in body mass due to age and/or other factors are not always linked to a proportional change in muscle weight but rather to a certain body composition [30].

The Weight Test evaluates the forelimb strength of rodents using a series of progressively heavier weights attached to a wire mesh ball. Mice are tested on their ability to hold each weight for three seconds, with rest periods in between attempts, to determine their maximum strength and endurance.

$\bullet$ Purpose: To assess forelimb strength in rodents.

$\bullet$ Materials:

- A Ball of fine wire mesh that will serve as an easy gripping object for mice.

- Chain links of increasing weight that can be connected to the ball of fine wire, ranging from 20 g to 98 g (Fig. 6).

$\bullet$ Procedure: The mouse is held from its tail vertically near the first weight allowing it to grasp the wire tightly raising it above the bench. If the mouse is able to hold the weight for 3 seconds, the experimenter will repeat the task with the second heaviest weight. Between the use of each weight the mouse rests for 10 seconds. After three weight fails the trial is ended and the maximum time holding the heaviest weight and the maximum weight achieved is recorded.

$\bullet$ Variables recorded:

- Time holding each weight: Duration (in seconds) that the mouse holds each weight.

- Maximum reached held weight: The heaviest weight (in grams) that the mouse can hold [31].

$\bullet$ Advantages:

- Simple and inexpensive material required.

- No training is required.

$\bullet$ Limitations: The variability in results due to individual differences in motivation and potential stress. Holding the mice longer before starting the trial may increase motivation to grasp the wire.

The Inverted Screen Test gauges rodents’ overall muscle strength, coordination, and fatigue by timing how long they can cling to an inverted wire mesh screen. This quick, cost-effective method provides a measure of motor function.

$\bullet$ Purpose: To evaluate the animals’ global muscle strength, coordination and fatigue [32].

$\bullet$ Materials:

- A 43 cm square of wire mesh composed by 12 mm squares of 1 mm diameter wire, bordered by a 4 cm wooden frame.

- A padded bench to ensure gentle falls (Fig. 7).

$\bullet$ Procedure: The mouse is placed in the centre of the screen which is rotated facing the padded bench in an interval of 2 seconds. Time is recorded from the moment the screen is inverted until the mouse falls.

$\bullet$ Variables recorded:

- Latency to Fall: The duration until the animal falls off the screen is scored as follows: Score 1: 1–10 s, Score 2: 11–25 s, Score 3: 26–60 s and Score 4: +60 s.

$\bullet$ Advantages:

- Fast and easy to perform.

- Requires simple and inexpensive materials.

$\bullet$ Limitations:

- Low sensitivity in detecting subtle motor deficits.

- Motivation and anxiety of the animals can influence the results [31].

The Wire Hang Test measures grip strength, endurance, and coordination in rodents by timing how long they can cling to a wire suspended above the ground. While easy to perform and requiring minimal equipment, the test’s results can vary due to the different ways animals can hang on the wire and the need for training to reduce individual variation.

$\bullet$ Purpose: To assess multiple aspects of locomotor ability, including grip strength, endurance, and body coordination [5, 33].

$\bullet$ Materials:

- A wire suspended 50–60 cm above the ground.

- Adhesive tape (for optional modifications) (Fig. 8).

$\bullet$ Procedure: The animal is placed on the wire which is suspended 50–60 cm above the ground. The time the animal spent on the wire is recorded. To obtain more objective and reliable results, the ways of hanging can be restricted, such as by covering the hindlimbs with adhesive tape.

$\bullet$ Advantages:

- The test is relatively easy to perform with low equipment requirements.

$\bullet$ Variables recorded:

The final reach and fall scores and measured for all animals tested. A survival curve can also be created using the timestamps for each instance of either the fall or reach score changing.

$\bullet$ Limitations:

- Training is required to reduce individual variation and obtain consistent results.

- The different ways animals can hang on the wire may affect their performance, introducing variability.

The Cylinder Test evaluates the sensorimotor function of the forelimbs and detects unilateral motor impairments by observing the use of forepaw paws during rearing. Rodents are placed in a cylinder, and their forelimb activity is recorded and analyzed, leveraging natural exploratory behavior for straightforward and minimally invasive assessment.

$\bullet$ Purpose: Assessment of sensorimotor forelimb area of the cortex and unilateral forelimb motor impairment.

$\bullet$ Materials:

- A cylinder: For rats: 20 cm diameter, 30 cm height. For mice: 15 cm diameter, 40 cm height.

- A mirror under the table to reflect the image of the cylinder.

- A camera for recording (Fig. 9).

$\bullet$ Procedure: The mouse is placed inside the cylinder for 10 minutes or until 20 rears take place. The mouse is expected to rear up using its forelimb paws to explore the walls of the cylinder. The activity with their forelimb paws is documented.

$\bullet$ Variables recorded:

- Ratio of left and right forepaw use. In cases of unilateral lesions, there is typically a decreased use of the contralateral forelimb paw during rearing.

$\bullet$ Advantages:

- Easy and fast to perform.

- Based on natural exploratory behaviour, requiring no animal training.

- Minimal equipment required [34, 35].

$\bullet$ Limitations:

- Not useful for models with global brain injury [33].

- Data analysis can be complex and time-consuming.

- Unconditional behaviors of mice, like remaining still or exploring the table’s edge, can lead to a low count of forepaw placements [5].

The Footprint Test assesses gait and motor coordination by analyzing the footprint patterns of rodents as they walk along a runway coated with non-toxic paint. This non-invasive method provides detailed information on stride length, base width, and gait symmetry, although it requires natural walking behavior and can involve time-consuming manual analysis.

$\bullet$ Purpose: To evaluate gait and motor coordination.

$\bullet$ Materials:

- Runway: A straight, narrow path with a darkened shelter at one end to encourage the rodent to walk through it.

- Non-toxic Paint: Different colours for hind paws and fore paws.

- Paper or Absorbent Surface: Placed on the floor of the runway to capture the footprints (Fig. 10).

$\bullet$ Procedure: The mouse walks over a white paper after coating its paws with ink or paint. The footprint pattern is studied.

$\bullet$ Variables recorded:

- Stride length: Distance between successive placements of the same paw.

- Front-base width: Distance between the left and right front paw prints.

- Hind-base width: Distance between the left and right hind paw prints.

- Gait Symmetry: Comparison of the left and right paw prints.

$\bullet$ Advantages:

- Non-invasive and easy to perform.

- Provides detailed information about gait and motor coordination.

- Can be used repeatedly to monitor changes over time.

$\bullet$ Limitations:

- Requires rodents to walk naturally, which may not always occur.

- Variability in individual rodent behavior can affect the consistency of results.

- Manual analysis of footprint patterns can introduce variability and human error.

- Manual analysis of footprints can be time-consuming.

- Inability to Capture Dynamic Parameters.

In recent years, automated systems have been developed to provide more detailed, accurate, and objective assessments of rodent gait patterns. These systems, such as CatWalk™, DigiGait™ and TreadScan™ use high-resolution video and specialized software to track the animal’s movements as it walks across a transparent or specialized platform [36].

CatWalk™: This system uses a glass walkway illuminated from below, with a high-speed camera capturing the footprints in real time. It measures not only static parameters like stride length and base width but also dynamic parameters such as swing duration, stance duration, and interlimb coordination, providing a more comprehensive analysis of gait [37].

DigiGait™: This system records the animal’s gait while walking on a motorized treadmill. The software generates a digital profile of each limb’s movement, offering detailed analysis of stride, step frequency, and paw placement. DigiGait™ is particularly useful for detecting subtle gait abnormalities that are difficult to observe with traditional methods [38, 39].

TreadScan™: Another automated system that uses treadmill-based gait analysis, TreadScan™ captures gait dynamics, including limb velocity, angle, and interlimb coordination. This method offers a highly sensitive assessment of gait irregularities and motor deficits in disease models like MS [40, 41, 42].

The Pole Test assesses rodent locomotion, motor coordination, and basal ganglia-related deficits, including bradykinesia. Mice are placed on a vertical pole to measure their time to turn and descend. This straightforward and cost-effective test effectively detects motor impairments but requires prior training and can be influenced by the rodent’s weight, motivation, and anxiety.

$\bullet$ Purpose: Assessment of overall locomotion and motor coordination, basal ganglia related movement deficits in rodents [43]. It can also be used to test bradykinesia, or slowness of movement [44].

$\bullet$ Materials:

- A vertical pole. Typically, 50–60 cm in length and 1–2 cm in diameter.

- A padded base platform to catch the rodent if it falls.

- Camera for recording the test to analyse the rodent’s movements (Fig. 11).

$\bullet$ Procedure: The mouse is placed with its head oriented upward on the top of a vertical pole. The mouse is allowed to descend the pole by turning its head downward. The latency to make the turn (Tturn) and the time to descend (TD) is recorded. If the mouse descends without turning its head downward, the TD is used to represent Tturn. The objective is to measure the time required for the mouse to descend the pole and reach the ground. If the mouse is unable to reach the ground within 60 seconds, the test is discontinued. If the mice pause during descending the trial is excluded and repeated. Prior to the actual test, mice undergo two training sessions to familiarize them with the task. The testing phase occurs on the third session. During the test, each trial is repeated twice consecutively, and the results from both trials are averaged to ensure accuracy and reliability [32, 43, 45].

$\bullet$ Variables recorded:

- Latency to Turn (Tturn): Time taken to turn from the head-up to the head-down position.

- Time to Descend (TD): Time taken to descend from the top to the bottom of the pole [44].

- Total Time: Sum of the turn time and descent time.

- Falls: Frequency and timing of falls during the test [46].

$\bullet$ Advantages:

- Sensitive to Motor Deficits. Effectively detects motor coordination and balance impairments.

- Simple and Inexpensive. Requires minimal equipment and is easy to set up.

$\bullet$ Limitations:

- Validity is questioned in MS mouse model. Scarce studies are published.

- Heavier rodents may not perform well in this test.

- Animal training is required.

- Motivational and anxiety factors may alter results.

This is one of the most frequently test used to detect cognitive dysfunction in mice involved in MS experimental models. The test is based on the rodent’s innate tendency to explore novel environments, which requires intact short term and spatial memory.

$\bullet$ Purpose: Assessment of short term spatial memory (hippocampus) and learning deficits or working memory function (prefrontal cortex) [47, 48].

$\bullet$ Materials:

- A Y-shaped maze with three arms of equal length, typically 30–50 cm each, oriented 120° from each other. Arms are labelled (e.g., A, B and C). The entry of each arm includes a divider or door that can be removed or placed according to the protocol being performed.

- Optional: Reward or food that will be placed at the end of some arms according to the protocol being performed.

- A camera to record the task (Fig. 12).

$\bullet$ Procedure:

This test can be carried out following two different protocols.

- Protocol 1:

Three arms remain open. The mouse is placed at a starting arm and is allowed to explore all three arms freely for 8 minutes without being previously familiarized with the maze. The behaviour of the mouse is recorded. A mouse with no lesions in the hippocampus and prefrontal cortex is expected to alternate between all three arms spontaneously and inspect all of them.

- Protocol 2:

This protocol includes two steps:

First, during training the mouse is placed in a starting arm. One of the entries of the three arms is blocked allowing the mouse to move freely between the other two arms for 5 minutes. One of the arms may include a reward to increase motivation during the exploration time. Afterwards, the mouse is returned to its cage and remains there for e.g., 2 hours (Inter Trial Interval, ITI). The task will be more challenging as the ITI increases since it will require greater memory skills. Meanwhile the maze is cleaned and prepared for the second step.

Second, during testing the mouse is placed again in the starting arm but this time is allowed to move freely between all three arms for 5 minutes and none is blocked. If spatial and short memory have no deficits, the mouse will remember which arm wasn’t entered yet and will be more prone to explore the novel arm (the arm that was blocked on the first step).

$\bullet$ Variables recorded:

- Number of Arm Entries: An arm entry is defined when all four limbs of the mouse are within the arm. Indicates the level of activity and exploration.

- Time Spent in Each Arm: Assesses preference or aversion, which can reflect anxiety or other behavioural changes.

- Spontaneous Alternation: The percentage of consecutive entries into all three arms without repetitions. A lower alternation rate may indicate working memory deficits. Alternation is defined by the following formula: % Alternation = Number of alternations/(Total number of arm entries –2) $\times$ 100 [49, 50].

$\bullet$ Advantages:

- The test is straightforward and easy to perform, requiring minimal training for the rodents.

- Basic material needed.

- The test is non-invasive since it leverages natural exploratory behaviour.

$\bullet$ Limitations:

- Data analysis is time consuming.

- Mouse behaviour can be confounding and play a role in the results.

- The test is sensitive to environmental external factors such as lighting and noise, which need to be strictly controlled.

- Primarily assesses working memory and spatial learning, and may not capture other cognitive impairments associated with MS [5].

The Novel Object Recognition (NOR) test evaluates visual non-spatial short-term recognition memory, primarily engaging the hippocampus. It involves a systematic procedure with an open field arena and distinct objects to gauge a rodent’s ability to recognize novel stimuli compared to familiar ones.

$\bullet$ Purpose: The NOR test is used to assess visual non-spatial short-term recognition memory, focusing on the hippocampus [51].

$\bullet$ Materials:

- An open field arena, which is typically a square or circular enclosure. Some protocols specify the arena to measure 60 $\times$ 60 $\times$ 20 cm, with the floor divided into 36 equal sections using gridlines. The walls should be sufficiently high to prevent the animal from escaping [5].

- Objects for the rodents to explore, usually two identical objects for the familiarization phase and a novel object for the test phase. The novel object should be similar in aspect and size but have different texture and shade to allow differentiation by the mice. It is not recommended to use objects of different colours due to mice limited ability to distinguish between colours [52, 53].

- A camera for recording purposes (Fig. 13).

$\bullet$ Procedure:

(1) The test is conducted in three steps: habituation, familiarization and testing.

(2) Habituation: The mouse is placed individually in the open field box without any objects and is allowed to explore the arena freely for 10 minutes. The mouse is returned to its cage.

(3) Familiarization: The day after, two identical objects are placed in the open field arena and the mouse is allowed to explore the area for 5–10 minutes. The mouse is returned to its cage. The time spent exploring each object is recorded.

Testing: After 4 hours, one of the objects is replaced by the novel item and the mouse is permitted to explore the arena for 5–10 minutes. Healthy rodents tend to notice new objects and inspect them for a longer period.

$\bullet$ Variables recorded:

- Exploration Time: The total time spent exploring both objects.

- Discrimination Index: (Time exploring the novel object – Time exploring the familiar object)/(Total time exploring both objects).

- Preference or recognition index: Time exploring the novel object/Total time exploring both objects. This provides a normalized measure of memory performance. A preference for the novel object indicates intact recognition memory [54].

Positive exploratory behaviour is defined by the times the nose or the forepaws of the mouse contact the object or by the times the nose of the mouse is up to 0.5 cm far from the object and is directed towards it. Behaviours such as sitting, standing, leaning on the object, or turning around are not considered an exploration.

Scoring can also be used as follows: the nose touch = 1 point; two paws on object = 2 points; four all paws on the top of object = 3 points. Points are tallied and touch to familiar versus unfamiliar objects are compared [55, 56].

$\bullet$ Advantages:

- Basic material required.

- Sensitive to cognitive impairments.

$\bullet$ Limitations:

- Data analysis is time consuming.

- Primarily assesses short-term recognition memory and may not fully capture other cognitive domains affected by MS.

- Rodents’ exploratory behaviour can be influenced by factors such as anxiety, motivation, and environment, which need to be carefully controlled.

The Open Field Test evaluates locomotor activity, exploratory behavior, and anxiety-like responses in rodents by utilizing their natural aversion to open, brightly lit environments. It involves observing the mouse’s behavior in a large, enclosed arena, providing insights into their motor functions and emotional state.

$\bullet$ Purpose: Assessment of locomotor activity, exploratory behaviour and anxiety-like behaviour [57].

$\bullet$ Materials:

- An Open Field Area. A large, enclosed space that can be circular or square. It must be sufficiently large for the mouse to feel exposed and with high walls to prevent escape.

-Recording camera [58].

-Software for data analysis (Fig. 14).

$\bullet$ Procedure: The mouse is placed in the open field box and is allowed to explore freely for 5–10 minutes while its spontaneous locomotor and exploratory activity is being recorded.

$\bullet$ Variables recorded:

Motor activity assessment:

- Horizontal Activity (Ambulation): Measured by the total distance travelled in cm [59].

- Vertical Activity (Rearing): Frequency of the mouse standing on its hind legs. This indicates exploratory behaviour and motor function.

Anxiety and Emotionality assessment:

- Zone Entries and Time Spent in Zones: Time spent in the centre versus outer zones ratio can be calculated. Healthy mice tend to enter the inner zones more frequently than anxious mice [8, 60].

- Thigmotaxis (Wall-Hugging): Tendency to remain close to the walls calculated as: (Total time spent close to the walls)/(Total test time) $\times$ 100. Anxiogenic behaviour can is correlated with higher thigmotaxis.

- Latency: Time taken to initiate movement. A shorter latency period is linked to higher exploratory behaviour and less anxiety [61].

- Number of defecations. Increased number of defecations indicates higher emotionality [57, 62].

$\bullet$ Advantages:

- Easy to set up and carry out.

- The test can analyse several parameters at the same time, which increases its efficiency. These parameters include motor activity, anxiety and emotionality [36].

$\bullet$ Limitations:

- Motor assessment can be confounded by anxiety or other psychological factors and vice versa. Psychological parameters registered that are concluded by tasks that rely on the mobility of the mouse can be confounded by locomotor deficits of the mouse [57, 61].

- Sensitive to external environmental factors such as variations on lighting and noise [62].

- High variability due to the availability of multiple protocols and different setup designs make it difficult to compare studies.

- Limited free available softwares for data analysis.

- Data analysis is time consuming.

The Elevated Plus Maze (EPM) Test assesses anxiety-like behaviors in rodents by exploiting their aversion to open spaces and heights alongside their curiosity for novel environments. By comparing their exploration of open versus enclosed arms, researchers can infer levels of anxiety based on the rodent’s preference for safe, enclosed spaces over open, elevated areas.

$\bullet$ Purpose: Assessment of anxiety-like behaviours in rodents.

$\bullet$ Materials:

- An apparatus with four arms (50 cm long $\times$ 10 cm wide) arranged in a plus (+) shape with two open arms (without walls) and two enclosed arms (with high walls, 40 cm high). The arms converge in a central area, and the apparatus is placed 50 cm above a padded base.

- A recording camera.

- Computer software for data analysis (Fig. 15).

$\bullet$ Procedure: The trial consists of two phases: habituation and testing. During the habituation phase, the mouse is placed in testing room for minimum 30 minutes for acclimatization. During the testing phase, the mouse is placed individually in the centre of the maze directed to the open arm and is allowed 5–10 minutes to explore the maze freely [63, 64].

$\bullet$ Variables recorded:

- Total distance travelled.

- Time spent in each type of arm, closed vs open: Reduced time in open arms and increased time in closed arms suggest heightened anxiety.

- Number of Arm Entries: More entries into open arms suggest lower anxiety [65].

$\bullet$ Advantages:

- Easy to perform.

- High validity and reliably results.

$\bullet$ Limitations:

- The test is sensitive to previous experiences of the mouse. In case mice are tested during the same day with different methods, the EPM should be performed first.

- Time spent on the central platform varies among trials and can significantly alter results. This time is difficult to interpret and reduces the time spent in open and enclosed arms.

- The exploratory behaviour and time spent in open arms decrease significantly after the first trial, known as the one-trial tolerance phenomenon [66].

- Unpredictable mouse behaviour can introduce confounding factors.

- Data analysis is time consuming.

The Elevated Zero Maze (EZM) evaluates anxiety-like behaviors in rodents by using a circular apparatus with both open and enclosed sections. Unlike the Elevated Plus Maze, the EZM’s continuous circular design avoids central platform ambiguities and does not exhibit the one-trial tolerance phenomenon, offering a clearer measure of anxiety through time spent and entries into open versus enclosed areas.

$\bullet$ Purpose: To evaluate anxiety-like behaviours [67].

$\bullet$ Materials:

- An annular (circular) apparatus elevated above the floor, divided into four equal quadrants with two open (without walls) and two enclosed (with high walls) sections.

- A recording camera (Fig. 16).

$\bullet$ Procedure: The test is carried out as previously described in the EPM. The starting test position of the mouse is in one of the two enclosed quadrants [63].

$\bullet$ Variables recorded:

- Time Spent in Open vs. Enclosed Sections: Increased time in open sections suggests lower anxiety, while more time in enclosed sections indicates higher anxiety.

- Number of Entries into Each Section: More entries into open sections suggest reduced anxiety.

- Total Distance Travelled: Can indicate overall activity levels.

$\bullet$ Advantages:

- The circular design eliminates the ambiguity of time spent in a central platform, unlike the EPM.

- The one-trial tolerance phenomenon is not described in the EZM [66].

$\bullet$ Limitations:

- Similar to the EPM.

The Marble Burying Test leverages the natural digging and burying behaviours of rodents to assess alterations in exploratory and anxiety-related behaviours. It is well-documented that mice with hippocampal lesions, such as those induced by cuprizone demyelination, perform poorly in this test. This poor performance is attributed to cognitive deficits, motivational issues, and increased anxiety, all of which are functions mediated by the hippocampus [68].

$\bullet$ Purpose: To evaluate anxiety-related and obsessive-compulsive behaviours in rodents.

$\bullet$ Materials:

- A cage filled with a 5 cm deep layer of wood chip bedding, lightly tamped to create a flat, even surface.

- 9 to 25 marbles, arranged according to the protocol used, placed evenly in the cage following a regular pattern, about 4 cm apart from each other [69] (Fig. 17).

$\bullet$ Procedure: The subject is placed in the cage with a layer of marbles, and its natural burying instinct is observed and recorded for 30 minutes. The primary measure is the number of marbles buried at the end of the test.

$\bullet$ Variables recorded:

- Number of Marbles Buried: This is a direct measure of digging behavior and is recorded using the following scoring system: Not buried: 0, Partially buried (up to 2/3 buried): 0.5, Buried (2/3 their depth or more): 1 [70].

$\bullet$ Advantages:

- Simple and cost-effective setup.

- Non-invasive method to assess anxiety and obsessive-compulsive behaviours.

$\bullet$ Limitations:

- Interpretation of results may be complicated by other variables affecting digging behaviour, such as general activity levels or motor function deficits.