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Appearance, body weight, temperature, spontaneous behavior, touch escape, object approach, trunk curl, whisker reflex, reaching reflex, Pinna reflex, provoked biting
Mice are housed in regular cages containing wireless running wheel (Med-Associated, St. Albans, Vermont) and activity patterns are monitored for days to weeks to examine how activity patterns vary under different light-dark cycles to analyze the animal’s circadian systems.
The locomotor activity monitoring is a common measure of exploratory behavior and general activity in both mice and rats. Animals are removed from the home cage and placed individually into a novel arena over a 10- to 120-min period. The activity is recorded by digital video or infrared beams. Field chambers of 22 x 42 cm are used for general assessment of locomotion.
The grip strength test can be used to measure forelimb/forepaw strength in rodents. Strength is measured with a semi-automated system (Animal Grip Strength System; San Diego Instruments). The entire test is approximately 10 seconds.
The rotarod is a standard test of motor coordination and balance in rodents. The rotarod apparatus (Ugo Basile) consists of a rotating cylinder (approximately 3 cm in diameter at a speeds ranging from 4 to 40 rpm); the animal is placed on the cylinder and must walk continuously to keep from safely falling. Latency at which mice fall off the rotating cylinder is measured. Maximal trial length is 300 sec. Rotarod performance may be assessed three times daily for 3 days.
The beam walking assesses a mouse’s ability to maintain balance while traversing a narrow beam to reach a safe platform. Measurements recorded include the time taken to cross the beam and the number of paw fault or slips. The entire test is approximately 60 seconds.
A digital video system that captures paw placements of mice during treadmill locomotion and generates standard gait measures from the video images (DigiGait, Mouse Specifics, Inc.) is as an additional measure of motor assessment.
Mouse is placed in a cylinder and its forelimb activity while rearing against the wall of the arena is recorded. Forelimb use is defined by the placement of the whole palm on the wall of the arena, which indicates its use for body support. Forelimb contacts while rearing are scored with a total of 5 min for each animal.
The pole test is used to estimate the bradykinesia. The mouse is placed head-upward on the top of a vertical rough-surfaced pole and the time until it descended to the floor is recorded.
The pattern of exploration of an open field is used in rodent studies as a measure of anxiety-like behavior, using the innate aversion of rodents to explore large, open areas. Similar to the activity monitory, activity is recorded by digital video or infrared beams for 10 min in an open field. The chamber in this test is 50 x 50 cm. The amount of time the rodent engages in thigmotaxis (the tendency of the animal to stay in contact with the wall) is determined. The test arena is divided into a center and periphery zone. The periphery is an 8 cm wide zone adjacent to the walls and is used to determined thigmotactic behavior.
The Elevated plus Maze is a widely used animal model of anxiety that is based on conflicting tendencies. The apparatus (Med-Associated, St. Albans, Vermont) consists of two open and two enclosed arms that form a “plus”. The mouse is given the choice of spending time in open, unprotected maze arms or enclosed, protected arms, all elevated from the floor. Mice are placed into the center of the apparatus at the beginning of the session and the number of arm entries and the amount of time spent in the open and closed arms are recorded (AnyMaze video-based system, Stoelting Co.) for 5 to 20 min.
Like the open field test, the light-dark box test uses an innate aversion of the animal to explore open, brightly lit areas as a test for anxiety-like behavior. In this task the animal is provided with an escape to a dark region of the test chamber. The overall test chamber is 22x42 cm, with the darkened chamber consisting of a 22x17cm section of that and the light chamber the remainder. The two chambers are connected by a dividing wall that contains a small portal at the base. Mice are place into the test chamber and activity is monitored for 10 min. Entrances in to the light box and time spent in the light box are the experimental measures.
In the social interaction test the dependent variable is the time pairs of male or female mice spend in social interaction, which decreases when mice are anxious. On the day of testing, pairs of mice are placed into a test chamber and observed for 10 minutes. All social interactions between mice are recorded (AnyMaze video-based system, Stoelting Co.).
The tail of the mouse is taped to the top of an open chamber so that it cannot escape. A mouse will try to escape from tail suspension by engaging in vigorous movements and then, after a few minutes, will become immobile. The mouse is suspended for 6 minutes and the amount of “immobility” is measured.
The barnes maze is a dry-land maze test for spatial learning and memory. It consists in a circular platform with 40 holes along the perimeter (Med-Associated, St. Albans, Vermont). During testing, animals receive reinforcement to escape from the open platform surface to a small, dark chamber located under one the holes called the “target box”. Latency, errors are recorded. Latency is defined as the time it takes to locate the target box.
The spontaneous alteration task is used to assess spatial “working memory” in rodents. The task is based on the innate, unlearned response of rodents to explore a previously unexplored arm of Y- or T-maze. Mice are placed in one arm of the Y-maze or T-Maze (Med-Associated, St. Albans, Vermont) and allowed to freely explore all three arms for 4-15 minutes session. The number and pattern of arm choices are recorded.
One arm is blocked. Starting from the stem, mice are allowed to explore the maze for 15 minutes. The rodent turns to the maze 1-4 hours later or after a delay of up to a week with all arms open and scored for 4-15 minutes. The first arm entered, the amount of time spent in each arm and the number of entries into each arm is recorded.
A positive reinforcer is placed in one of the arms, since the mice need to learn to locate the food reward, they were food deprived to 85% of ad libitum body weight and maintained at this weight throughout the experiment. Mice are habituated to the maze and arm preference is determined. Before training, food- and water-deprived mice are habituated to the maze for three 30-min sessions during which the reinforcer is intermittently available in the recessed wells. After the third day of habituation, mice are run individually on the T-maze task, 6-12 trials per day, with a 45-s intertrial interval. Before each trial mice are confined to either the left or right arm of the T-maze. At the beginning of each trial, the Plexiglas panel is lifted to allow access to the entire maze. Mice have the opportunity to earn two reinforcers per trial, by running into the stem first and then into the opposite arm, where mice are confined until the start of the next trial. Mice are run on the task until reaching a predetermined criterion, which typically takes 12-20 sessions. Mice are then tested periodically, up to 10 sessions per month, to assess changes in spatial working and reference memory.
The radial maze task has been used most extensively used to investigate specific aspects of spatial working and reference memory. This task is based upon the premise that animals have evolved an optimal strategy to explore their environment and obtain food with the minimum amount of effort. The radial maze (Med-Associated, St. Albans, Vermont) is made with eight arms. Reinforcers are the same as for the T-maze rewarded alternation task. A recessed well is located at the end of each arm for the delivery of reinforcers. Before training, food- and water-deprived mice are habituated to the maze for three 30-min sessions during which the reinforcer is intermittently available in the recessed wells. After the third day of habituation, mice are run individually on the radial maze task, 1-12 trials per day, with a 45-s intertrial interval. At the beginning of each trial, mice are placed on the central platform, and allowed to explore the maze. Depending on the experimental parameters, some or all of the arms may be reinforced, and some or all of the arms may be accessible to the mice at any given moment. A trial lasts until all the available reinforcers are consumed or 5 min., whichever comes first.
The social recognition test is designed to investigate social learning and memory. Animals are tested for their ability to remember conspecifics over various time intervals. Social familiarization is quantified as the total amount of time engaged in olfactory investigation. Recognition is measured as a significant decrease in time spent engaged in olfactory investigation in repeated encounters. The most common protocol for assessing the social recognition examines repeated olfactory investigation of the same stimulus animal in two 5 minutes trials separated by inter-trial intervals of varying length.
The recognition test is based on the natural tendency of rodents to investigate a novel object instead of a familiar one. The choice to explore the novel object reflects the use of learning and (recognition) memory processes. In the two-trial novel object recognition test, a rodent is placed in an enclosure and exposed for a set length of time to two identical objects that are located a specified distance from each other. The rodent is then removed from the environment and a predetermined amount of time is allowed to pass. The subject is then retested in the same environment except that one of the two previously used (familiar) objects is replaced with a novel object that differs from the familiar object in shape, texture and appearance and the rodent behavior is recorded.
This widely used associative learning task involves measuring a behavioral fear response (i.e. time spent while motionless or “freezing”) to a conditioned stimulus (cue and context) that predicted a mild foot-shock (0.1-0.5 mA) during training trials. We will use a one-trial protocol where mice are trained on Day 1 and tested 24 hrs later on Day 2. The training trial consists of a 2 min acclimatization period (used to determined baseline freezing) followed by presentation of the auditory cue for 30 seconds and then immediate presentation of the foot-shock stimulus for 2 sec. The mice are removed from the test chamber and returned to their home cage. On day 2 mice are re-exposed to the test chamber (no cue presentation) and context-dependent freezing is recorded. Mice are then moved to a test chamber containing an altered context and the auditory cue is presented to induce cued freezing. Data collection and analysis are semi-automated via a video-monitoring fear-conditioning apparatus (Med Associates, Inc.). We use this task because: 1) The freezing response involves no complex motor action, limiting the impact of confounding motor abnormalities. 2) It provides a means of distinguishing hippocampal-dependent associative learning deficits (context conditioning) from amygdala-dependent deficits (cue conditioning) 3) A single trial can produce robust learning in wildtype mice making this procedure optimal for high-throughput analysis of multiple transgenic mouse lines.
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