Several customized behavioral tests are offered to phenotype mouse and rat models and evaluate effects of compounds in different in vivo models.
New behavioral tests are continuously developed and validated.
Barnes Maze Test
The Barnes maze is a standard behavioral test to investigate spatial learning and cognition in rodents.
The test consists of a circular arena with equally distributed holes on its periphery with one hole serving as target zone that is equipped with the animal’s home cage. The animal can use proximal or distal visual cues to locate and remember the target zone. Automated recordings of consecutive trials provide details on the rodent’s navigation in the arena and thus delivers information about their learning and memory performance.
Figure 1: Experimental set-up of the Barnes Maze test. Learning and reversal learning of the hippocampal learning protocol. The test can also be performed with a striatal learning protocol (not shown).
Figure 2. Barnes Maze test in 5xFAD transgenic mice. Latency of the first contact with the escape hole (A) and latency of the first contact with the target whole during the probe trial (B). n = 16 per group. Two-way ANOVA with Bonferroni post hoc test (A) and Mann-Whitney U-test (B); **p<0.01; ***p<0.001.
Contextual Fear Conditioning Test
This test allows the simultaneous assessment of associative learning by simple cues and/or learning by complex stimuli such as context.
Animals are trained and tested on 2 consecutive days. On the training day animals are placed in a chamber and receive a conditioned stimuli / unconditioned stimuli package (tone/foot shock). Contextual memory is tested 24 hours after training. To do so the animal is placed into the same chamber as during the training and freezing behavior is automatically recorded. Cued memory is scored one hour after the contextual test in a novel, modified chamber where the animal receives the auditory cued stimulus and freezing behavior is again automatically recorded.
The time spent freezing during the contextual test and tone-dependent test are defined as index of fear-related learning and memory. Cued and contextual fear conditioning is a task that measures the ability of the animal to learn and remember an association between an aversive experience and environmental cues. All parameters of the test are measured automatically with a tracking system.
Contextual fear conditioning test. Percentage of freezing of MK-801- and sham-treated animals during the contextual phase of the CFC. A: Freezing behavior over a 5 minutes testing period; B: Total freezing time. n = 10 per group. Mean ± SEM, Two-way ANOVA with Bonferroni’s post hoc test (A), unpaired t-test (B); **p<0.01; ***p<0.001.
Morris Water Maze Test
The Morris water maze is widely used to study spatial learning and memory. Animals are placed in a pool of water and have to swim to a hidden platform. Each mouse has to perform three trials on each of four consecutive days. After the last trial on day 4, mice have to fulfill a probe trial. During the probe trial, the platform is removed from the pool and the number of crossings over the former target position is recorded together with the abidance in the target quadrant.
Assessment of spatial learning of APPSL transgenic mice. MWM learning curves showing distance traversed and escape latencies during 4 testing days of 6 (A, D), 9 (B, E) and 12 month (C, F) old animals. 6 month old APPSL n = 19, ntg n = 21; 9 month old APPSL n = 21, ntg n = 19; 12 month old APPSL n = 13, ntg n = 22. Two-way ANOVA with Bonferroni ‘s post hoc test compared to ntg. *p<0.05; **p<0.01; ***p<0.001.
Novel Object Recognition Test
This test is used to assess memory of interactions with novel objects. Rodents tend to spend more time interacting with a new object rather than with an already known object. Acquisition phase: on the first day the animal is habituated to the test chamber. On the second day the animal is placed in the box and allowed to explore two identical objects for a set period of time. Retention phase: on the third day, the animal is returned to the box where one of the familiar objects has been replaced with a novel object. The time exploring the novel object relative to the time spent exploring the familiar object is measured to evaluate memory.
Passive and Active Avoidance Test
The passive avoidance behavior is based on negative reinforcement and examines long-term memory and emotional learning.
In this task, the animals learn to refrain from stepping through a door to an apparently safer but previously punished dark chamber. A light and a dark chamber are connected in an apparatus separated by a door.
Acquisition: The mouse is placed into the illuminated chamber of the apparatus upon entering the dark chamber, the door will close and an electrical stimulus is applied. Retention test: 24 hours after acquisition, the animal is placed back into the illuminated chamber. The time until the animal visits the dark chamber (latency) is measured and serves as an index of emotional memory. All parameters are measured using an automated tracking system.
Passive Avoidance Test in male Scopolamine-treated Sprague Dawley rats at the age of 10 weeks. Animals were treated once with 1 mg/kg Scopolamine. n = 14 per group. Mean + SEM. Mann-Whitney U-test; **p<0.001.
Two Choice Swim Test
An unique feature of this test is the possibility to test spatial learning abilities also in modestly motor-impaired animals, since the choice accuracy is not influenced by motor deficits.
The water filled corridor-like tank is illuminated at one end. The hidden platform is positioned either at the dark or lit end of the tank. The mouse is placed in the center of the tank. During acquisition of the task, each animal performs several training trials by swimming towards or away from the light to find the platform. Following the acquisition phase, the platform is switched towards the opposite end of the corridor, while the light source remains at the former position to test for reversal learning. In all trials, both choice and latency to reach the platform are recorded.
Figure 2. Two Choice Swim Test of 12 week old R6/2 mice. Number of wrong choices compared to non-transgenic mice. Mean ± SEM. n = 14 per group. Two-way ANOVA; **p<0.01.
The Y-Maze Test is based on the natural behavior of rodents to explore new environments. Rodents typically prefer to investigate new environments rather than familiar ones, so animals will explore a new arm of a maze rather than returning to the one that was previously visited. Many parts of the brain including the hippocampus, septum, basal forebrain, and prefrontal cortex are involved in this task.
Spontaneous Alternation test
Testing occurs in a Y-shaped maze with three plastic arms oriented in a 120° angle. After placing the mouse in the center of the maze, the animal is allowed to freely explore the three arms, where it should show a tendency to enter a less recently visited arm. This test is used to quantify cognitive deficits in transgenic animals and evaluate novel compounds for their effects on cognition.
Y-Maze Novelty and Memory
To test whether rodents prefer to spend time in new or familiar areas, one arm of the Y-maze is blocked and the animal is allowed to explore the other two arms. The rodent is then placed in the start arm and the blocked arm is opened, so the animal should show a tendency to enter the formerly blocked arm more frequently. The Y-maze test is particularly useful as an initial test of memory function in mice.
Y-Maze, longitudinal evaluation of 9 month old 5xFAD transgenic and non-transgenic littermates. Longitudinal evaluation of the alternation rate (A) and number of arm entries of testing 1 and 2 in the Y-Maze (B) of 5xFAD and ntg mice; n = 8 per group, mixed gender. Mean ± SEM. Two-way ANOVA. **p<0.01.