All animals will undergo testing for developmental neurological/neurobehavioral abnormalities prior to behavioral testing as a criterion for inclusion. For a description of these studies, please visit the Locomotor/Neuromotor Function page.
Rationale: The elevated plus maze is used to assess emotionality, reactivity, exploration and activity levels. All these measures are strongly modified by aging.
Instrument: Elevated Plus Maze apparatus from San Diego Instruments.
Measures: The test setting consists of a plus-shaped apparatus with two open and two enclosed arms, each with an open roof, elevated from the floor. Animals are allowed to freely explore. Basal anxiety levels are indicated by the proportion of time spent in the closed arms (time in closed arms/total time of exploration). Total number of arm entries provide a measure of general activity.
Data Inference: Time spent in open arms is expected to be lower than time spent closed arms since open and elevated spaces are aversive to mice.
Controls: A minimum of 20% trial time spent exploring the maze is used as threshold for inclusion to rule out confounds related to lack of motivation or low activity levels.
Rationale: Hippocampal dependent contextual memory of an aversive event can be tested in the fear conditioning task.
Instrument: Coulbourn isolated behavioral test chambers for mice, and Coulbourn FreezeFrame 4 Software for data recording and quantification.
Measures: Fear conditioning is conducted over 2 days. On the first day, mice receive 2 pairings of a 30 second auditory stimulus (white noise), which co-terminates with a 2 second mild foot shock. 24 hours later, hippocampal dependent contextual memory is assessed during a 5 minute exposure to the context itself, in the absence of any tone or shock. The length of time an animal exhibits freezing behavior during the contextual test is an indicator of hippocampal dependent memory for the association between the context and the aversive stimulus.
Data Inference: Contextual fear conditioning measures the strength of the association between the aversive stimulus and the context in which it occurred, which involves hippocampal integration of signals from the amygdala. Longer times spent freezing during the contextual test indicates that hippocampal contextual memory is intact.
Controls: Freezing behavior during the initial acquisition trial is monitored to ensure equivalent learning among all groups. If differential learning is coupled with contextual memory deficits, results can be reported as a deficit in learning and memory (in contrast to only memory impairment). Animals will be excluded from the final data analysis if they exhibit less than 7% freezing behavior during the initial acquisition session. Additionally, as fear conditioning and the freezing response to contextual cues also relies on the amygdala, amygdala function will be confirmed via freezing response to an acoustic stimulus which was previously paired with the mild foot shock. The presence of equivalent amygdala function among all groups confirms that any deficits in contextual fear memory are due to hippocampal dysfunction.
Rationale: The forced swim test measures motivation in rodents.
Instrument: Transparent Plexiglas cylinders and video recording software.
Measures: Mice receive a single 6 minute session, divided into 2 minutes pre-test and 4 minute testing phase. Rats receive 2 sessions separated by 24 hours: a 15 minute pre-test and a 5 minute test. The length of time spent immobile/floating, struggling/climbing, and swimming are quantified during the testing phase. Immobility scores, defined as the lack of movement beyond those necessary for balance to keep the animal’s head above water, are indicative of depressive-like behavior in rodents.
Animals are monitored at all times when they are in the water. If an animal appears to be in serious distress, the animal is removed from the water and excluded from the study.
Data Inference: The forced swim test is based on the assumption that after a period of time, animals will forgo escape efforts and exhibit immobility, which is indicative of behavioral despair.
Controls: Ability to swim unencumbered (criterion for inclusion).
Rationale: The complete Morris water maze task tests spatial and non-spatial learning and memory, which are impaired with aging.
Instrument: Morris water maze pool, visual cues, platform, and HVS AND ANY-maze software.
Measures: The test is conducted over 2.5 weeks. We routinely run the task to include reversal training, which measures cognitive flexibility (or lack thereof, ‘perseverance’), a cognitive deficit commonly associated with aging. Animals are monitored daily, and their weights are recorded weekly during the course of testing. Our studies are run in conditions that minimize floating and preclude hypothermia.
Data Inference: Time and path taken to reach a hidden escape platform while relying on extra-maze cues are measures of learning. Time spent in the former location of the escape platform is one measure of memory.
Controls: Capacity for unencumbered swim to a platform in a linear swimming chamber, and visual competence, are some key criteria for inclusion in MWM studies.
Rationale: Novel object recognition is used to evaluate non-spatial recognition memory, which decreases with aging.
Instrument: For mice a large rodent cage (16”x9”x5.5”) is used. FreezeFrame, Ethovision Software.
Measures: The test in conducted over a period of 3 consecutive days. The same test cage is used throughout with each mouse having their own individual cage. The mouse is habituated on the first day and allowed to explore for 5 minutes. On the second day two identical objects are placed on one side of the cage and the mouse is allowed to explore for 5 minutes. On the third day one object from the previous day and one new object are placed and the mouse is allowed to explore for 5 minutes.
Data Inference: The introduction of novelty in a set of objects of equivalent interest is expected to result in increased exploration if recognition memory is intact. A minimum of 20% trial time spent exploring the stimuli is used as threshold for inclusion to rule out confounds related to lack of motivation or low activity levels.
Controls: Animals have to be physically interacting with the object or showing direct attention towards the object to be counted as time spent.
Rationale: Long-term memory of an aversive event can be tested using inhibitory avoidance tasks. The passive avoidance test is routinely used for testing for contextual conditioning.
Instrument: Passive Avoidance System from San Diego Instruments.
Measures: The passive avoidance assay is performed over a period of three days. On Day 1 the mouse is habituated in the right chamber of the apparatus by leaving the mouse in the dark for 30s. After the 30s has passed a light comes on in the compartment the mouse is in and the gate separating the compartments is opened to reveal the dark chamber on the left. The time to move from the lighted to the dark compartment is recorded, with a maximum time limit of 5 minutes. The mouse is immediately taken out of the dark chamber once it crosses over. On Day 2, the mouse is placed in the right chamber as before and left in the dark for 30s. The gate opens after 30s to allow the mouse to move to the dark chamber. Once the animal crosses over a shock (0.2mA) is given for 2s. The mouse is left in the dark chamber for an additional 30s and then removed from the dark chamber. On Day 3, the protocol is the same as for Day 1. Latency to cross over to the dark compartment is recorded, with an upper limit of 5 minutes.
Data Inference: Latency to enter an environment associated with an unconditioned aversive stimulus (brief mild electric shock) should be increased if memory of the aversive stimulus is intact.
Controls: Minimal shock intensity needed to motivate animals is determined prior to testing.
Rationale: Spatial novelty is used to evaluate spatial recognition memory, which decreases with aging.
Instrument: The spatial novelty task is performed in an opaque white chamber measuring 11 x 11 x 8 in (W x D x H) using Actimetrics (Limelight) Software.
Measures: This task comprises habituation, training, and testing. During habituation, mice are allowed to freely explore an empty chamber featuring prominent visual cues for 5 minutes. Twenty-four hours later mice are returned to the chamber for presentation with 3 distinct objects in that context. Total time of exploration and percent time spent exploring each of the objects is recorded. Testing involves the displacement of one object and can be performed at different times after training as needed to test different mechanisms of memory formation and consolidation. Exploration is defined as the animal orienting toward an object and within 1cm. Total time exploring and percent time exploring each object is recorded.
Data Inference: The introduction of a novel location for one familiar object among a set of objects of equivalent interest is expected to result in increased exploration if recognition memory is intact. A minimum of 20% trial time spent exploring the stimuli is used as threshold for inclusion to rule out confounds related to lack of motivation or low activity levels.
Controls: This task requires careful set up as it is important to determine whether the objects that are presented are of equal value (appetitiveness/aversiveness) for the specific strains/ages/genotypes to be tested.
Rationale: The tail suspension test measures motivation in mice.
Instrument: Opaque chambers fitted with a suspension bar, tape, climbstoppers (clear polycarbonate tubing placed around the tail to prevent tail climbing), and video recording software.
Measures: Mice are suspended by the tail from the suspension bar for 6 minutes. The time spent mobile and immobile are calculated for the entire session. Higher immobility scores are reflective of reduced motivation to escape, interpreted as depressive-like behavior.
Data Inference: The tail suspension test measures behavioral despair in response to an inescapable, but moderately stressful situation. Therefore, increased immobility scores are indicative of greater depressive-like behavior.
Controls: Certain mice strains are known for climbing their tail during this procedure which can confound the results of the tail suspension test, therefore, we place “climbstoppers” made from clear polycarbonate tubing on the tails of all mice to prevent tail climbing behaviors.
Rationale: The Y-maze is used to evaluate activity levels, exploratory drive and working memory in rodents. Exploratory drive and working memory are decreased with aging.
Instrument: Y-maze apparatus from San Diego Instruments.
Measures: Exploration of a Y-shaped maze is expected to follow a pattern of alternating entries in all three arms of the maze if short-term memory of the last arms entered is intact.
Data Inference: Percent alternations are used as an index of short-term memory. An alternation is defined as the mouse entering different arms of the maze three times in succession as a result of consecutive arm entering. The number of overlapping entrance sequences is defined as the number of alternations.
Controls: A minimum of 10 total arm entries is used as threshold for inclusion to rule out confounds related to lack of motivation or low activity levels.