Behavioral analysis of Cofilin 2 Nestin and ADF Cofilin 2 Nestin animals

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resistance that reaches at least 1GΩ. During the sealing process a negative voltage close to the cells resting potential was applied to hold the cell later at -70mV. To break through the membrane a short suction pulse was applied. When the membrane was opened the program Clampfit was used to acquire 1minute recordings. For every patched cell at least 10 minutes were recorded.

3.6. Behavioral analysis of Cofilin 2 Nestin and ADF Cofilin

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one mouse can be placed so that four mice can be analyzed in parallel. The open field was divided with the AYN-maze software into two different zones: the center area and the periphery and different paradigms were recorded with the software for every zone, like zone entries, time spent there and the freezing- and immobility-times. Therefore the animal was placed in the center of the apparatus and was allowed to freely explore the apparatus for a test session of 60min, while tracking the midpoint of the animal as center point. During the test session repetitive behaviors, like rearing in the center or at the wall and the number of grooming performances were manually counted by the experimenter. To assess the number of freezing episodes and the time spent freezing the detection of freezing was based on a minimum freeze duration of 250ms and a freezing rate of 6 to 16%. The minimum immobility was based on 50% and minimum immobility duration was set to 1000ms. The locomotor activity was measured in the total distance travelled during the 60min test session. For assessing exploratory behavior the 60min test session was divided into 5min bins and the number of center entries and the time spent there were examined in these 5min bins and compared between wt and mt animals. A session lasting longer than 30min allows also the detection of habituation to an increasingly familiar chamber environment, which is due to a synaptic depression of connections involved in exploratory behavior. Therefore the total distance travelled and the immobility time was examined in 5min bins for the 60min test session and changes between wt and mt were analyzed. The open field enables also an initial screening for anxiety-like behavior which is influenced by two factors during the session: first is social isolation resulting from the physical separation from cage mates while performing the test; second is the stress created by the brightly lit and unprotected center area. Therefore the freezing rates in the center and the periphery and the number of center entries were examined. Mice will typically spend significantly more time exploring the periphery of the open field than the unprotected center area. An increased time in the center area would reflect a reduced anxious-like baseline behavior. For the Cofilin 2 Nestin mouse line nine mice per genotype were analyzed and for the ADF Cofilin 2 Nestin mouse line 10 animals per genotype.

3.6.2. Elevated Plus Maze

The elevated plus maze takes advantage of an approach-avoidance conflict between the natural tendency of mice to explore a novel environment and an anxiety evoking stimulus.

Therefore this test can be used to study anxiety-like behavior in rodents. The apparatus (figure 18) consist of two sets of opposing arms with a length of 37cm and width of 5cm extending from a central area (5x5cm). During a test session of 5min mice were allowed to freely explore the maze and decide between spending time in enclosed protected arms, or in open unprotected arms. Thereby the maze was also lifted 1m above the ground. Mice tend to

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avoid the open arms since they induce an anxiolytic stimulus and stay in the protected enclosed arms. At the beginning of the experiment the mouse was placed in the central region and the number of open and closed arm entries and the time spent there was determined. Additionally also the number of stretch-attend postures were counted manually by the experimenter, since they are risk-assessment behaviors which indicate that the animal is hesitant to move to a new position. A high frequency of these postures indicates a higher level of anxiety. To exclude the possibility that a higher number of open arm entries was only related to an increased locomotor activity also the total number of arm entries was compared between wt and mt animals. To compare the time wt and mt animals stayed immobile the immobility detection was set to 70% with a minimum immobility period of 2000ms. Nine mice per genotype were analyzed for the Cofilin 2 Nestin mouse line, and ten mice per genotype for the ADF Cofilin 2 Nestin mouse line.

Fig. 18: Elevated plus maze (EPM): a behavioral device to analyze anxiety-like behavior in animals. The EPM consists of four arms: two which are enclosed and protected arms, and two open and unprotected arms. The EPM is elevated 1m above the ground. Mice were placed in the middle area and could freely explore the apparatus for 5min. Thereby mice tend to avoid the open arms and mice that spent more time exploring the unprotected area reflect a reduced anxiolytic behavior.

http://www.stoeltingco.com/anymaze/mazes/anxiety-depression/elevated-plus-maze.html

3.6.3. Y-maze

Mazes are experimental devices employed for evaluation of spatial memory in rodents. The Y-maze analysis spontaneous alternations, which are a reference procedure to assess the working memory. Thereby the working memory is a type of short-term memory that allows the animal to remember which arm it had visited before. This memory function depends on the hippocampus since place cells in the hippocampus fire whenever the animal enters a specific location. The Y-maze consists of three arms with a length of 35cm and 5cm width.

For this test mice were placed at the end of the X- arm and were allowed to freely explore the maze for 10min. When walking out of an arm of the maze, the animal remembers from which arm it came and explores the third arm finalizing a complete alternation or sequence (X-Y-Z).

If the animal revisits the arm it had encountered before the actual one, the animal displays a failed sequence (X-Y-X). The animal could also stayed in one arm, go back to the center area and then visited exactly the same arm it had just left, which was marked as same arm

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return (X-center-X). During the exploration of the maze the sequence of arm visits was written down by the experimenter and analyzed afterwards for the occurrence of sequences, fails and same arm returns. The total number of arm visits was calculated as 100% and the relative number of sequences, fails and same arm returns were calculated for every animal.

An average was calculated from nine mice per genotype of the Cofilin 2 Nestin and for ten mice per genotype of the ADF Cofilin 2 Nestin mouse line. The standard error was calculated and the significance was tested in a two-tailed independent T-test.

Fig. 19: Working memory was tested in a Y-maze. A behavioral test to assess working memory is the Y-maze device, which is used to count the numbers of spontaneous alternations. Therefore mice were placed in the X- arm and could freely explore the maze for 10mins. The sequence of visited arms was noted by the experimenter and afterwards analyzed for the occurrence of sequences (X-Y-Z), failed sequences (X-Y-X) and same arm returns (X-Center-X).

http://sbfnl.stanford.edu/cs/bm/lm/bml_ymaze.html

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