Morris Water Maze studies

2.9.1 Testing equipment

The Morris water maze (MWM) used in this study consisted o f a white circular tank. The diameter o f the swimming pool was 1.5 m. The pool was placed on a table high enough to allow the experim enter to rem ain unseen by the experim ental animal throughout testing. The pool was surrounded by white curtains at a distance o f 0.55 m from the edge on three sides (N, W and E). Several distinct three-dimensional cues were placed in front o f the curtains, including an orange plastic chair, a set o f coloured plastic footballs in a net and cues made from black and white cardboard. On the south side the

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curtains were kept open to present the door o f the room as a cue (fig. 2-1). The room was dimly lit by indirect light. The MWM was filled with water to approximately 0.19 m below the rim of the pool and the water was made opaque through the addition of white non-toxic paint (1:1800 dilution. Ready mix paint from ‘The Early Learning Centre’, Swindon, UK). A platform was placed 0.3 m from the edge o f the pool. The platform diameter was 0.1 m and it was hidden 5 mm below the surface of the water. The position o f the platform was SW or SE (fig. 2-1). Water temperature was kept at 23 ± r c throughout the experiment. The swimming behaviour o f the animals was monitored via a camera mounted on the ceiling above the pool. The camera was connected to a PC via a tracker system (HVS2020 Tracker, HVS Image Ltd.). The HVS Water 2020 program (HVS Image Ltd.) was used to analyse the behavioural data.

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F ig . 2-1 M orris water maze setup

A 1.5 m pool an d 0.1 in p la tfo n n w ere used. T he p latfo rm w as in p o sitio n SW (b la c k ) for g ro u p s 1/3 and in po sitio n SH (red ) fo r g roup 2. S tartin g p o sitio n s varied p se u d o -ra n d o m ly from trial to trial (N ,E ,S o r W ). P robe trials w ere started slig h tly offsid e from p o sitio n s N or W (arro w s).

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2.9.2 Testing procedure

Male and female Camkk2 null mutant animals and controls were analysed in a total of three experimental groups (group 1: 4 WT males, 2 mut males; 4 WT females, 6 mut females; group 2: 5 WT males, 6 mut males; 3 WT females, 1 mut female; group 3: 4 WT males, 5 mut males; 8 WT females, 9 mut females). Camkk2 floxed mutant mice were analysed in one group. Conditions were kept constant for all groups. Before the onset o f training the mice were handled for six days. Each animal was individually placed on the hand o f the experimenter and allowed to explore for two min. During this time, the animals were lifted by the tail and replaced for several times. In this way they became used to being handled during the experiment. This reduces stress during testing and improves performance in the MWM task.

After handling, the mice were tested with four trials per day for ten days. Mice were brought into the MWM room at least 30 min before the onset o f training. Home cages were positioned under the pool. Animals were tested individually. On the first day the animals were habituated to the pool. They were allowed to sit on the platform for 30 s followed by 30 s swimming and guidance to the platform from three different angles. For testing, the animals were put onto the platform for 60 s before the first trial. Animals were then placed into the MWM facing the pool-wall and allowed to search for the platform. Starting positions (N, S, W and E, fig. 2-1) varied pseudo-randomly from trial to trial. A trial was ended and the escape latency was scored when the animal had found and climbed onto the platform. The maximal trial length was 90 s and the inter­ trial interval was 60 s, during which the animal was allowed to rest on the platform. If an animal did not find the platform within 90 seconds it was placed onto the platform m anually and a latency o f 91 seconds was scored. If an animal jum ped from the platform it was picked up from the w ater and placed back. A fter the last trial the animals were returned to their home cage. Animals that showed extensive floating behaviour (more than 25% o f time spent floating on at least two trials per day,

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com m encing training day six) were excluded from the experiment. Furthermore, animals that would repeatedly jum p from the platform were excluded. In total, one male WT and two male mutant mice, as well as two female WT and four female Camkk2 null mutant mice were excluded from the experiment.

To assess the animal’s search strategy, 60 s probe trials were given at the end o f training days six and ten. In order to test for retention o f spatial memory, an additional probe trial was given seven days after the end o f training. First, the animal was placed onto the platform and allowed to rest for 60 s. Then, the platform was removed and the animal was placed into the MWM slightly offside to the opposite platform position (fig. 2-1) and allowed to search for 60 s.

Video tracking and the HVS water programme were used to analyse search strategies. The following parameters were used to assess search strategies:

mean swimming speed (m/s)

thigmotaxis (% time swimming within 7.5 cm from the pool wall) time spent searching in the quadrants (% in NE, SE, SW, NW quadrant) crosses through the original platform position and equivalent positions in the other quadrants

mean cumulative distance to the centre o f the platform and the equivalent positions in the other quadrants (m)

2.9.3 Statistical analysis

Two-way analysis o f variance (ANOVA) was used to analyse average escape latencies for each day o f the acquisition phase o f the MWM (days 1-6). One-way ANOVAs and Tukey post-hoc tests were used to analyse probe trial data. If the data was not normally distributed, Kruskal-Wallis one-way ANOVAs on ranks were used. Data from groups

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