Behavioral experiments

All behavioral experiments were conducted in the animal facility of the University Medical Center of Bonn. 8-10 week old C57BL/6 wild type animals, CB₁^ko/ko andDaglα^ko/ko animals on C57BL/6 background were bred in-house. Both male and female animals were tested.

3.6.6.1 Open field test

The open field test is a behavioral paradigm commonly used to assess locomotor activity and exploratory behavior of rodents. The set up shown in Figure 4 consists of an infrared beam-operated open field arena (45 x 45 x 23 cm), which is placed in a sound-attenuated environment. The infrared-beam enables the tracking of the animal’s movement. At the beginning of a test session, the animal was placed in a corner of the arena. Subsequently, the movements of each animal were recorded for 30 min. The test was performed under dim-light conditions. Generally, 4 parameters are evaluated at the same time. These

parameters include the time spent moving (s), the distance travelled (m) by the animal during the test-session, the number of rearings (n) and the time spent (s) in the center of the arena. The analysis of the distance travelled and time spent moving reveals potential deficits in locomotor activity, whereas the time spent in the center of the arena is commonly interpreted as an indicator for anxious behavior. Thus, anxious animals would spend more time at the walls or in the corners than exploring the open area.

Figure 4: Top view on an infrared beam-operated open field arena. The animal’s locomotor activity was tracked for 30 min in a sound-attenuated environment under dim-light conditions The behavior of Daglα^ko/ko animals was tested in the open field. C57/BL6 wild type animals served as controls.

3.6.6.2 Hot plate test

The hot plate test is used to analyze the acute pain reaction of rodents to a noxious heat stimulus. As shown in Figure 5, an animal was placed into an acrylic glass cylinder on a heated surface (52°C). The latency (s) from time point zero (placing the animal on the surface) to the first pain reaction (shaking of the hind-paw) was measured. If the animal did not show a pain reaction, the animal was removed from the heated plate after 30 s. In order to reveal potential differences in pain sensitivity, Daglα^ko/ko mice were tested in this paradigm. C57BL/6 wild type mice served as controls.

Figure 5: Hot plate test device. The animal was placed on a heated surface (52°C). Next, the latency to the first pain reaction (shaking of the hind-paw) was measured

3.6.6.3 Fear conditioning paradigm

Fear extinction is a process, which leads to a decline of conditioned fear responses. In order to study this phenomenon, a behavioral paradigm based on the principle of Pavlovian fear conditioning was used. In this paradigm, a neutral auditory stimulus (tone, conditioned stimulus) is paired with an aversive stimulus (electric foot-shock, unconditioned stimulus). Animals exposed to these stimuli will learn to associate the tone with the unpleasant experience of the shock and will later express fear, even when the tone is presented alone. In rodents, freezing behavior indicates fear. Freezing is defined as a “cessation of all bodily movements except those required for respiration” (Myers &

Davis 2007). A decline in fear response and thus in freezing behavior is achieved, when the tone is presented several times without application of another foot-shock. This effect is referred to as fear extinction. The paradigm conducted in this work consisted of a fear conditioning trial and several extinction trials, as schematically illustrated in Figure 6. Fear conditioning was performed on day 0. The subsequent extinction trials (E1-E3) were performed at 3 consecutive days starting 24 h after the conditioning trial. No extinction trial was performed on day 4 and 5. Extinction trial E6 was conducted on day 6 after fear conditioning. Another extinction trial, termed recall was performed on day 7. Therefore, the animal was placed into the cage where it had received the foot-shock. During the recall it received 180 s of tone presentation after a habituation of 300 s.

Figure 6: Experimental set up of the fear extinction paradigm. Fear conditioning was performed on day 0. Fear extinction training started 24 h later and was performed at the first 3 consecutive days, as well as on day 6 after fear conditioning. On day 7, a recall was performed The fear conditioning at day 0 was performed as follows. The test animal was placed in a small cage with a metal grid and transferred into a sound-isolated box (Figure 7) to avoid disturbances from the environment. After a habituation period of 300 s, a tone of 9 kHz, (conditioned stimulus) was presented for 20 s with an intensity of 80 dB. Between the 18^th and 20^th second, a brief electric foot-shock of 0.7 mA was applied (unconditioned stimulus). The shock was followed by a relaxing phase of 60 s. Subsequently, the animal was returned to the home cage for 24 h.

For the extinction trials E1-E6, the animal was placed into a glass beaker and re-transferred into the sound-isolated box. The habituation period (300 s) was now followed by 180 s of tone presentation (9 kHz, 80 dB). During extinction, no foot-shock was applied. Movements of mice were measured by vibration-sensitive transducer platforms.

For the recall trial performed at day 7, the animal was placed into the metal cage that had been used for fear conditioning at day 0. The animal was re-transferred into the sound-proof box and an extinction trial, consisting of 300 s habituation and 180 s of tone presentation, was performed.

Figure 7: Startle response box used for the fear conditioning paradigm. The set up consisted of a sound-protected box equipped with an electric device for the application of the foot-shock, loud speakers for tone presentation and a vibration-sensitive platform, which detected the animal’s movements

Vibrations caused by the movements of the test animal were recorded during all extinction trials and the recall trial. Periods of immobility that exceeded 2 s were considered as freezing behavior. The percentage of time that the animal spent freezing during a test session was calculated for each trial and served as an indicator for the decline in fear response from one session to the next (between-session extinction). In addition, the course of freezing behavior during a single extinction trial was analyzed. Therefore, the 180 s of tone presentation was divided into 60 s intervals. (0-60 s, 61-120 s, 121-180 s).

The percentage of time spent freezing was calculated for each interval separately. The decline in freezing response over the whole period of tone presentation within one session served to analyze within-session extinction. Both, CB₁^ko/ko animals as well as Daglα^ko/ko animals were analyzed in this paradigm. C57BL/6 wild type animals served as control animals, respectively.