Anxiety and Stress

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3.4.1. Chronic Mild Stress

1. Rats or mice, males or females, are used in this paradigm.

2. For each species there will be a control group and an experimental group. The control groups will be given food and drink at all times, as well as comfortable cage surroundings.

3. The experimental groups will be subjected to different kinds of timed stressors such as tilting and wetting their cages, fasting, and discomforts for 4 wk.

4. These stressors are applied alternately and unpredictably for 4 wk. The CMS animals are housed separately from the controls. Weight changes are monitored throughout the studies in both controls and CMS animals.

Fig 2. Setup of the elevated plus-maze test of anxiety. A computerized setup that automatically monitors the time spent and number of entries into the open, center, and closed arm of the plus-maze.

4. These stressors are applied alternately and unpredictably for 4 wk. The CMS animals are housed separately from the controls. Weight changes are monitored throughout the studies in both controls and CMS animals.

5. Once a week a measure of anhedonia is obtained by monitoring consumption of 2% sucrose for a de ned time.

6. After exposure to the CMS procedure, the pharmacological effects of cannabinoid ligands are investigated in this animal model of depression to test the hypothesis that depression may play a role in substance abuse. Other animals of varying genetic background can be subjected to CMS for the determination of novel targets that are involved in depression and cannabinoid action, for example, using the CBir and CB2r and VRir mutant mice in the CMS model.

1. The mouse is placed in the central platform and allowed to freely explore the maze for 5 min (see Note 7).

2. The behavior can be videotaped or scored by one or two observers, or automated versions are computer linked for data acquisition, storage, and retrieval (Fig. 2).

3.4.3. Ultrasonic Vocalizations (USVs)

1. After the application of the stress stimulus and/or the drug under study (see Note 8), the animal is placed in a small test chamber (6.5 x 10 x 7 cm high).

2. The bat detector output is digitized and may be recorded by various devices. We have used the Ultravox hardware and software (Noldus, Wageningen, the Netherlands), alternatingly (every 3 min) recording at 25, 30,60, and 70 kHz, starting before stress, for a duration of 65 min.

3. After experimenting with various types of bedding of the test chamber (sawdust, cotton, rubber, sponge), we now use cotton wool as bedding because it creates the least amount of interference.

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