Materials and Reagents

Male albino Wistar rats (250-270 g)

Equipment

Glass tank (44 x 33 x 30 cm)
Plastic tube (21 x 6 cm, 6 cm diameter)
Individual cages (47 x 32 x 20 cm)
Cold room or refrigerator (4 °C)

Procedure

The stressors schedule used in this protocol is listed in Table 1. Application of stress starts at a different time every day to minimize its predictability (from 8:00 a.m. to 8:00 p.m.).

Table 1. Schedule of stressors used during chronic variant stress treatment
----------------------------------------------
Day   Stressor           Time
----- -----------------------   ------------
1    Forced swimming 10 min
2     Restraint                 3 h
3 Water deprivation   24 h
4    Restrain at 4 °C      90 min
5 Isolation                 24 h
6 Food deprivation     24 h
7    Water deprivation   24 h
8 Restrain at 4 °C     2 h
9 Food deprivation    24 h
-----------------------------------------------

Day 1: Forced swimming

Fill a glass tank 22 cm deep with water at 23 ± 2 °C (Figure 1).
Place the animal in the glass tank for 10 min. While the animal is swimming it may try to get out of the tank, so to prevent escape it may be necessary to carefully close it with a heavy lid.
Return the animal to a clean and dry cage with fresh bedding in order to avoid chills and colds.

Figure 1. Picture of a typical glass tank filled with cold water

Day 2: Restraint

Place the animal in a 21 x 6 cm plastic tube; adjust it with plastic tape on the outside so the animal is unable to move (Figure 2). The tube must have a 6 cm hole at the far end to allow regular breathing. To place the animal in the plastic tube, it is necessary to place the head of the animal close to the entrance, after which it should enter by itself (Figure 3).

Figure 2. Picture of the tube with tape and animal inside

Figure 3. Pictures showing the restraint procedure using a plastic tube
Wait for 3 h. Although the plastic tube should be sufficient to prevent the animal coming out is desirable to leave the immobilizer inside the cage.
Return the animal to its cage. The best way to extract the animal is to make a sudden movement downwards, dropping it into the cage. Try to avoid pulling from the tail.

Day 3: Water deprivation

Remove the bottle of water from the cage during 24 h. If the animal house is supervised by staff, indicate that the water/food must not be replaced in that cage.
Place the bottle of water back after the time point is reached.

Day 4: Restraint at 4 °C

Place the animal in the above-mentioned plastic tube.
Place the tube with the animal inside a cold room or a refrigerator at 4 °C for 90 min. Cold is a well-documented stressor (Krishnamurthy et al., 2011). Therefore, it is necessary to find a place with these features to put the animals in.
Return the animal to its cage at room temperature.

Day 5: Isolation

Place the animal alone in a new cage.
Return the animal to the cage with their mates.

Day 6: Food deprivation

Remove the food from the cage for 24 h.
Place the food back.

Day 7: Water deprivation

Remove the water container from the cage for 24 h.
Place the bottle of water back.

Day 8: Restraint at 4 °C

Put the animal in the plastic tube described above.
Place the tube with the animal inside a cold room or a refrigerator at 4 °C for 2 h. It has been observed that after the first cold-environment exposure, 90 min at 4 °C is not enough time any more to reach adequate stress levels, that is why 2 h at 4 °C is more convenient.
Return the animal to its cage at room temperature.

Day 9: Food deprivation

Remove the food from the cage for 24 h.
Place the food back.

Representative data

Validation of the stress model: Changes in body and adrenal glands weight, in the blood levels of corticosterone, and in dopamine (DA) and 3,4-Dihydroxyphenylacetic acid (DOPAC) levels in the prefrontal cortex are typical effect of stress and are used as methods to assess stress models. With this chronic variant stress protocol, the body weight of the animals decreases, whereas adrenal weight and the blood levels of corticosterone increase (Figure 4). It has also been found that increased levels of dopamine and DOPAC in the prefrontal cortex are observed (Table 2).
Notes about reproducibility and variability: Stress perception is very subjective and each animal, just as each person, is stressed to a different degree. Therefore, it is normal that the results present some variability. To avoid this, glucose preference test is recommended (Hu et al., 2010); then, only animals that have been actually stressed must be included in the study.

Table 2. DA and DOPAC amount in brain cortex in control and stressed rats

Animals were killed at different time points after treatment (0, 2, 4, 8, and 10 days respectively), and the prefrontal cortex was harvested and processed for DA and DOPAC quantification by HPLC as described in de Pablos et al. (2006). Numbers are expressed as nanograms per gram of wet tissue and are Mean ± SD of five independent experiments. *p<0.05, **p<0.01, statistical significance (Student’s t test) compared with control animals.

Figure 4. A. Body weight gain (g); B. Adrenal glands weight (mg, bars) and ratio between adrenal glands weight and body weight gain (scatter plot and line). C. Serum corticosterone (percentage of control animals). Statistical signification: Student’s t test comparing before C and after 10 days of variate stress (S10d); *, p< 0.05; **, p< 0.01; #, p< 0.01 (for the ratio adrenal glands weight/body weight gain). One-way ANOVA followed by the LSD post hoc test for multiple range comparisons, p< 0.01; *, compared with the control; a, compared with the previous time point (S1d to S10d indicate the days subjected to variate stress).

Acknowledgments

Chronic-variable stress was adapted from other models of variable stress (Gamaro et al., 2003; Konarska et al., 1990; Murua and Molina, 1992; Muscat et al., 1992; Papp et al., 1991; Willner et al., 1987) with significant modifications. This work was supported by grant SAF-2012-39029 from the Spanish Ministry of Economy and Competitiveness and P10-CTS-6494 (Proyecto de Excelencia of Junta de Andalucia).

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