Models of Depression: Unpredictable Chronic Mild Stress in Mice

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

Major depression is a complex psychiatric disorder characterized by affective, cognitive, and physiological impairments that lead to maladaptive behavior. The high lifetime prevalence of this disabling condition, coupled with limitations in existing medications, make necessary the development of improved therapeutics. This requires animal models that allow investigation of key biological correlates of the disorder. Described in this unit is the unpredictable chronic mild stress mouse model that is used to screen for antidepressant drug candidates. Originally designed for rats, this model has been adapted for mice to capitalize on the advantages of this species as an experimental model, including inter?strain variability, which permits an exploration of the contribution of genetic background, the ability to create transgenic animals, and lower cost. Thus, by combining genetic features and socio?environmental chronic stressful events, the unpredictable, chronic mild stress model in mice can be used to study the etiological and developmental components of major depression, and to identify novel treatments for this condition. Curr. Protoc. Pharmacol. 61:5.65.1?5.65.17 © 2013 by John Wiley & Sons, Inc.

Keywords: major depression; animal model; antidepressants

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Introduction
Basic Protocol 1: Unpredictable Chronic Mild Stress (UCMS) Test in Mice
Basic Protocol 2: Assessment of Self‐Directed Activity and Anhedonia in Mice
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Unpredictable Chronic Mild Stress (UCMS) Test in Mice

Materials

BALB/c mice (64), aged 8 weeks at the beginning of the experiment (e.g., Elevage Janvier)
Food pellets and water ad libitum
Stressors (see Table 5.65.1 ) including:
- CD of various recordings of birds of prey cries lasting ∼10 min (at ambient level); these CDs can be purchased from a variety of commercial suppliers, such as http://www.wildtones.com or http://www.amazon.com
- Soiled sawdust of rats from the same or previous day (with feces and urine)
- Clock for changing the light/dark cycle for the stressed animals with a minimum accuracy of 30 min
- Spruce sawdust
Fluoxetine (e.g., Sequoia Research Products, cat. no. SRP01950f) or imipramine (e.g., Sigma‐Aldrich, cat. no. I0899) diluted in saline solution (NaCl, 9 g/liter)

At least two different sound‐proof rooms in animal facilities with a stable environment (inverted 12 hr light/dark cycle, temperature 22°C ± 1°C, humidity 55 ± 10%); one room is used to house stressed mice and one room for nonstressed, control mice
Behavioral testing room
32 individual cages for the stressed mice
Six to eight cages for the nonstressed mice (four to five mice per cage) with two shelters and one small tube (10‐cm length × 6.5‐cm i.d.) in each cage
Plastic restraint tubes closed at each end but enabling air circulation via small holes (length 6.5 cm × internal diameter 3.7 cm; see Fig. )
Environmental enrichment (shelters and tubes)
Precision balance 0.1 mg

1‐ml syringes with 26‐G. 1/2‐in. needles for intraperitoneal (i.p.) injections

Table 5.5.1 MaterialsExperimental Stressors

Stressor	Description
Social stress	Each mouse is placed in an empty cage previously occupied by another individual
Cage change	Each mouse is placed in the empty cage of another individual, and then returned to its original cage
Sawdust change	In the beginning of the UCMS protocol, the sawdust is changed 2 to 3 times per 24 hr, and up to 6 times per 24 hr at the end of the UCMS regimen. It is also possible to replace clean sawdust by soiled sawdust coming from control mice. The volume of each sawdust change is 250 ml (measured with a beaker).
Without sawdust	The sawdust is removed during 1 to 6 hr
Damp sawdust	Place 125 ml water in each cage. The period of damp sawdust can range from 1 to 6 hr.
“Bath”	The sawdust of each cage is removed and replaced by about 125 ml water at 20°C (about 1 cm water) for 15 to 30 min.
Cage tilting	Tilt the cages backwards (45 degrees) during 1 to 4 hr
Rat feces	About 60 ml of rat sawdust is deposited in each cage for a period of 1 to 2 hr
Restraint stress	The mice are kept in closed and ventilated tubes (6.5‐cm length × 3.7‐cm i.d.) for 15 to 30 min (mice have the possibility to turn themselves back into the tube)
Predator sounds	Broadcast a recording of birds of prey cries during 10 min
Cycle disturbances	Change of the light/dark cycle (e.g., complete reversal of the light/dark cycle, division of the light/dark cycle into four periods of 6 hr, or one to several illumination periods from 30 min to 2 hr during the dark phase and vice versa)

Basic Protocol 2: Assessment of Self‐Directed Activity and Anhedonia in Mice

Materials

Four experimental groups of mice from the previous subdivisions in protocol 1 (non‐UCMS/vehicle, non‐UCMS/antidepressant, UCMS/vehicle, UCMS/antidepressant; sixteen mice per group)
Shortbread cookies [e.g., Pepito (LU) buttery shortbread cookies topped with chocolate, or Oreo (Kraft Foods)] cookies with two chocolate wafers separated by cream filling; the cookie must be appetent for mice in being somewhat crispy and sweet
70% alcohol
10% sucrose solution (elaborated with white sugar sold in conventional retail stores)

Device containing three aligned chambers (20‐cm length × 20‐cm width × 20‐cm height); only the colors of the walls and the floor are different between the chambers: white for the first one, gray for the second, and black for the third. The three chambers are linked by two gates with a door controlled by the experimenter (Fig. )
Light dimmer and luxmeter
Stopwatches
32 individual cages for non‐UCMS mice (for the nest building test and the splash test)
Cotton nestlets (5 × 5 cm, 2 to 3 g; e.g., SERLAB, D0009 or LBS Biotech Nestlets)
One cage for the splash test
1‐liter sprayer (e.g., garden hand sprayer) for the splash test
Lamp with red bulb

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Figures

Figure 5.65.1 Experimental design. Four groups of mice ( n = 16 mice per group) were created based on the environment (non‐UCMS/UCMS) and the treatment (vehicle/fluoxetine). The UCMS regimen lasted 9 weeks. The coat state and the body weight were assessed weekly by two experimenters blind to the treatment. Fluoxetine (20 mg/kg/day) and vehicle (NaCl, 9 g/liter) intraperitoneal administration began after two weeks of UCMS and continued until the end of the experiment. On the seventh week, behavioral tests (cookie test, nest building test and splash test) were initiated.

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Figure 5.65.2 Example of a plastic restraint tube designed, built, and used in our laboratory. This restraint tube (6.5‐cm length × 3.7‐cm i.d.) is closed at one end by a plastic wall pierced with a hole, allowing the animal to put its snout outside. The other end is closed by a rotating lid pivoting around a screw. Small holes on the tube enable air circulation.

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Figure 5.65.3 Assessment of the coat state for (A ) non‐UCMS control mouse and (B ) UCMS‐subjected mouse. The coat state score results from a qualitative scoring of different parts of the body including the head, the neck, the forepaws, the back, the abdomen, the hindpaws, and the tail. Each zone is scored 0 if in a good state (the fur is smooth and shiny, with no tousled, spiky patches), 0.5 if in moderately bad state (the fur is slightly fluffy with some spiky patches), and 1 in bad state (the fur is dirty and fluffy on most of the body with slight staining). On the figure, the non‐UCMS control mouse would be scored 0.5 given the presence of a slight degradation of the coat on the neck. The UCMS mouse would be scored 1 on the head, 1 on the neck, 1 on the back and 0.5 on the abdomen (not visible on the picture), and thus obtain a global score of 3.5. Mice were marked with picric acid in these pictures.

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Figure 5.65.4 Example of the apparatus designed, built and used in our laboratory. This device consist of three aligned chambers (20‐cm length × 20‐cm width × 20‐cm height) communicating by two gates (doors are controlled by the experimenters). Only the colors of the walls and the floor are different between the chambers: white for the first one where the mouse is placed, gray for the second, and black for the third chamber where the food is available.

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Figure 5.65.5 Example of nests. (A ) Score 1: the mouse did not use the cotton nestlet, which is intact. (B ) Score 2: the mouse partially used the cotton nestlet. (C ) Score 3: the mouse scattered the cotton, but there is no nest. (D ) Score 4: the mouse gathered the cotton to form a flat nest. (E ) Score 5: the mouse gathered the cotton to form a “comfortable” nest with walls and a small entry. It is possible to assign an intermediate score for nests that do not fully satisfy the scoring criteria. Mice were marked with picric acid in these pictures.

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Figure 5.65.6 Example of a “staircase‐shaped” protocol with three sets of mice that started the UCMS protocol with 5 days lag. Each set of mice experiences the exact same schedule of stressors, but not at the same time. After week 7 of UCMS, mice are subjected to three days of behavioral testing (BT) in the morning (respectively BT 1, BT 2, BT 3, and BT 4), and subjected to usual stressors (St) in the afternoon (respectively St 1, St 2, St 3, and St 4). Animals are then sacrificed (Sc), which allows minimization of the number of mice involved in the second phase of behavioral tests for the second set.

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Figure 5.65.7 Effects of unpredictable chronic mild stress (UCMS) and 6‐week fluoxetine treatment (20 mg/kg/day, i.p.) on physical state. (A ) The UCMS induced a significant deterioration of the coat state, as demonstrated by increasing coat state scores (non‐UCMS/vehicle group versus UCMS/vehicle group, *** p <0.001). Drug treatment initiated in the beginning of the third week of the UCMS exposure reversed this deterioration after 3 weeks of fluoxetine administration (UCMS/vehicle group versus UCMS/fluoxetine group, ## p <0.01 and ### p <0.001). No significant difference was observed between the two non‐UCMS groups. (B ) The UCMS significantly disrupts the body weight gain (non‐UCMS/vehicle versus UCMS/vehicle, * p <0.05, ** p <0.01 and *** p <0.001), whereas chronic treatment with fluoxetine counteracted this disruption (UCMS/vehicle versus UCMS/fluoxetine, # p <0.05, ## p <0.01 and ### p <0.001) (mean ± standard error; n = 16 mice per group).

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Figure 5.65.8 Effects of unpredictable chronic mild stress (UCMS) and 6‐week fluoxetine treatment (20 mg/kg/day, i.p.) on food consumption in the cookie test. (A ) As a control experiment, the consumption of a regular food pellet (number of bites) was quasi‐null during the four sessions for all groups. (B ) Compared to the UCMS/vehicle group, the consumption of the cookie increased in both non‐UCMS/vehicle group ~undefined p <0.05 and ** p <0.01) and UCMS/fluoxetine group (# p <0.05 and ## p <0.01) during the third and the fourth session. (C ) No significant difference of the latency to chew the regular food pellet was observed between the experimental groups over the four sessions. (D ) Compared to the UCMS/vehicle group, the latency to chew the cookie decreased in both non‐UCMS/vehicle group ~undefined p <0.05 and ** p <0.01) and UCMS/fluoxetine group (# p <0.05 and ## p <0.01) during the third and the fourth session (mean ± standard error; n = 8 mice per group).

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Figure 5.65.9 Effect of unpredictable chronic mild stress (UCMS) and 6‐week fluoxetine treatment (20 mg/kg/day, i.p.) on behavior in the nest building and splash tests. (A ) In the nest building test, nesting behavior was decreased in UCMS/Vehicle mice compared to non‐UCMS/vehicle group ~undefined p <0.05), while chronic treatment with fluoxetine reversed this UCMS‐induced alteration (data show the score of the nest quality at the end of the 24‐hr nesting period; UCMS/vehicle group versus UCMS/fluoxetine group, * p <0.05). (B, C, D ) In the splash test, UCMS induced an increase in the latency to groom, and a decrease in grooming frequency, as well as the amount of time spent grooming (non‐UCMS/vehicle group versus UCMS/vehicle group, ** p <0.01), which was reversed by chronic treatment with fluoxetine (UCMS/vehicle group versus UCMS/fluoxetine group, ** p <0.01) (mean ± standard error; n = 16 mice per group).

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Videos

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Internet Resources
	http://www.affective.disorders.sciences.univ‐tours.fr
	Our laboratory Web site providing pictures and videos of the cookie test, the nest building test, and the splash test.

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Models of Depression: Unpredictable Chronic Mild Stress in Mice

Abstract

Table of Contents

Materials

Basic Protocol 1: Unpredictable Chronic Mild Stress (UCMS) Test in Mice

Basic Protocol 2: Assessment of Self‐Directed Activity and Anhedonia in Mice

Figures

Videos

Literature Cited