Chronic unpredictable mild stress (CUMS) is a widely used model to study stress-coping strategies in rodents. Different factors have been shown to influence whether animals adopt passive or active coping responses to CUMS. Individual adaptation and susceptibility to the environment seem to play a critical role in this process. To further investigate this relationship, we examined the effects of CUMS on Carioca high- and low-conditioned freezing rats (CHF and CLF, respectively), bidirectional lines of animals selected for high and low freezing in response to contextual cues that were previously associated with footshocks. For this purpose, the behavior of CHF and CLF animals was evaluated in the contextual fear conditioning, open field, elevated T maze, and forced swimming tests before and after 21 days of CUMS. Animals kept under standard housing conditions were used as controls. For all tests, CHF rats were more susceptible to the effects of CUMS compared to CLF and control groups. CHF animals exposed to CUMS displayed a reduction in freezing behavior, decreased number of entries and time spent in the center of the open field, greater latencies to become immobile, and increased avoidance and escaping behaviors in the elevated T maze. Overall, these findings support the hypothesis that a heightened susceptibility to the environment exerts a strong influence on coping responses to chronic stress.

Animals and housing

A total of 108 Wistar rats were obtained from the Laboratory of Behavioral Neuroscience (LANEC) of the Pontifical Catholic University of Rio de Janeiro. Two inbred strains were used, CHF and CLF, that were selectively bred for high- and low-conditioned freezing responses to contextual cues as previously described (de Castro Gomes & Landeira-Fernandez, 2008). A total of 18 rats were used per experimental group, with the exception of the T-maze test (n = 8-9/group). Except as specified below, the animals were kept on a 12 h/12 h light/dark cycle (lights on at 7:00 AM, lights off at 7:00 PM) at a controlled temperature (24°C ± 1°C) with free access to food and water. The behavioral experiments were conducted between 9:00 AM and 1:00 PM and performed in accordance with the Declaration of Helsinki and Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health.

Chronic unpredictable mild stress

Stress was applied for a total of 21 days based on procedures that were previously described and reviewed by Willner (2017). Briefly, the weekly stress regimen consisted of a period of some of the following stressors: restraint (4 h), immersion in cold water (18°C, 5 min), soiled cage (24 h), low-intensity stroboscopic lighting (150 flashes/min, 12 h), light/dark cycle inversion (24 h), uninterrupted lighting (24 h), water deprivation (24 h), tilted cage (45º, 24 h), low temperature (4°C, 1 h), food deprivation (24 h), and excessive grouping (24 h). There was also a rest period. Before the behavioral tests, the animals in the CUMS group remained stress-free for 24 h. A control group (CON), comprising half of the total number of subjects, was kept under standard maintenance procedures.

Behavioral tests

Contextual fear conditioning

Before the 21-day CUMS protocol, all of the animals were tested in conditioning chambers to assess contextual fear. On the first day of conditioning, the animals were exposed to the conditioning chamber for an 8 min baseline period (pre-shock period). Immediately afterward, they were exposed at fixed 20-s intervals to three unavoidable electric footshocks (0.6 mA, 1 s), followed by an additional 2-min period (post-shock period) without any aversive stimulation. At the end of this period, the animals were returned to their home cages. On the second day of conditioning, the animals were subjected for 8 min (trial 1) to the same experimental context, but no electric footshock was delivered during the session. Another trial session (trial 2) was conducted after the 21-day period. Freezing behavior (i.e., the complete absence of movements except those related to breathing; Fanselow, 1980) was recorded every 2 s. A more descriptive procedure can be found in the literature (Baldi, Lorenzini, & Bucherelli, 2004; Macedo-Souza, Maisonnette, Filgueiras, Landeira-Fernandez, & Krahe, 2019).

Open field test

After the experimental protocol, all of the animals were tested in an open field arena that was flanked by walls (60 cm × 60 cm). For the analysis, the area was delimited into nine isometric squares. An area entry was defined as the rat having entered a square with all four legs. The following parameters were evaluated over a total of 300 s: number of crossings between the eight peripheral squares, number of entries into the central square, and percentage of time spent in the central square. Forced swim test Two trials were conducted before (trial 1) and after (trial 2) the 21-day stress protocol. A cylindrical plastic container (60 cm) was filled with 18°C water to 80% of the total volume of the container so that when the rat was placed in the container, it could not easily touch the bottom or escape over the edges (Porsolt, Anton, Blavet, & Jalfre, 1978). The rat was positioned in the center of the water, and the following parameters were evaluated during the 300 s trial: latency to the first pause (i.e., when exploration stopped) and the sum of all pause periods.

Elevated T-maze

The elevated T-maze was an apparatus that was similar to the elevated plus maze, with the exception that one of the closed arms was obstructed at its interface to the central area. The procedure, as described previously (Teixeira, Zangrossi, & Graeff, 2000), consisted of a series of three trials to measure avoidance or escaping behaviors. In each of these trials, the animal was positioned at the end of the closed (avoidance) or open (escape) arms, facing the center of the apparatus. The time taken for the rat to leave the initial arm and enter the central area was recorded. A time limit of 300 s was set for each trial. To enhance the efficiency of escape behavior, all of the animals underwent a habituation phase 24 h before the test phase that consisted of 30 min exposure to the open arm that was blocked at its interface with the central platform.

Data_Stress
Excel table file containing raw data from Figures 1-4, divided in sheets, of the related paper. 
Figure 1: Fear conditioning in Carioca High-conditioned Freezing (CHF) and Carioca Lowconditioned Freezing (CLF) rats
Figure 2: Behavioral patterns in the open field test, showing entries into (a) and the time spent in (b) the central area and crossings in the periphery (c)
Figure 3: Activity in the forced swim test. (a) Latency to first pause. (b) Total immobility time during the 300 s test.
Figure 4: Elevated T-maze, showing (a) avoidance behavior, (b) escape from the open arms, and (c) freezing during escape trials. 

Groups were coded as:
CUMS - with stress intervention
CON - without stress intervention
Trial 1 - before CUMS or CON
Trial 2 - after CUMS or CON
CHF - High-conditioned Freezing rats
CLF - Low-conditioned Freezing rats