DOI: 10.5061/dryad.73n5tb2vm
title: Behavioral effects of chronic stress in Carioca High- and Low-conditioned Freezing rats

description: 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.
description: 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 (<i>n</i> = 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.
description: Data_Stress<br> Excel table file containing raw data from
 Figures 1-4, divided in sheets, of the related paper. <br> Figure 1:
 Fear conditioning in Carioca High-conditioned Freezing (CHF) and Carioca
 Lowconditioned Freezing (CLF) rats<br> 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)<br> Figure 3:
 Activity in the forced swim test. (a) Latency to first pause. (b) Total
 immobility time during the 300 s test.<br> 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:<br> CUMS - with stress intervention<br> CON - without
 stress intervention<br> Trial 1 - before CUMS or CON<br> Trial
 2 - after CUMS or CON<br> CHF - High-conditioned Freezing
 rats<br> CLF - Low-conditioned Freezing rats