Background

The bidirectional selection of high and low anxiety-like behavior is a valuable tool for understanding the neurocircuits that are responsible for anxiety disorders. Our group developed two breeding lines of rats, known as Carioca High- and Low-conditioned Freezing (CHF and CLF), based on defensive freezing in the contextual fear conditioning paradigm. A random selected line was employed as a control (CTL) comparison group for both CHF and CLF lines of animals. The present study performed Fos immunochemistry to investigate changes in neural activity in different brain structures among CHF and CLF rats when they were exposed to contextual cues that were previously associated with footshock.

Results

The study indicated that CHF rats expressed high Fos expression in the locus coeruleus, periventricular nucleus of the hypothalamus (PVN), and lateral portion of the septal area and low Fos expression in the medial portion of the septal area, dentate gyrus, and prelimbic cortex (PL) compared to CTL animals. CLF rats exhibited a decrease in Fos expression in the PVN, PL, and basolateral nucleus of the amygdala and increase in the cingulate and perirhinal cortices compared to CTL animals.

Conclusions

Both CHF and CLF rats displayed Fos expression changes key regions of the anxiety brain circuitry. The two bidirectional lines exhibit different pattern of neural activation and inhibition with opposing influences on the PVN, the main structure involved in regulating the hypothalamic–pituitary–adrenal neuroendocrine responses observed in anxiety disorders.

Animals

All animals were bred in the animal facilities of the Psychology Department, Pontifical Catholic University (PUC-Rio), Rio de Janeiro, Brazil. We used male adult rats selectively bred for high (CHF, n = 10) and low (CLF, n = 10) contextual fear conditioning according to previously described procedures (Castro-Gomes and Landeira-Fernandez, 2008). Non-selectively bred Wistar rats were used as a control group (CTL, n = 12). All animals were born and maintained in the colony room of the PUC-Rio Psychology Department under controlled room temperature (24°C ± 1°C) and a 12 h/12 h light/dark cycle (lights on 7:00 AM–7:00 PM). The rats were housed in groups of five to seven per polycarbonate cage (18 cm × 31 cm × 38 cm) according to their respective lines with food and water available ad libitum. The experiment was conducted during the light phase of the light/dark cycle. The rats were tested at 3-4 months of age and weighed 190-330g. The experimental procedures were performed in accordance with the guidelines for experimental animal research that were established by the Brazilian Society of Neuroscience and Behavior (SBNeC) and National Institutes of Health Guide for the Care and Use of Laboratory Animals. Animal handling and the methods of sacrifice were reviewed and approved by the Committee for Animal Care and Use of the Pontifical Catholic University of Rio de Janeiro (PUC-Rio) protocol no. 20/2009.

Apparatus

The contextual fear conditioning protocol was conducted in four observation chambers (25 cm × 20 cm × 20 cm), each placed inside a sound-attenuating box. A red-light bulb (25 W) was placed inside the box, and a video camera was mounted on the back of the observation chamber to observe the animal’s behavior on a monitor that was placed outside the experimental room. A ventilation fan that was attached to the box supplied 78 dB background noise (A scale). The floor of the observation chamber consisted of 15 stainless-steel rods (4 mm diameter) that were spaced 1.5 cm apart (center-to-center), which were wired to a shock generator and scrambler (Insight, São Paulo, Brazil). An interface with eight channels (Insight) connected the shock generator to a computer, which allowed the experimenter to apply an electric footshock. Ammonium hydroxide solution (5%) was used to clean the chamber before and after each subject.

Procedure

The contextual fear conditioning procedure consisted of an acquisition and a test session. During acquisition, each animal was placed in the observation chamber for 8 min. At the end of this period, three unsignaled 0.5 mA, 1 s electric footshocks were delivered with an intershock interval of 20 s. Three minutes after the last footshock, the animal was returned to its home cage. The contextual fear conditioning test session was conducted approximately 24 h after training. This test consisted of placing the animal for 8 min in the same chamber where the three footshocks were delivered the previous day. No footshock or other stimulation occurred during this period.

Fos protein immunochemistry

Two hours after the conditioned fear test (i.e., the interval that is required for the synthesis and accumulation of Fos protein; Morgan and Curran, 1991), the animals were deeply anesthetized with an overdose of urethane (1.25 g/kg, intraperitoneal; Sigma-Aldrich, St. Louis, MO, USA) and intracardially perfused with 0.1 M phosphate-buffered saline (PBS) followed by 4% paraformaldehyde in 0.1 M PBS (pH 7.4). The brains were removed and stored in 30% sucrose in 0.1 M PBS for cryoprotection. The brains were then frozen in isopentane (-40°C) and sliced in a cryostat (-19°C). As described in our previous studies (Albrechet-Souza et al., 2009; Almada et al., 2013; Reis et al., 2016), coronal 40 μm cryostat sections were collected in 0.1 M PBS and subsequently processed free-floating according to the avidin–biotin system using the Vectastain ABC Elite peroxidase rabbit IgG kit (Vector, USA). All reactions were carried out under agitation at 23±1 °C. The sections were first incubated with 1% H₂O₂ for 10 min, washed four times with 0.1 M PBS (5 min each), and incubated overnight with rabbit polyclonal primary IgG against Fos (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The next day, the sections underwent a series of three 5-min washes and were then incubated for 1 h with secondary biotinylated anti-rabbit IgG (H+L; Vectastain, Vector Laboratories). After another series of three washes in 0.1 M PBS (A and B solution, ABC kit, Vectastain, Vector Laboratories), the sections were incubated for 1 h with the avidin-biotin-peroxidase complex in 0.1 M PBS and then washed again three times in 0.1 M PBS. Fos immunoreactivity was revealed by the addition of the chromogen 3,3’-diaminobenzidine (DAB; 0.02%, Sigma, St. Louis, MO, USA) to which 0.04% hydrogen peroxide was added before use. Finally, the sections were washed twice with 0.1 M PBS.

Tissue sections were mounted on gelatin-coated slides and dehydrated, and Fos-positive neurons were counted by bright-field microscopy (Olympus, BX-50, 100´ magnification, coupled to a Leica DFC320 video camera. The anatomical localization of Fos-positive cells was determined based on the Paxinos and Watson (1998) stereotaxic rat brain atlas. The images were scanned and analyzed using ImagePro Plus 6.2 software (Media Cybernetics, Bethesda, MD, USA). The system was calibrated to ignore background staining. All brain regions were counted bilaterally (7-12 animals per region), and the mean was calculated for each structure.