DOI: 10.7910/dvn/1raqon
title: Effects of footshock stress on social behavior and neuronal activation in the medial prefrontal cortex and amygdala of male and female mice

description: ABSTRACT: Social behavior is complex and fundamental, and its deficits are common pathological features for several psychiatric disorders including anxiety, depression, and posttraumatic stress disorder. Acute stress may have a negative impact on social behavior, and these effects can vary based on sex. The aim of this study was to explore the effect of acute footshock stress, using analogous parameters to those commonly used in fear conditioning assays, on the sociability of male and female C57BL/6J mice in a standard social approach test. Animals were divided into two main groups of footshock stress (22 male, 24 female) and context exposed control (23 male and 22 female). Each group had mice that were treated intraperitoneally with either the benzodiazepine-- alprazolam (control: 10 male, 10 female; stress: 11 male, 11 female), or vehicle (control: 13 male, 12 female; stress: 11 male, 13 female). In all groups, neuronal activation during social approach was assessed using immunohistochemistry against the immediate early gene product cFos. Although footshock stress did not significantly alter sociability or latency to approach a social stimulus, it did increase defensive tail-rattling behavior specifically in males (p = 0.0022). This stress-induced increase in tail-rattling was alleviated by alprazolam (p = 0.03), yet alprazolam had no effect on female tail-rattling behavior in the stress group. Alprazolam lowered cFos expression in the medial prefrontal cortex (p = 0.001 infralimbic area, p = 0.02 prelimbic area), and social approach induced sex-dependent differences in cFos activation in the ventromedial intercalated cell clusters (p = 0.04). Social approach following stress-induced cFos expression was positively correlated with latency to approach and negatively correlated with sociability in the prelimbic area and multiple amygdala subregions (all p &lt; 0.05). Collectively, our results suggest that acute footshock stress induces sex-dependent alterations in defensiveness and differential patterns of cFos activation during social approach.
description: Materials and methods

Animals
2–4-month-old male and female C57BL/6J mice were obtained from the Jackson Laboratory (Bar Harbor, ME, Stock No: 000664) and housed on a 12 h light/dark cycle with ad libitum access to water and chow under standard laboratory conditions. Mice were individually housed for 7 days before the start of and all throughout the experiments, since single housing avoids intermale aggression and social dominance-induced behavioral changes (24). Experiments were performed during the light phase. All animal procedures were performed in accordance with institutional guidelines and were approved by the Institutional Animal Care & Use Committee of Tulane University (ethics approval protocol ID – 1013). Unfamiliar strain-, sex- and age-matched mice (N = 33) were used as stimulus mice during social approach tests. 

Groups
A total of 45 males and 46 females were separated into the following groups:
1) Control males treated with vehicle, N = 13; 2) Control females treated with vehicle, N = 12; 3) Control males treated with alprazolam, N = 10; 4) Control females treated with alprazolam, N = 10; 5) Stressed males treated with vehicle, N = 11; 6) Stressed females treated with vehicle, N = 13; 7) Stressed males treated with alprazolam, N = 11; 8) Stressed females treated with alprazolam, N = 11.

Footshock stress exposure
Footshock exposure or control context exposure was conducted in standard mouse operant conditioning chambers (ENV-307W, Med Associates, Inc., St. Albans, VT) enclosed within sound- and light-attenuating cubicles (ENV-022MD, Med Associates, Inc., St. Albans, VT). The chambers were connected to a computer through an interface and controlled by MED-PC software. The chamber was equipped with a grid floor and a house light, which was cleaned using 70% ethanol. 

Seven days after single housing, mice underwent footshock exposure for two consecutive days. Each of the shock sessions included five 1 s, 0.9 mA footshocks presented with a 120 s average pseudorandom intertrial interval (range 90−150 s), totaling 800 s in the chamber. The intensity of footshock was chosen based on previous studies (25,26). Mice in the control group were exposed to the same chambers for the same period but did not experience footshock. 

Social approach test
The day after footshock exposure, the mice underwent the social approach test in a square 46 x 46 X 38 cm arena constructed from sheets of white plexiglass. Behavioral videos were recorded using a digital camera (Allied Vision “Pike” camera, Germany) and Plexon Studio tracking software (Plexon, Dallas, TX). Tests were conducted under dim (10.6 lux) white fluorescent lighting. Stimulus mice were single housed for 3 days before tests. Each of the stimulus mice interacted with three experimental mice with at least 30 min between tests. Experimental mice were perfused 90 min after the test to assess cFos expression.

An indirect social interaction method was chosen to avoid physical aggression between male mice. For the first 3 min, mice were allowed to explore the open arena with two rectangular (15 X 5 X 6 cm) or circular (8 cm diameter, 10 cm high) metallic mesh boxes located in opposing corners 5 cm away from the walls. After the initial exploration, an unfamiliar, untreated stimulus mouse was put underneath one of the boxes. Behavior was recorded for an additional 5 minutes, and sociability was scored using time spent sniffing the mesh box containing the stimulus mouse as a percentage of total box interaction time (mouse preference, %), the latency to approach the stimulus mouse, and the number of defensive tail rattles. Total exploration of the mesh boxes was also scored in seconds to assess general activity. All behavioral measurements were scored by an observer blinded to condition. Consistent with other social approach scoring protocols (27), sniffing directed to the upper and top part of the mesh boxes, sniffing of feces, bar biting and circulating around the corral without sniffing, were not scored as social approach. 

Alprazolam treatment
Alprazolam (Sigma-Aldrich, St. Louis, MO) was dissolved in a drop of Tween 80 (Merck, Germany) and saline was added to make a final dose of 0.25 mg/kg. This dose was shown to have anxiolytic effects (28) with minimal motor impairment in C57BL/6J mice (29). Tween 80 + saline solution was used for vehicle injections. Solutions were administered at 10 ml/kg volume, intraperitoneally, 30 min before social approach tests.

Histology
Following testing, mice were anesthetized with 2,2,2-tribromoethanol (240 mg/kg, ip, Sigma) and subsequently transcardially perfused with 4% paraformaldehyde in phosphate-buffered saline (PBS). Subjects for cFos analysis were chosen randomly from the respective behavioral cohorts and balanced across the groups. cFos expression was assessed in mice that were perfused 90 min after the social approach test. Fixed brains were cut on a compresstome vibrating microtome (Precisionary, Greenville, NC) in 80 μm coronal slices.

Antibody staining was performed on free-floating tissue sections. After 3 x 10 min washes with 0.5% PBST slices were put in 5% donkey serum for 2 hours. Sections were then incubated overnight in primary rabbit anti-cFos antibody (dilution 1:1500; #226 003, Synaptic Systems, Germany) at 4°C. On the next day sections were washed in 0.5% PBST (3 X 10 min), and then went through a 2 hr incubation with secondary donkey anti-rabbit antibody AlexaFluor 488 (dilution 1:500; #A-21206, Thermo Fisher Scientific, Waltham, MA) at 4°C. After 3 x 10 min washes in PBS slices were mounted with mounting medium with DAPI (Biotium, Fremont, CA).
 
Images were obtained using an AxioScan.Z1 slide-scanning microscope (Zeiss, Germany) and a Nikon A1 Confocal microscope (Nikon, Japan). cFos-positive nuclei were quantified using Fiji ImageJ software (NIH, Bethesda, USA), and averaged for each animal. A blinded observer quantified cFos expression in 2-5 slices per structure per mouse.