Effects of footshock stress on social behavior and neuronal activation in the medial prefrontal cortex and amygdala of male and female mice

Mariia Dorofeikova; Chandrashekhar D Borkar; Katherine Weissmuller; Lydia Smith-Osborne; Samhita Basavanhalli; Erin Bean; Avery Smith; Anh Duong; Alexis Resendez; Jonathan P Fadok

doi:10.1371/journal.pone.0281388

. 2023 Feb 9;18(2):e0281388. doi: 10.1371/journal.pone.0281388

Effects of footshock stress on social behavior and neuronal activation in the medial prefrontal cortex and amygdala of male and female mice

Mariia Dorofeikova ^1,^2,^#, Chandrashekhar D Borkar ^1,^2,^#, Katherine Weissmuller ³, Lydia Smith-Osborne ^1,⁴, Samhita Basavanhalli ³, Erin Bean ³, Avery Smith ³, Anh Duong ^1,³, Alexis Resendez ^1,², Jonathan P Fadok ^1,^2,^*

Editor: Alexandra Kavushansky⁵

PMCID: PMC9910713 PMID: 36757923

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 < 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.

Introduction

Social behavior is important for survival, and social deficits are common pathological features for a variety of mental illnesses including social anxiety disorder, depression, and posttraumatic stress disorder [1, 2]. More women than men suffer from these disorders, yet there is a paucity of data on sex differences in social behavior after stress [3]. There are data suggesting that childhood trauma leads to more aggressive behavior in men and more social withdrawal and avoidance in women [4], and women with social anxiety disorder report greater clinical severity, which may be associated with stressful life experiences [5]. Therefore, understanding sex differences in the effects of traumatic stress on social behavior, as well as the underlying neural substrates that potentially control this behavior, has important translational relevance.

Animal models of stress and trauma show alterations in many aspects of behavior [6]. In general, both acute and chronic stress have been found to lead to social fear and withdrawal in rodents [1, 7]. One common model of traumatic stress in rodents is footshock exposure [8]. However, there is a lack of data investigating whether footshock stress influences mouse social behavior in a sex-dependent manner. In rats, footshock stress has been shown to induce social avoidance in animals with elevated levels of fear generalization [9], and impair the behavioral response to a social-paired compartment [10]. Moreover, intense footshock (2 mA, 10 s), followed by situational reminders, elicits impairments in social interaction in female rats [11].

It is also unclear whether there are sex differences in the activation of brain regions responsive to social encounters following acute stress. Several lines of evidence suggest that neural activity in the medial prefrontal cortex (mPFC) is important for social behavior and subregions of the mPFC are differentially activated following social interaction. Neurons in the infralimbic cortex (IL) are preferentially activated in response to social cues compared to neurons in the prelimbic cortex (PL) [12], and a social neural ensemble within the IL may contribute to the social buffering of fear after fear conditioning [2, 13].

Several subnuclei of the amygdala have also been implicated in social behavior. The role of the basolateral amygdala in social interaction following different stress paradigms has been well-established [14–16]. For example, activation of the basolateral amygdala leads to reduced social interaction in a social interaction test [3, 17]. The medial nucleus of the amygdala (MeA) is also involved in both social behaviors and responses to stressors [18]. cFos expression in the intercalated nucleus of the amygdala (ITC) is increased during social buffering in rats [19], and social interaction is impaired in mice with altered migration and differentiation of ITCs [20]. Among the amygdala regions implicated in social behavior, the central nucleus of the amygdala (CeA) is relatively unexplored. There is recent evidence, however, that CeA circuits may be linked to sociability, and some manipulations of CeA activity impact social behavior [21–23].

Thus, multiple brain regions are involved in social cognition [12, 14], but the differences in social approach or avoidance behavior following acute footshock stress are still poorly understood. In the current study, our goal was to assess the extent to which there are sex differences in sociability using a social approach test that eliminates the possibility of direct physical aggression. We hypothesized that two days of footshock stress would negatively affect sociability, and that those changes might depend on sex and be reversible with the fast-acting benzodiazepine alprazolam, which is used in short-term management of anxiety disorders. Additionally, we aimed to determine patterns of neuronal activation in the mPFC, CeA, MeA, and ventral ITC associated with social behaviors using expression of the immediate early gene cFos.

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.

Statistical analysis

Data were analyzed using Prism 9 (GraphPad Software, San Diego, CA). The definition of statistical significance was p ≤ 0.05. Two male mice that showed no interaction with either mesh box during the social approach test were excluded from analysis. Because the data were noncontinuous, tail-rattling was analyzed using Fisher’s exact test. To assess the interaction of factors, a 3-way ANOVA was used. If a significant effect was detected, Sidak’s multiple comparisons test was used, because it assumes independent comparisons and has more power than the Bonferroni method. Correlations between cFos expression and behavior were analyzed for control (N = 16) and stressed (N = 16) mice using pooled data from alprazolam and vehicle treatment groups of both sexes. The mice having cFos data for all brain regions were randomly selected for correlations with their behavior. Using the Kolmogorov-Smirnov test, the data were determined to be non-parametric, so Spearman’s correlation coefficient was used for this analysis. Because there was a Drug x Sex interaction and a main effect of Drug in cFos expression in the IL, we did not include these data in the correlation analysis. For the sake of clarity, we report the results of the interaction tests, the significant simple main effects, and the significant post-hoc tests in the main text. The results of all tests are reported in Table 1. All statistical tests were two-tailed.

Table 1. Results of statistical analyses.

Effects of alprazolam x footshock stress x sex: 3-way ANOVA
Source			Sum of squares	DFn	DFd	F	p
Fig 1A Sociability (Mouse preference, %)
AlprazolamStressSex			23.8	1	83	0.059	0.80
Alprazolam*Stress			757	1	83	1.88	0.17
Alprazolam*Sex			373	1	83	0.92	0.33
Stress*Sex			292	1	83	0.72	0.39
Alprazolam			5.22	1	83	0.013	0.90
Stress			83.2	1	83	0.20	0.65
Sex			75.4	1	83	0.18	0.66
Fig 1B Latency to approach
AlprazolamStressSex			1765	1	83	0.24	0.62
Alprazolam*Stress			4503	1	83	0.61	0.43
Alprazolam*Sex			8301	1	83	1.14	0.28
Stress*Sex			11760	1	83	1.61	0.20
Alprazolam			1570	1	83	0.21	0.64
Stress			15040	1	83	2.06	0.15
Sex			7434	1	83	1.02	0.31
Fig 1D Exploration
AlprazolamStressSex			5.7	1	83	0.03	0.86
Alprazolam*Stress			15	1	83	0.09	0.77
Alprazolam*Sex			104	1	83	0.60	0.44
Stress*Sex			23	1	83	0.13	0.71
Alprazolam			146	1	83	0.85	0.36
Stress			0.91	1	83	0.005	0.94
Sex			55	1	83	0.32	0.57
Fig 1C Tail rattling
Contingencies			Yes	No		Fisher’s exact test
Male vehicle control vs			1 vs 8	12 vs 3		p < 0.0022*
Male vehicle stress			1 vs 8	12 vs 3		p < 0.0022*
Female vehicle control vs			4 vs 5	7 vs 8		p > 0.9999
Female vehicle stress			4 vs 5	7 vs 8		p > 0.9999
Male vehicle control vs			1 vs 4	12 vs 7		p < 0.1421
Female vehicle control			1 vs 4	12 vs 7		p < 0.1421
Male veh stress			8 vs 2	3 vs 9		p < 0.0300*
Male alp stress			8 vs 2	3 vs 9		p < 0.0300*
Female veh stress			5 vs 4	8 vs 7		p > 0.9999
Female alp stress			5 vs 4	8 vs 7		p > 0.9999
Effects of alprazolam x footshock stress x sex on cFos: 3-way ANOVA
Source			Sum of squares	DFn	DFd	F	p
Fig 2C IL PFC
AlprazolamStressSex			579981	1	41	2.43	0.13
Alprazolam*Stress			172328	1	41	0.72	0.40
Alprazolam*Sex			1378855	1	41	5.78	0.02*
Stress*Sex			169279	1	41	0.71	0.40
Alprazolam			3014936	1	41	12.6	0.001*
Stress			2231	1	41	0.01	0.92
Sex			538912	1	41	2.26	0.14
Fig 2D PL PFC
AlprazolamStressSex			657.3	1	33	0.008	0.93
Alprazolam*Stress			322417	1	33	3.86	0.06
Alprazolam*Sex			169139	1	33	2.03	0.16
Stress*Sex			7228	1	33	0.09	0.77
Alprazolam			546812	1	33	6.55	0.02*
Stress			3566	1	33	0.04	0.84
Sex			29411	1	33	0.35	0.56
Fig 2E Capsular CeA
AlprazolamStressSex			24040	1	34	0.55	0.46
Alprazolam*Stress			19319	1	34	0.45	0.51
Alprazolam*Sex			54202	1	34	1.25	0.27
Stress*Sex			45335	1	34	1.05	0.31
Alprazolam			38512	1	34	0.89	0.35
Stress			36099	1	34	0.83	0.37
Sex			82985	1	34	1.91	0.18
Fig 2F Medial CeA
AlprazolamStressSex			5538	1	34	0.92	0.34
Alprazolam*Stress			3419	1	34	0.57	0.46
Alprazolam*Sex			8969	1	34	1.49	0.23
Stress*Sex			8175	1	34	1.35	0.25
Alprazolam			15566	1	34	2.59	0.12
Stress			307.6	1	34	0.05	0.83
Sex			24836	1	34	4.11	0.05
Fig 2G Lateral CeA
AlprazolamStressSex			6744	1	34	0.38	0.54
Alprazolam*Stress			10219	1	34	0.58	0.45
Alprazolam*Sex			762.9	1	34	0.04	0.84
Stress*Sex			11.84	1	34	0.0005	0.98
Alprazolam			11499	1	34	0.65	0.42
Stress			3797	1	34	0.22	0.64
Sex			69608	1	34	3.97	0.05
Fig 2H ITC
AlprazolamStressSex			56271	1	34	0.12	0.73
Alprazolam*Stress			46481	1	34	0.10	0.75
Alprazolam*Sex			153940	1	34	0.33	0.57
Stress*Sex			14920	1	34	0.03	0.86
Alprazolam			19359	1	34	0.04	0.84
Stress			9053	1	34	0.02	0.89
Sex			2104455	1	34	4.53	0.04*
Fig 2I MeA
AlprazolamStressSex			291674	1	34	1.87	0.18
Alprazolam*Stress			4035	1	34	0.03	0.87
Alprazolam*Sex			278969	1	34	1.79	0.19
Stress*Sex			103189	1	34	0.66	0.42
Alprazolam			38692	1	34	0.25	0.62
Stress			6305	1	34	0.04	0.84
Sex			147128	1	34	0.95	0.34
Fig 3A: Control group correlations (N = 16; Spearman’s coefficient)
		PL	CeC	CeL	CeM	ITC	MeA
Sociability	r	-0.121	-0.571	-0.231	0.003	-0.406	0.144
Sociability	p	0.656	0.023*	0.386	0.993	0.120	0.594
Exploration	r	0.132	0.081	-0.131	0.272	0.397	0.206
Exploration	p	0.625	0.765	0.627	0.305	0.129	0.443
Tail rattling	r	0.018	0.143	-0.124	0.442	-0.228	0.138
Tail rattling	p	0.950	0.600	0.650	0.096	0.417	0.625
Latency to approach	r	0.153	0.407	-0.165	0.427	-0.032	0.445
Latency to approach	p	0.570	0.118	0.538	0.100	0.908	0.086
Fig 3B: Footshock stress group correlations (N = 16; Spearman’s coefficient)
		PL	CeC	CeL	CeM	ITC	MeA
Sociability	r	-0.756	-0.843	-0.673	-0.349	-0.576	-0.762
Sociability	p	0.001**	0.00008***	0.005**	0.185	0.022*	0.001**
Exploration	r	0.162	-0.394	-0.183	0.150	-0.182	-0.232
Exploration	p	0.549	0.131	0.496	0.576	0.498	0.385
Tail rattling	r	-0.175	-0.122	-0.268	0.046	-0.273	-0.200
Tail rattling	p	0.513	0.649	0.313	0.865	0.303	0.455
Latency to approach	r	0.617	0.568	0.502	-0.091	0.487	0.558
Latency to approach	p	0.013*	0.023*	0.049*	0.735	0.057	0.027*

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Results

Effects of footshock stress and alprazolam treatment on social behavior

Following two days of footshock stress or context exposure, male and female mice were allotted to the vehicle and alprazolam treatment groups. These mice were then subjected to a social approach test designed to measure sociability (Fig 1A and 1B). A three-way ANOVA revealed that there was no statistically significant interaction between the effects of either stress, alprazolam, sex or their interactions on sociability (Fig 1C;Sex x Stress x Drug, F_(1,83) = 0.059; p = 0.80) or latency to approach the social stimulus (Fig 1D; Sex x Stress x Drug, F_(1,83) = 0.24; p = 0.62).

Fig 1 — A. Experimental timeline. B. Schematic of the social arena. C. There was a significant interaction between the drug and stress condition, but there were no significant results from post hoc multiple comparison tests. D. Latency to approach was not significantly affected by stress or alprazolam treatment. E. The % of male mice show tail rattling behavior increased by stress and was significantly reduced by alprazolam. F. Exploratory behavior was not significantly altered by stress or drug treatment. Data are presented as means ± SEM. *p<0.05; *p<0.01 post hoc tests.

Further, we applied Fisher’s exact test to analyze noncontinuous tail-rattling data. Interestingly, significantly higher number of stressed males displayed tail-rattling behavior than control males during the social approach test (p = 0.002), while females in both groups displayed equivalent levels of tail rattling (Fig 1E; p > 0.99). On the other hand, alprazolam treatment significantly reduced the number of males showing tail-rattling behaviors (p = 0.03) but did not affect tail-rattling in females. In the control vehicle condition, females show higher tail-rattling than males, although this difference did not reach statistical significance (p = 0.14). Exploratory behavior, measured as total interaction time with both mesh boxes, was unaffected by footshock stress or alprazolam treatment (Fig 1F; 3-way ANOVA, sex X stress X drug, F _{(1, 83)} = 0.03, p = 0.86).

cFos expression analysis

We next quantified expression of cFos in several brain regions involved in the regulation of social behavior (Fig 2A and 2B). A three-way ANOVA was performed to test for the effects of sex, stress, and alprazolam treatment on cFos expression in the IL, PL, CeA, ventromedial ITC, and MeA (N = 13 control vehicle (5 male, 8 female), N = 10 control alprazolam (5 male, 5 female), N = 14 stress vehicle (5 male, 9 female), N = 12 stress alprazolam (6 male, 6 female). There was no significant three-way interaction between the effects of these variables on cFos expression in any of the brain areas analyzed (see Table 1). There was a significant sex by alprazolam interaction effect on cFos expression in IL (Fig 2C; F_(1,41) = 5.78, p = 0.02) and a main effects analysis showed that alprazolam treatment significantly reduced cFos expression in the IL (drug effect, F_(1,41) = 12.6, p = 0.001). Post hoc analysis showed that control females injected with alprazolam had significantly fewer cFos+ cells in the IL compared to vehicle-injected female controls (Sidak’s multiple comparisons test, p = 0.03). There was a main effect of drug on cFos expression in the PL (Fig 2D; drug effect, F_(1,33) = 6.55, p = 0.02). There were no significant simple main effects of sex, stress, or drug on cFos expression in the capsular subdivision of the CeA (Fig 2E), the medial (Fig 2F, sex, F _{(1, 34)} = 4.11, p = 0.0505) or lateral subdivision of the CeA (Fig 2G, sex, F _{(1, 34)} = 3.96, p = 0.0546), or in the medial amygdala (Fig 2I). There was a significant effect of sex on cFos expression in the ventromedial intercalated nucleus of amygdala, with greater expression levels in males (Fig 2H; sex effect, F_(1,34) = 4.53, p = 0.04).

Correlations between cFos levels and behavior following the social approach test

Spearman’s correlation coefficient was used to assess the relationship between behavioral variables and cFos expression levels (Fig 3 and Table 1). These correlation analyses were performed for the control and stressed groups. For the brain areas in which we did not find any significant effect of drug x sex, we pooled the data from their respective vehicle or alprazolam treatment groups together for analysis. In controls (Fig 3A), there was a significant negative correlation between CeC cFos expression and sociability (r = -0.57, p = 0.023). In the stress group (Fig 3B), there was a significant negative correlation between sociability and cFos expression in numerous areas, with more socially active mice demonstrating less neuronal activation in the PL (r = -0.75, p = 0.001), capsular (CeC, r = -0.84, p = 0.00008) and lateral (r = -0.67, p = 0.005) subdivisions of CeA, as well as in the ventromedial ITC (r = -0.58, p = 0.02) and medial amygdala (MeA, r = -0.76, p = 0.001).

Latency to approach the social stimulus positively correlated with the number of cFos+ cells in the PL (r = 0.62, p = 0.01), CeC (r = 0.568, p = 0.023), CeL (r = 0.502, p = 0.050) and MeA (r = 0.55, p = 0.027), with a trend to significance in ITC (r = 0.49, p = 0.06; Fig 3B). Distribution of data points and regression lines for the respective brain area for sociability and latency is shown in Fig 3C and 3D.

Discussion

Exposure to footshock is a commonly used model of acute traumatic stress to assay core features of stress disorders such as social withdrawal [30]; however, sociability after footshock stress has been investigated predominantly in male rodents and most other studies that have focused on the effects of stress on social behavior have largely employed chronic stress models which typically lead to social withdrawal [7]. In the current study, several measurements of social behavior were taken in male and female mice to investigate potential sex differences in the impact of footshock stress on sociability (Fig 1) and neuronal activation in the mPFC, CeA, and MeA (Fig 2).

Although the footshock stress parameters we used did not significantly alter sociability, tail rattling behavior was significantly different between groups during social approach in a sex-dependent manner, being affected by stress and diminished by alprazolam only in males (Fig 1). Tail rattling is elicited due to territorial aggression in male mice [31, 32] and it has been suggested that it can be a measurement of threat-induced defensive aggression [26]. Our results are therefore comparable to other mouse sociability studies in which footshock stress produced an enhancement of aggression in male mice [32]. Interestingly, female mice exhibit more tail rattling than males during fear conditioning [26], suggesting that sex differences in tail rattling behavior are both stress- and context-dependent. The significant reduction of tail rattling by alprazolam further supports the link between this behavior and negative valence states. Overall, the observed sex differences reinforce the idea that sex is a crucial factor that should be considered in stress-related studies.

There is evidence that social interaction itself is anxiogenic, even under control conditions [33]. This is supported by our finding of a negative correlation between sociability and cFos expression in the CeA (Fig 3). Our results also show that cFos expression in the ITC is higher in males compared to females after social interaction. Given the known role of these amygdala regions in mediating threat responses [21, 34], these data suggest that social interaction may induce a higher level of defensiveness in male mice. Consistent with this hypothesis, previous work has shown that CeA neuronal activation is associated with male mouse aggression during social interaction in the resident intruder assay [18, 35]. These findings emphasize the need to study sex differences while deciphering the relationship between stress and social behaviors. Further investigations should be conducted into sex differences in different neuronal populations of CeA, expressing molecular markers such as somatostatin or corticotropin-releasing factor. This is especially relevant given that there are more corticotropin-releasing factor receptor 1-containing neurons in the male CeA compared to female, and they have different neuronal excitability in response to corticotropin-releasing factor [36].

cFos expression in PL and IL mPFC in our study was not significantly affected by prior footshock stress exposure; however, PL and IL cFos levels were decreased due to alprazolam treatment both in stressed and control mice, which is consistent with the observed decrease in mPFC activation in humans given benzodiazepines [37]. Acute restraint stress has been shown to activate PL, but not IL or CeA, and social interaction following stress enhances PL activation without affecting CeA activation [38]. The cFos expression patterns observed in our study may be due to the delay (at least 24 hr.) between stress exposure and sacrifice of the animals.

Among control vehicle-injected mice, females had more cFos+ cells in IL. Tan et al. obtained comparable results in the mPFC of females after chronic adolescent social isolation stress, with pyramidal neurons being less activated during sociability tests [39]. Alprazolam significantly lowered the number of cFos+ cells in the IL compared to vehicle-injected female controls, consistent with the benzodiazepine effects observed in humans: women demonstrate decreased activity in frontal regions after treatment, while an opposite effect is present in males [40]. This effect could be related to females having significantly higher GABA-A benzodiazepine receptor availability [41].

The MeA has been linked to a wide variety of social behaviors, such as aggression, mating, and parenting [42]. Here, we find that MeA cFos expression induced by the social approach test is not affected by sex, stress, or alprazolam. Prior studies have demonstrated that socially defeated females housed with aggressive male residents exhibit increased cFos activation in the MeA [43], and the MeA is more responsive to aggressive than to benign social interaction [44]. We designed our experiments to exclude the possibility of aggressive interactions, therefore free interactions between the experimental and stimulus mice were not possible. Future studies could investigate the effects of stress and sex on MeA activation in free interaction paradigms.

In stressed animals, sociability was negatively correlated with cFos expression in the PL, CeC and CeL, as well as in the ventromedial ITC and MeA. In non-stressed animals, MeA activation has been demonstrated previously because of social interaction [45], and an opposite relationship has been shown between CeA cFos expression and sociability after anxiogenic synthetic amphetamine treatment [46]. Because mPFC activation has been demonstrated to suppress social behaviors [47], the negative correlation observed in the current study could be expected, although enhanced activity of a subset of mPFC neurons was correlated with social approach behavior previously [48]. In controls, there was a significant negative correlation between sociability and CeC cFos expression, and an opposite correlation between the latter and the latency to approach. Latency to approach the social stimulus also positively correlated with the number of cFos+ cells in the PL and MeA.

In conclusion, we found that two days of footshock exposure at levels commonly used in fear conditioning did not elicit changes in mouse sociability. However, we did find sex differences in defensive tail rattling, the effects of alprazolam on defensive tail rattling, and cFos expression during the social approach assay. The social consequences of stress have been extensively studied using the social defeat stress model that entails exposure to emotional / psychological stress and leads to depression-related outcomes [7]. After the social defeat procedure, most mice develop a decreased drive to approach and interact with the social target [49]; however, social defeat stress is difficult to achieve in female mice. Acute stress paradigms utilizing footshock could facilitate investigations into sex differences in social behavior following trauma, which would be valuable in the search for sex-specific mechanisms involved in the pathophysiology of post-traumatic stress disorder (PTSD) and could facilitate the development of personalized therapeutic interventions. Future work should therefore define the optimal conditions, such as footshock intensity or lighting conditions, that influence sociability after stress. It could be especially valuable to develop paradigms that stratify mice as resilient and susceptible to further validate acute footshock stress as a tool for PTSD research.

Data Availability

Here is the DOI for the data in our manuscript: https://doi.org/10.7910/DVN/1RAQON.

Funding Statement

This work was supported by Tulane University, the Louisiana Board of Regents through the Board of Regents Support Fund (LEQSF(2018-21)-RD-A-17), and the National Institute of Mental Health of the National Institutes of Health under award number R01MH122561 to JPF. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. There was no additional external funding received for this study.

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PLoS One. doi: 10.1371/journal.pone.0281388.r001

Decision Letter 0

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PONE-D-22-19470Effects of footshock stress on social behavior and neuronal activation in the medial prefrontal cortex and amygdala of male and female micePLOS ONE

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Reviewer #1: 1. Main theme in summary

Authors aimed to demonstrate the sex-dependent effects of acute traumatic stress on mouse sociability using two-days of footshock stress followed by social interaction test, and its correlation with cFos expression in the medial prefrontal cortex and amygdala. The reversibility effects of acute benzodiazepine alprozalam treatment on these behavioral and neuronal changes were also investigated. Study findings do offer a potential correlation of sex differences in defensive behavior in male mice, but not sociability, and the underlying cFos activation, as well as the reversibility effects with acute alprazolam treatment.

2. Strengths

Overall, this is quite an interesting study which has generated good results to describe sex differences in the effects acute traumatic stress on social behaviors and the underlying neural substrates involvement.

3. Limitation

The study design (i.e., the groups allocation and treatment) are unclear. Require elaboration and clarification - elaborated further in methods section.

4. Detailed remarks

Abstract - needs revision for the following:

• The problem statement cannot be clearly seen from the abstract.

• Authors should add number of animals (i.e., N total, N for each group), the route of alprazolam administration, p-value

• Authors should also highlight on the reversal effects of alprazolam treatment in abstract conclusion.

Introduction is satisfactory overall, with additional info necessary to improve clarity to the research background as below:

• Para 2, Line 47: “One common model of traumatic stress in rodents is footshock exposure” – Authors should briefly describe previous studies using footshock exposure for social behaviors

• Para 5: Authors should briefly describe on benzodiazepine alprozalam and its common indication (e.g: anxiolytic? Antidepressant effects?)

• Para 5, Line 74,75: “Therefore, we performed social interaction tests 24 hours after two consecutive days of footshock stress” – More suitable to be in the Methodology sections and therefore to be removed from Introduction.

Materials and Methods - some missing info needed to be updated, and additional clarity as follow:

• Ethics approval protocol number should be added in the text.

• Line 87-88: “Unfamiliar strain-, sex- and age-matched mice were used as the passively interacting counterparts (stimulus mice) during social interaction tests.” – Author should disclose the number of stimulus mice used for this study.

• The assignment of groups and treatment are unclear. I suggest to include a Grouping subsection in the Methodology, instead of in the Results section. Summarised on the male vs female, footshock vs control context exposure, vehicle vs alprozalam group allocations, and the total number of animals (i.e., N total, N for each group). It would be helpful to include one figure that summarised the overall study design (i.e., the treatment groups, schedules up to whole brain collection) for better clarity of the group allocations.

• Is the behavioral study design (ie., duration, intensity of footshock exposure, design of social interaction test etc) based on previous protocols? If yes, please cite the previous study(s).

Results - Overall is satisfactory and met study objectives with one minor remark as follow:

• The quality of Figure 1C-F can be further improved – in present form, hard to read and appreciate the results.

Discussion - overall is satisfactory with some clarifications/additions suggested below:

• Para 3, Line 280-281 “…which mostly employed chronic stress models.” – should briefly describe the effects of chronic stress on social behaviour from previous animal/rodent studies.

• Para 5, Line 302-304: “…which may occur due to the delay between stress exposure and sacrifice of the animals.” – What is the estimated duration between stress exposure and animal sacrifice in your study? Is it based on previous protocol? Suggestion of duration for future studies?

• Concluding paragraph: Authors should briefly describe overall findings from this study, highlighting on the potential sex differences in stress-related studies. Emphasise on the need to understand these mechanisms in both sexes, which will provide new insights into the sex dimorphism documented in the pathophysiology of PTSD and possibly help facilitate the development of sex-specific therapeutic interventions.

Reviewer #2: Male and female mice underwent footshock stress for 2 days and were then exposed to a novel stimulus mouse. Half of the mice received alprazolam and half received vehicle. cFos protein expression was assessed in subregions of the prefrontal cortex and amygdala. No effects of stress were observed on social approach, but tail rattling was increased somewhat in males. There were some effects of sex and alprazolam on cFos but no straightforward main effects or interactions involving stress.

The main concern with this experiment is that five 1 sec footshocks appear insufficiently stressful to elicit long-lasting changes in social interaction behavior.

No effects of stress on social interaction were observed in males or females, but there was increased tail rattling in males exposed to footshock stress. Why should we care about the effects of stress on cFos expression when there are no robust behavioral effects on social interaction per se, or on latency to interact. Similarly the effects of alprazolam would be more interesting if there were robust behavioral effects of stressor exposure using these procedures.

Other minor concerns are listed below.

Why were the experimental mice singly housed for 7 days? There is no rationale provided for this.

Mice were exposed to a conspecific in a mesh box and there was no physical interaction, so it may better be described as social approach. Since there was no actual interaction.

Experiments were done during the light phase, the inactive phase in rodents. Time of day has profound effects on behavior, and results may be more clinically relevant when experiments are done during the active phase.

As noted by the authors on line 341, “Future work should define the optimal conditions, such as footshock intensity or lighting conditions, that influence sociability after stress.” Those conditions should have been defined using pilot studies before this experiment was performed.

Reviewer #3: Overall, this is a straightforward experiment and the research question was clear - to determine whether acute stress (2 days of inescapable footshock) changed social behavior in a sex-dependent manner and whether these effects were reversible with the anxiolytic, alprazolam. The manuscript is well written, and overall find the authors to have sufficiently summarized and integrated the existing literature into their intro and discussion. With that said, I have some issues with the study design, statistics and reporting of significant effects which are enumerated below:

1. The authors state that "male (N = 18 control, N = 22 stress) and female (N = 17 control, N = 24 stress) mice were allotted to the vehicle (N = 49, 24 males and 25 females) and alprazolam (N = 32, 16 males and 16 females) treatment groups" but do not explicitly state how many vehicle were from stress or control conditions, i.e., female-control-vehicle = x, male-control-vehicle = y, so it is clear how the groups are balanced. Best I can tell (but should not have to guess or count, please spell this out) there are:

Male-Control-Vehicle = 13

Male-Stress-Vehicle = 11

Male-Control-Alprazolam = 5

Male-Stress-Alprazolam = 11

Female-Control-Vehicle = 12

Female-Stress-Vehicle = 13

Female-Control-Alprazolam = 5

Female-Stress-Alprazolam = 11

Thus it appears that the groups are very unbalanced, and statistically this is problematic because of the inherent variability this introduces, and the likelihood that this is violating the equal variances assumptions for all the ANOVAs performed. For example, the authors report a significant interaction between stress and alprazolam, which is clearly being driven by 3 extreme cases across the male and females in the control condition where they have grossly under-sampled and thus extreme data points have large influence. The authors need to increase the animal numbers in their control-alprazolam conditions in order to make this data interpretable. This applies to every statistical comparison made in this manuscript.

2. For the tail rattling analysis, the Fisher's exact test is, from my understanding, a test that can explore a 2 x 2 interaction, not the three way they have here. So this test may not be the most appropriate. However, probability stats should be reported along with p values at a minimum, along with a clear explanation of which factors were compared that produced such probability values.

3. Related to comment 1, when we get to the cFos analysis, group numbers change with no explanation as to how they selected the animals they analyzed:

Male-Control-Vehicle = 13 -> 5

Male-Stress-Vehicle = 11 -> 5

Male-Control-Alprazolam = 5 -> 5

Male-Stress-Alprazolam = 11 -> 6

Female-Control-Vehicle = 12 -> 8

Female-Stress-Vehicle = 13 -> 9

Female-Control-Alprazolam = 5 -> 5

Female-Stress-Alprazolam = 11 -> 6

This is less unbalanced, but without a clear explanation of how animals were selected, the process could have been purposefully or accidentally biased in some manner, and may largely skew the correlational analyses.

3. The authors include animals in their cFos graphs that seemingly did not go through stress or social interaction procedures. These appear to not be included in any analyses or methodological description and their inclusion should be described and either properly included in the analyses or excluded from the manuscript.

4. In the correlational analysis for cFos against sociability and latency to approach, it is unclear if the same animals are used across all of the brain regions. For example, if all the same animals were used for the cFos analysis you would have cFos data counts in sets of 5 for each sociability score, which you can see often, as in Fig 3C, data points for each brain region hovering at ~94% sociability you can clearly see one set of 5, but if you look just to the left at ~90%, there is only one orange square at this sociability score, so I am just trying to understand how these data were derived and how animals were included for some brain regions and not others, some animals with extreme scores were included and other excluded from this analysis. This occurs in the latency graph (3D) as well.

Overall, the authors are not making any grandiose conclusions from their data, but I believe more controls need to be run and more careful attention paid to statistical analyses. As well, explicit criteria for inclusion or exclusion in subsequent cFos analyses should be given.

**********

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PLoS One. 2023 Feb 9;18(2):e0281388. doi: 10.1371/journal.pone.0281388.r002

Author response to Decision Letter 0

20 Dec 2022

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Partly

________________________________________

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

________________________________________

3. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

________________________________________

4. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

________________________________________

5. Review Comments to the Author

We thank the reviewers for their thoughtful critiques of our manuscript. We have addressed all the reviewer’s comments with additional experiments, data analysis, statistical testing, and major revisions to the text. We feel the revised manuscript is improved.

Our point-by-point responses are in bold text below and the changes made to the manuscript are highlighted in the Revised Manuscript with Track Changes file.

Reviewer #1: 1. Main theme in summary

2. Strengths

We thank the reviewer for their positive feedback on our manuscript.

3. Limitation

The study design (i.e., the groups allocation and treatment) are unclear. Require elaboration and clarification - elaborated further in methods section.

We have added clear information about group allocation and treatment in the revised manuscript under the Groups subheading in the Methods.

4. Detailed remarks

Abstract - needs revision for the following:

• The problem statement cannot be clearly seen from the abstract.

We clarified the intention of the study by stating in the Abstract, “The aim of this study was to explore the effect of 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 interaction test.”

• Authors should add number of animals (i.e., N total, N for each group), the route of alprazolam administration, p-value

We have added the total number of animals as well as the group composition to the abstract. We also added the route of drug administration (i.p.), and the p-values for the significant mentioned results.

• Authors should also highlight on the reversal effects of alprazolam treatment in abstract conclusion.

We have highlighted the effects of alprazolam by stating “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 interaction induced sex-dependent differences in cFos activation in the ventromedial intercalated cell clusters (p = 0.04).”

Introduction is satisfactory overall, with additional info necessary to improve clarity to the research background as below:

• Para 2, Line 47: “One common model of traumatic stress in rodents is footshock exposure” – Authors should briefly describe previous studies using footshock exposure for social behaviors

We have added the previous literature on footshock exposure induced traumatic stress as follows.

“In rats, footshock stress has been shown to induce social avoidance in animals with elevated levels of fear generalization [Dong et al., 2020] and impair the response to the social-paired compartment which was reversed by antidepressant treatment [Daniels et al. 2021]. Moreover, intense footshock (2 mA, 10 s) followed by three weekly situational reminders elicit long-term impairments in social interaction in female rats [Louvart et al., 2005]. ”

• Para 5: Authors should briefly describe on benzodiazepine alprozalam and its common indication (e.g: anxiolytic? Antidepressant effects?)

We have specified that alprazolam is used in short-term management of anxiety disorders.

We have modified the relevant statement as “We hypothesized that two days of footshock stress would negatively affect sociability, and that those changes might depend on sex and be reversible with the fast-acting benzodiazepine alprazolam, that which is used in short-term management of anxiety disorders.”

Materials and Methods - some missing info needed to be updated, and additional clarity as follow:

• Ethics approval protocol number should be added in the text.

We have added the IACUC ethical approval protocol number (Protocol ID-1013).

We have used a total of 33 mice as the stimulus mice. This information is included in the revised version of the manuscript.

We have added the following information in the revised manuscript.

A total of 45 males and 46 females were separated into the following groups:

Control males treated with vehicle, N = 13,

Control females treated with vehicle, N = 12,

Control males treated with alprazolam, N = 10,

Control females treated with alprazolam, N = 10,

Stressed males treated with vehicle, N = 11,

Stressed females treated with vehicle, N = 13,

Stressed males treated with alprazolam, N = 11,

Stressed females treated with alprazolam, N = 11.

• Is the behavioral study design (ie., duration, intensity of footshock exposure, design of social interaction test etc) based on previous protocols? If yes, please cite the previous study(s).

We used footshock duration and intensity based on our previous study [Borkar et al. 2020] and others [Bali and Jaggi, 2015]. We employed the method of the social interaction scoring from Macbeth et al., 2009 [27]. We have incorporated this information in the manuscript.

Results - Overall is satisfactory and met study objectives with one minor remark as follow:

• The quality of Figure 1C-F can be further improved – in present form, hard to read and appreciate the results.

As suggested, we have revised Figure 1 to be of higher quality.

Discussion - overall is satisfactory with some clarifications/additions suggested below:

• Para 3, Line 280-281 “…which mostly employed chronic stress models.” – should briefly describe the effects of chronic stress on social behaviour from previous animal/rodent studies.

We have rewritten the Discussion to convey a clearer message overall. We have added the following clarification. “most other studies that have focused on the effects of stress on social behavior have largely employed chronic stress models which typically lead to social withdrawal [Toth and Neumann, 2013; Louvart et al., 2005].”

There was ~ 48 hours delay from the first footshock session and ~ 24 hours delay from the last footshock session to the experimental endpoint of the study. We have clarified this in the Discussion. Our overall goal was to assay cFos following social interaction (as opposed to stress itself) in the separate groups of animals. Therefore, we sacrificed subjects 90 minutes following social interaction, following common convention in the field. (For example, Zhong, J. et al., 2014, https://doi.org/10.1186/s13041-014-0066-x).

In addition, we have added the following statements regarding future directions. “Future work should define the optimal conditions, such as footshock intensity or lighting conditions, that influence sociability after stress. It could be especially valuable to develop paradigms that stratify mice as resilient and susceptible to further validate acute footshock stress as a tool for PTSD research.”

• Concluding paragraph: Authors should briefly describe overall findings from this study, highlighting on the potential sex differences in stress-related studies. Emphasize on the need to understand these mechanisms in both sexes, which will provide new insights into the sex dimorphism documented in the pathophysiology of PTSD and possibly help facilitate the development of sex-specific therapeutic interventions.

As suggested, we summarize the main findings in the concluding paragraph, and we have added the following statement. “Acute stress paradigms utilizing footshock facilitate investigations into sex differences in social behavior following trauma, which would be valuable for the search of sex-specific mechanisms involved in the pathophysiology post-traumatic stress disorder (PTSD) and could facilitate the development of personalized therapeutic interventions.”

The main concern with this experiment is that five 1 sec footshocks appear insufficiently stressful to elicit long-lasting changes in social interaction behavior.

The aim of this study was to elucidate the effects of two days of acute footshock stress on behavior during the social interaction assay, using shock parameters commonly used in Pavlovian fear conditioning studies. We use routinely use these parameters (0.9 mA, 1 s duration) to elicit a complex array of defensive responses to threat (see Fadok et al. 2017, Borkar et al. 2020 for examples). Because these shock parameters induce robust escape behavior, including jumping, we hypothesized that they would be strong enough to induce changes in social interaction. Although our data did not support this hypothesis, we believe it is important to publish these negative data as they provide vital information for others in the community who may be interested in conducting similar studies. The omission of negative data in neuroscience publications is a fundamental problem that can lead to wasted resources, time, and animal life. Additionally, although we did not see a difference in the shock-exposed group in social interaction or latency to approach, we did find sex-dependent differences in defensive tail rattling behavior, as well as sex-dependent differences in the effect of alprazolam on tail rattling behavior. These data will be of interest to researchers interested in social valence and sex differences.

We present the cFos data because it provides the reader with an assessment of the extent to which neuronal activation in subdivisions of the medial prefrontal cortex and amygdala is impacted by social interaction, previous stress exposure, sex, and alprazolam. We find that alprazolam reduces cFos in the PL and IL regions of the medial prefrontal cortex, and there is a significant reduction in cFos in the IL of control females. We also found a significant effect of sex on cFos expression in the ventromedial intercalated nucleus of amygdala, with greater expression in males. These sex-dependent effects will be of potential interest to researchers interested in sex differences in brain function and the effects of drugs on brain activation.

Other minor concerns are listed below.

Why were the experimental mice singly housed for 7 days? There is no rationale provided for this.

We used single housing to avoid potential aggression in male mice exposed to footshock. This is customary practice in our laboratory during fear conditioning studies. Moreover, single housing for 1 week is a relatively short duration and may not be as stressful as longer durations of social isolation (e.g. 5-11 weeks; Buckinx et al., 2021, PMID: 34776890). We have added this justification in the Methods section.

Mice were exposed to a conspecific in a mesh box and there was no physical interaction, so it may better be described as social approach. Since there was no actual interaction.

Although the mice were able to freely sniff one another, we agree that full physical interaction was not possible. We have changed the language to reflect this through the manuscript.

We agree that time of day has a profound impact on behavior. Because we were interested in observing the effects of shock based on fear conditioning parameters, we performed our experiments during the light phase, which is when our lab and many others perform fear conditioning studies.

We have modified the last two paragraphs of the Discussion to clarify the intention of our study and to emphasize that although the parameters we used did not affect social approach, future studies should determine the optimal conditions because of the potential translational relevance.

Male-Control-Vehicle = 13

Male-Stress-Vehicle = 11

Male-Control-Alprazolam = 5

Male-Stress-Alprazolam = 11

Female-Control-Vehicle = 12

Female-Stress-Vehicle = 13

Female-Control-Alprazolam = 5

Female-Stress-Alprazolam = 11

We thank the reviewer for their positive feedback and careful review of our manuscript. We appreciate the reviewer’s concerns about group balance, statistics, and better reporting of group composition. To address this concern, we added 5 mice to the male-control-alprazolam group and 5 mice to the female-control-alprazolam group. We then retested all the data using ANOVAs. There are no significant differences in sociability, latency to approach, or exploration time.

We have added the following group breakdown to the Methods section:

“Groups

A total of 45 males and 46 females were separated into the following groups:

Control males treated with vehicle, N = 13,

Control females treated with vehicle, N = 12,

Control males treated with alprazolam, N = 10,

Control females treated with alprazolam, N = 10,

Stressed males treated with vehicle, N = 11,

Stressed females treated with vehicle, N = 13,

Stressed males treated with alprazolam, N = 11,

Stressed females treated with alprazolam, N = 11.”

We agree that the Fisher’s exact test is designed to test 2 x 2 contingency tables and not three-way comparisons. This was the way we tested the data (between group comparisons with rattle versus no rattle), but we presented the data as counts in the figure. We realize that this was not the best way to present the data in the figure. We have changed the panel to reflect that we tested the contingencies using the proportion of mice in each group that tail rattled or not.

3. Related to comment 1, when we get to the cFos analysis, group numbers change with no explanation as to how they selected the animals they analyzed:

Male-Control-Vehicle = 13 -> 5

Male-Stress-Vehicle = 11 -> 5

Male-Control-Alprazolam = 5 -> 5

Male-Stress-Alprazolam = 11 -> 6

Female-Control-Vehicle = 12 -> 8

Female-Stress-Vehicle = 13 -> 9

Female-Control-Alprazolam = 5 -> 5

Female-Stress-Alprazolam = 11 -> 6

We apologize for not clearly stating how mice were selected for the cFos analysis. All included mice were randomly selected from the separate groups for histological analysis. All tissue processing, imaging, and analysis was performed by investigators blinded to experimental conditions. We have ensured that this information is included in the Methods.

The “No SI” group was a home cage control and was included to serve as a qualitative measurement of cFos changes in the different brain areas. Because there were only 2 males and 3 females in the No SI group, we did not include these data in the quantitative analysis. We apologize for not describing this group in the methods. We have excluded these data from the revised manuscript.

We thank the reviewer for highlighting the issue regarding the variable number of animals included in the correlation analysis. We have revised Figure 3 considering this. In the revised version we have used cFos and behavioral data from 8 males and 8 females randomly selected from the Alp and Veh treated groups (which have cFos data for all brain nuclei) to analyze the correlations in mice subjected to the stress and control group. The data points represent the correlation of sociability or latency to approach in 5 brain regions from the respective animal.

As suggested, we added 5 males and 5 females in the control+Alprazolam treatment group to balance the group composition and we retested the data statistically. In addition, we have clarified the inclusion criteria used for the selection of mice for cFos analyses.

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PLoS One. doi: 10.1371/journal.pone.0281388.r003

Decision Letter 1

Alexandra Kavushansky

23 Jan 2023

Effects of footshock stress on social behavior and neuronal activation in the medial prefrontal cortex and amygdala of male and female mice

PONE-D-22-19470R1

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Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: (No Response)

**********

6. Review Comments to the Author

Reviewer #1: (No Response)

**********

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Reviewer #1: Yes: Mustapha Muzaimi

**********

PLoS One. doi: 10.1371/journal.pone.0281388.r004

Acceptance letter

Alexandra Kavushansky

30 Jan 2023

PONE-D-22-19470R1

Effects of footshock stress on social behavior and neuronal activation in the medial prefrontal cortex and amygdala of male and female mice

Dear Dr. Fadok:

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Data Availability Statement

Here is the DOI for the data in our manuscript: https://doi.org/10.7910/DVN/1RAQON.

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PERMALINK

Effects of footshock stress on social behavior and neuronal activation in the medial prefrontal cortex and amygdala of male and female mice

Mariia Dorofeikova

Chandrashekhar D Borkar

Katherine Weissmuller

Lydia Smith-Osborne

Samhita Basavanhalli

Erin Bean

Avery Smith

Anh Duong

Alexis Resendez

Jonathan P Fadok

Roles

Abstract

Introduction

Materials and methods

Animals

Groups

Footshock stress exposure

Social approach test

Alprazolam treatment

Histology

Statistical analysis

Table 1. Results of statistical analyses.

Results

Effects of footshock stress and alprazolam treatment on social behavior

Fig 1. Effects of acute stress and alprazolam on behavior in the social approach test.

cFos expression analysis

Fig 2. cFos expression patterns following social approach.

Correlations between cFos levels and behavior following the social approach test

Fig 3. Correlations between behavioral variables and cFos expression.

Discussion

Data Availability

Funding Statement

References

Decision Letter 0

Alexandra Kavushansky

Roles

Author response to Decision Letter 0

Decision Letter 1

Alexandra Kavushansky

Roles

Acceptance letter

Alexandra Kavushansky

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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