Sex Differences in the Expression of c-fos in a Rat Brain after Exposure to Environmental Noise

Abstract

Noise is an inarticulate stimulus that threatens health and well-being. It compromises audition and induces a strong stress response that activates the brain at several levels. In the present study, we expose male and female rats to environmental noise in order to investigate if acute or chronic stimulation produces differential brain activation patterns. The animals were exposed to a rat’s audiogram-fitted adaptation of a noisy environment and later sacrificed to quantify the expression of the brain activity marker c-fos. Additionally, the serum corticosterone (CORT) levels were measured to elucidate possible the stress-related effects of noise. It was found that environmental noise differentially increased the serum CORT levels in male and female rats. We identified 17 brain regions outside the classical auditory circuits with a high expression of c-fos, including the hypothalamus, prefrontal cortex, habenular complex, septum, cingulate cortex, nucleus accumbens, insular cortex, amygdala, and hippocampus. Overall, we evidenced that females exhibit less intense c-fos expression in most of the examined areas. We concluded that females might be less affected by the changes produced by environmental noise.

1. Introduction

Noise is one of the most important pollutants in the modern world [1]. Undesired sounds are everywhere. They derive from work environments, loud music, traffic (airway, railway, and roadway), or domestic appliances that exceed tolerable thresholds [2]. When sounds are excessive, they become annoying and interfere with adaptation [3]. While the effects of noise on auditory organs have been well attended, changes in brain circuitries outside audition remain less understood.

It is known that the brain is the most adaptable organ in the body, but also a major target for environmental threats. It integrates information and coordinates complex responses to ensure adaptation. The auditory system is a clear example of this. It obtains information about the environment from complex sound waves reaching the ears [4]. After reaching the ears, sounds follow the classical auditory pathway that connects inputs from the auditory nerve with serial stations in the brainstem (cochlear nucleus, inferior colliculus, geniculate body, and superior olivary complex) and the auditory cortex [5]. For an optimal adaptation, sounds may follow alternative pathways involving deep brain structures that enrich the sound experience. In undesired or traumatic sounds, stimuli could take these alternative pathways and even separate at the cochlear level [6]. Then, noxious sounds activate neural structures outside the classic auditory circuits, including the neuroendocrine stress regulatory system. Stress hormones (i.e., corticosterone) may exert numerous effects in the central nervous system, since many brain areas possess receptors to these hormones.

Immediate early gene (IEG) expression may be a powerful tool to identify extra-auditory structures activated by noise. The IEG c-fos and its protein product, c-fos, are reliable markers for neuronal activity [7]. It is expressed in low amounts during basal activity. However, it progresses after neural activity generated by extensive signals of physiological or environmental stimuli, inducing a rapid (~2 h) or long-lasting up-regulation of the c-fos protein members (c-fos, fos B, fra-1, and fra-2) [8]. Immunohistochemical identification of c-fos may help to reveal complementary brain regions activated by noise and to establish differences related to how individuals process undesired sounds [9].

Gender has been recognized as one of the most significant modulators of brain activity. It is known that females differ from males in a variety of conditions. Stress response and sound processing are well-documented examples of sex driven differences [10,11]. For instance, human females have better pure-tone thresholds at frequencies above 1–2 kHz, whereas males may be better below these frequencies [12]. Similarly, female auditory organs seem to be more resistant to deterioration over the years [13]. Consequently, environmental stressors could also trigger different activation patterns in brains of both genders. To gain knowledge on this, the present study employs the c-fos protein mapping to investigate the differences on how male and female brains respond to environmental noise.

2. Materials and Methods

2.1. Animals

The experiments were performed in 90-days-old Wistar rats. A total of 20 males and 20 females weighing 300.2 g ± 27.16 g were randomly divided and divided into 4 groups: acute effect of noise throughout 2 h (males n = 4; females n = 4) and 12 h (males n = 4; females n = 4) of exposure, and chronic effect of noise throughout 7 days (males n = 4; females n = 4) and 21 days (males n = 4; females n = 4) of exposure. Additionally, a non-exposed group was used as a control (males n = 4; females n = 4). All groups were maintained in a 12:12 light–dark cycle with lights on at 06:00 h. The temperature in the experimental room was maintained at 24 ± 2 °C and humidity at 70%. We guaranteed free access to tap water and balanced food. The cages were always changed in the testing room. All animal experiments obeyed the National Institute of Health guide (NIH Publications No. 8023, revised 1978) for the care and use of laboratory animals, and were approved by the health sciences ethics, biosafety and scientific board at the University of Guadalajara (CI. 068-2014). Figure 1 illustrates the procedure.

2.2. Noise Exposure

To produce a noisy environment, we exposed the animals of both sexes to an audiogram-fitted device provided with representative sounds of urban environments (i.e., turbines, hooters, horns, and others), as described by Rabat [14]. The administered sounds considered the rats’ lower capacity to detect low frequencies (under 500 Hz) and their improved capacity to perceive high frequencies (over 8000 Hz). We used metal grid cages to avoid sound refraction and housed the animals in groups of 4 in a soundproofed room. Professional tweeters (Yamaha, Inc. Osaka, Japan) were placed 1 m above the cages and connected to a Mackie amplifier (Mackie M1400; freq. 20 Hz to 70 kHz; 300 W at 8 Ω). The speakers’ characteristics and the tweeter allowed the sounds to be reproduced at frequencies between 20 and 50,000 Hz. Recordings of noisy environments were played randomly with noise exposures (18–39 s of a turbine, hooter, or horn sounds) and intervals of silence ranging from 20 to 165 s. Sound intensities were presented in a range of 70 to 105 dB to avoid cochlear damage. This protocol was offered for 2 h to the acute group 1, and for 12 h to the acute group 2; the chronic exposed groups remained in the noise chamber for 7 days (chronic group 1) and 21 days (chronic group 2) (Figure 1). A sound level meter was placed at every corner of the cages to ensure that all the animals received the same exposure dose. To avoid housing confounds, all the rats were transferred to the testing room 48 h before the start of the stimuli.

2.3. c-Fos Immunohistochemistry

For the neuronal activity evaluation of the exposed rats, the protein product (c-Fos) of the proto-oncogene, c-Fos, was immunohistochemically analyzed. We initiated exposures at 6:00 a.m. and perfused the animals when the time intervals (2 h, 12 h, 7 d, and 21 d) were completed. Immediately after exposure to the environmental noise, the rats were anesthetized with sodium pentobarbital (60 mg/kg) and transcardially perfused with 0.1 M phosphate-buffered saline (PBS), followed by 3.8% paraformaldehyde (PFA) in PBS pH 7.4. The brains were removed and postfixed in 3.8% paraformaldehyde for 2 h. Sections were cut at 40 μm in the coronal plane using a vibratome Leica VT1000E. Every third section (120 μm intervals) spanning from Bregma 2.6 mm to Bregma −3.8 mm [15] was collected in PBS. Endogenous peroxidase activity was blocked by incubating the sections in 3% hydrogen peroxide and PBS for 30 min. The sections were incubated in citrate buffer (10 mM citric acid, 0.05% Tween 20, pH 6.0) for 20 min at 85 °C. The blocking sections were incubated with 10% normal goat serum (NGS) in Tris-buffered saline (0.05 M, 0.9%, pH 7.4) plus 0.3% of Triton X-100 (TBST) for 30 min at 37 °C and 30 min at room temperature (25–28 °C). The sections were incubated in TBST + 1% NGS at 4 °C that contained a c-fos rabbit polyclonal antibody (Santacruz Biotechnology, Santa Cruz, CA, USA, polyclonal E-8, cat: sc-166940) for 18 h at 4 °C. Incubation was stopped and washed 3 times for 10 min with TBST, and sections were incubated in biotinylated a goat anti-rabbit antibody (1:500; Vectastain, Vector Laboratories Burlingame, CA, USA, cat: BA-1000) for 2 h at room temperature. The sections were then washed and incubated in the avidin-biotin–peroxidase complex (1:100, Vectastain Elite Kit, Vector Laboratories, cat: PK-6200) TBST solution for 1 h at room temperature. The reaction was visualized using diaminobenzidine 0.05% (Sigma Aldrich, Darmstadt, Germany; 500 μg/mL). The sections were washed and mounted with Permount mounting medium. The sections from experimental and control rats were processed simultaneously to ensure access to the same sets of solutions.

2.4. c-Fos+ Cell Analysis

The stained tissues from all of the groups were photographed with a Leica DFC 7000T camera attached to a DMi8 microscope. All the slices in every group were first checked to identify the regions that expressed the c-fos mark (from Bregma +2.6 mm to Bregma −3.10 approx. 1.8 cm of the brain). In every region identified, we counted the c-fos+ cells on an average of 8 to 10 coronal slices. Both hemispheres were quantified for every region expressing the mark. The 10X (0.864 μm/px) objective was used to obtain 2 photographs per region in an 8 bit grayscale. No adjustments (contrast, intensity, or gamma correction) were applied to the images. Every photograph represented a microscopic field, in which the dimensions of each region were completely covered. Image analysis was performed using ImageJ software and the plug-in: Cell Counter Notice (https://imagej.nih.gov/ij/ (accessed on 8 November 2021), 15 August 2018, ver.1.44, National Institutes of Health, Bethesda, MD, USA). The results were averaged and compared between the groups.

2.5. Corticosterone Assays

The blood samples were obtained from the animal’s tail and were gently warmed for 1 min in water at 40 °C. The tail was dried and cut with a razor blade, and an approximate 200 μL volume was collected in Eppendorf 1.5 mL tubes. The blood was allowed to clot for 3 min and centrifuged at 1000–2000× g for 10 min in a refrigerated centrifuge. Serum samples were frozen at −80 °C until all groups were collected. The serum corticosterone levels were measured using an enzyme immunoassay kit (Enzo Life Sciences Catalog No. ADI-900-097) following the manufacturer’s instructions. Blood samples were collected throughout exposure to noise at 2 h, 12 h, 7 d, and 21 days (always between 07:00 and 08:00 h, except for the 12 h group). Our corticosterone kit’s reported sensitivity was established at 27.0 pg/mL (range: 32–20,000 pg/mL).

2.6. Data Analysis

All the results were expressed as the mean ± standard error of the mean (SEM). The c-fos expression and serum levels of CORT data were analyzed by using a two-way ANOVA (sex (male/female) × noise condition (0 h, 2 h; 12 h; 7 d; 21 d)). The Dunn–Šidák correction and post hoc test were employed to evaluate the simple main effects. Values of * p < 0.05 (** p < 0.01, *** p < 0.001) were considered statistically significant. The statistical calculations were performed using SPSS software version 25 (SPSS Inc. Chicago, IL, USA) and, for the design and export of figures, we used GraphPad software Inc. Prism Version 7, San Diego, CA, USA.

3. Results

3.1. Corticosterone Analysis

The two-way ANOVA analysis showed no significant effect of sex on the serum CORT levels (main effect of sex, F_{(1, 110)} = 0.4425, p = 0.5073). Nevertheless, the time of exposure significantly altered the CORT levels (main effect of time, F_{(4, 110)} = 5.738, p = 0.0003). This effect differed between the males and females (sex × time interaction, F_{(4, 110)} = 5.488, p = 0.0003). Post hoc comparisons showed that noise-exposed males increased their CORT levels on day 7 (p < 0.001) and 21 (p < 0.009). On the other hand, female differences were observed only after 2 h of exposure (p < 0.001). Between the sexes, differences were significant at 2 h when exposed females outpointed exposed males (p < 0.01). Figure 2 illustrates the results of the CORT analysis.

3.2. Brainwide Quantification of c-Fos+ Cells

First, we compared the total number of c-fos+ cells across the entire brain of the exposed subjects (Bregma +2.6 mm to Bregma −3.10). Two-way ANOVA evidenced that c-fos expression did not differ as a function of sex (main effect of sex, F(_{1, 30}) = 1.730; p = 0.1984), but was significantly affected by the time (main effect of time, F(_{4, 30}) = 29.61; p < 0.0001) and interaction (sex × time interaction, F_{(4, 30)} = 6.787, p = 0.0005). Post hoc comparisons showed that males exposed to noise increased their expression levels at 2 h, 12 h, 7 d, and 21 d (p < 0.0001). Females also increased their expression levels at 2 h, 12 h, 7 d, and 21 d (p < 0.0001). The differences between the sexes were registered at 2 h, and 21 d when males showed higher c-fos expression levels than females (p < 0.01); and at 12 h when females expressed higher levels than males (p < 0.01). Figure 3 and Figure 4 illustrate this.

Next, we identified the regions in which the c-fos changes were pronounced. Regions that increased the c-fos expression included the caudate nucleus (CPu); lateral septum (LSV); claustrum (VCL); olfactory bulb (AOM); amygdala (MLA); habenula (Hb); dentate gyrus (DG); Cornu Ammonis (CA3, CA2, CA1); prelimbic cortex (PrL); infralimbic cortex (IL); cingulate cortex (Cg); insular cortex (INS); piriform cortex (Pir); secondary auditory cortex (AuD); primary auditory cortex (AU1); the paraventricular nucleus of the hypothalamus (PVN); ventromedial nucleus (VMH); and paraventricular nucleus of the thalamus (PV). Figure 4 illustrates the key regions that show c-fos expression after noise exposure. The results of the c-fos measures are summarized in

Table 1. Summary of the immunohistochemical quantification of c-fos expression after exposure to environmental noise. The data represent the mean number (±SEM) of c-fos+ cells in every region. Regions that reached statistical significance according to the two-way ANOVA are signaled with (*). Multiple means comparisons: (ᶲ) control vs. exposed males; (ˠ) control vs. exposed females; and (Ⱡ) exposed males vs. exposed females. Statistically significant at a value * p < 0.05 (** p < 0.01, *** p < 0.001).

Summary of Immunohistochemical Quantification of Brain c-Fos Protein Expression in Male and Female Wistar Rats
Brain Region	Gender	Control	Acute Noise Exposure		Chronic Noise Exposure
		Experimental Group Means (± SEM)
		0 h	2 h	12 h	7 days	21 days	Factor	F	df	p
Forebrain
Caudate nucleus *	Male	0.7 (0.08)	72.47 (10.6) ᶲ	28 (0.8) ᶲ	32.93 (1.8) ᶲ	31.43 (2.9) ᶲ	Sex	43.11	4, 290	0.0001
	Female	0.7 (0.08)	53.47 (4.2) ˠ	73.13 (1.8) ˠⱠ	69.87 (2.6) ˠⱠ	21.63 (4.7) ˠ	Time	50.11	4, 290	0.0001
							Interaction	40.11	4, 290	0.0001
Lateral septum *	Male	1 (0.03)	139.70 (15.3) ᶲⱠ	61.03 (4.7) ᶲ	58.53 (2.5) ᶲⱠ	42.60 (7.0) ᶲ	Sex	18.48	1, 290	0.0001
	Female	1 (0.03)	64.2 (4.4) ˠ	95.30 (5.4) ˠⱠ	19.63 (1.9)	33.27 (8.0) ˠ	Time	70.97	4, 290	0.0001
							Interaction	19.84	4, 290	0.0001
Claustrum *	Male	0.5 (0.09)	79.40 (6.1) ᶲⱠ	19.40 (1.6) ᶲ	56.70 (1.7) ᶲⱠ	46.23 (3.3) ᶲ	Sex	0.3096	1, 290	0.5783
	Female	0.5 (0.09)	58.6 (3.4) ˠ	71.93 (1.4) ˠⱠ	29.43 (1.2) ˠ	36.60 (4.0) ˠ	Time	141.6	4, 290	0.0001
							Interaction	58.33	4, 290	0.0001
Olfactory bulb *	Male	1.1 (0.06)	201 (26) ᶲⱠ	74.97 (3.0) ᶲ	107.90 (8.5) ᶲⱠ	45.80 (2.7) ᶲ	Sex	7.771	1, 290	0.0057
	Female	1.1 (0.06)	109.6 (13.4) ˠ	154.67 (6.2) ˠⱠ	51.60 (2.1) ˠ	24.77 (4.2) ˠ	Time	73.84	4, 290	0.0001
							Interaction	20.51	4, 290	0.0001
Amygdala complex *	Male	0.6 (0.08)	52.27 (3.7) ᶲ	35.50 (1.8) ᶲ	44.13 (1.7) ᶲ	36.40 (3.6) ᶲⱠ	Sex	39.77	1, 290	0.0001
	Female	0.6 (0.08)	60.53 (3.5) ˠ	55.77 (1.9) ˠⱠ	77.87 (2.3) ˠⱠ	23.40 (2.3) ˠ	Time	195.4	4, 290	0.0001
							Interaction	26.64	4, 290	0.0001
Habenular complex *	Male	0.5 (0.09)	35.27 (4.9) ᶲ	15 (1.0) ᶲ	21.60 (1.5) ᶲ	14.23 (1.6) Ⱡ	Sex	0.2757	1, 290	0.5999
	Female	0.5 (0.09)	28.4 (2) ˠ	30.77 (1.3) ˠⱠ	27.50 (1.0) ˠ	2.60 (0.4) ˠ	Time	86.58	4, 290	0.0001
							Interaction	15.26	4, 290	0.0001
Hippocampus
Dentate gyrus *	Male	0.2 (0.07)	12.77 (1.2) ᶲ	18.53 (0.8) ᶲ	38.87 (1.0) ᶲⱠ	42 (3.5) ᶲⱠ	Sex	19.14	4, 290	0.0001
	Female	0.2 (0.07)	13.4 (0.6) ˠ	28.60 (1.2) ˠⱠ	31.60 (1.6) ˠ	16.97 (1.7) ˠ	Time	160.4	4, 290	0.0001
							Interaction	35.78	4, 290	0.0001
CA3 *	Male	0.2 (0.07)	15.87 (0.8) ᶲⱠ	09.57 (0.8) ᶲ	22.17 (0.6) ᶲ	27.20 (2.2) ᶲⱠ	Sex	31.19	4, 290	0.0001
	Female	0.2 (0.07)	8.8 (0.6) ˠ	19.93 (0.9) ˠⱠ	16.53 (0.9) ˠ	7.60 (2.4) ˠ	Time	73.71	4, 290	0.0001
							Interaction	38.95	4, 290	0.0001
CA2	Male	0.1 (0.06)	17.8 (0.8) ᶲⱠ	6.47 (0.4) ᶲ	14.50 (0.4) ᶲⱠ	5.70 (0.7)	Sex	54.82	4, 290	0.0001
	Female	0.1 (0.06)	6.8 (0.6) ˠ	11.23 (0.5) ˠⱠ	6.87 (0.4) ˠ	2.53 (0.4)	Time	94.84	4, 290	0.0001
							Interaction	36.39	4, 290	0.0001
CA1 *	Male	0.1 (0.09)	24.03 (4.4) ᶲⱠ	2.23 (0.2)	10.63 (0.5) ᶲ	9.30 (1.04) ᶲⱠ	Sex	6.362	1, 290	0.0001
	Female	0.1 (0.09)	4.5 (0.7) ˠ	7.07 (0.7)	6 (0.6) ˠ	1.67 (0.2) ˠ	Time	19.02	4, 290	0.0001
							Interaction	14.83	4, 290	0.0001
Cortex
Prelimbic *	Male	0.8 (0.6)	113.9 (13.3) ᶲ	20.17 (1.0)	75.30 (5.3) ᶲ	38.60 (5.6) ᶲ	Sex	0.2748	1, 290	0.6005
	Female	0.8 (0.6)	88.8 (8.4) ˠ	92.67 (1.7)	20.47 (1.1) ˠ	57.80 (13.5) ˠ	Time	51.18	4, 290	0.0001
							Interaction	23.18	4, 290	0.0001
Infralimbic *	Male	0.7 (0.07)	115.1 (11.7) ᶲ	30.57 (1.3) ᶲ	58.50 (5.1) ᶲ	32.87 (4.5) ᶲ	Sex	0.1482	1, 290	0.7005
	Female	0.7 (0.07)	81 (6.4) ˠ	93.30 (2.0) ˠ	18.57 (0.8)	51.10 (9.4) ˠ	Time	78.01	4, 290	0.0001
							Interaction	27.04	4, 290	0.0001
Cingulate *	Male	1.1 (0.05)	154.3 (19.1) ᶲ	41.57 (1.2)	153.57 (6.6) ᶲⱠ	92.90 (15.0) ᶲ	Sex	58.64	1, 290	0.4445
	Female	1.1 (0.05)	132.7 (14.8) ˠ	146.73 (5.1) ˠⱠ	59.33 (2.8) ˠ	76.13 (19.6) ˠ	Time	42.87	4, 290	0.0001
							Interaction	20.00	4, 290	0.0001
Insular *	Male	0.5 (0.09)	40.1 (2.7) ᶲ	27.57 (1.0) ᶲ	49.40 (2.5) ᶲⱠ	18.83 (3.0) ᶲ	Sex	5.034	1, 290	0.0256
	Female	0.5 (0.09)	41.6 (4.2) ˠ	59.97 (3.0) ˠⱠ	23.50 (0.9) ˠ	30.47 (4.8) ˠ	Time	81.16	4, 290	0.0001
							Interaction	29.07	4, 290	0.0001
Piriform *	Male	1 (0.1)	159.5 (23.2) ᶲ	90.10 (2.4) ᶲ	82.13 (2.4) ᶲ	64.63 (5.8) ᶲ	Sex	2.027	1, 290	0.1556
	Female	1 (0.1)	139.3 (16.1) ˠ	225 (5.5) ˠⱠ	42.30 (1.1)	32.53 (4.8) ˠ	Time	101.4	4, 290	0.0001
							Interaction	28.90	4, 290	0.0001
Temporal auditory * (secondary)	Male	0.6 (0.08)	122.33 (10) ᶲⱠ	128.73 (5.5) ᶲ	77.03 (4.0) ᶲ	69.00 (9.9) ᶲⱠ	Sex	0.2242	1, 290	0.6362
	Female	0.6 (0.08)	75.3 (6.9) ˠ	153.67 (3.0) ˠ	129.37 (6.1) ˠⱠ	29.70 (4.2) ˠ	Time	164.2	4, 290	0.0001
							Interaction	24.55	4, 290	0.0001
Temporal auditory * (primary)	Male	0.7 (0.08)	129.8 (10.8) ᶲⱠ	132.33 (4.7) ᶲⱠ	61.73 (1.8) ᶲ	61.83 (9.2) ᶲⱠ	Sex	16.62	1, 290	0.0001
	Female	0.7 (0.08)	85.13 (6.2) ˠ	88.33 (3.1) ˠ	115.60 (5.8) ˠⱠ	24.00 (3.6)	Time	137.4	4, 290	0.0001
							Interaction	27.91	4, 290	0.0001
Hypothalamus
Paraventricular nucleus *	Male	1.2 (0.1)	118.3 (14.7) ᶲ	33.73 (1.3) ᶲ	91.47 (2.4) ᶲ	63.17 (5.4) ᶲⱠ	Sex	0.7501	4, 290	0.3872
	Female	1.2 (0.1)	127.6 (10.9) ˠⱠ	100.03 (2.2) ˠ	63.67 (2.9) ˠ	30.07 (5.6) ˠ	Time	94.12	4, 290	0.0001
							Interaction	19.89	4, 290	0.0001
Ventromedial nucleus *	Male	0.5 (0.09)	114.2 (14.6) ᶲⱠ	22.67 (1.4)	45.23 (1.5) ᶲ	22.70 (3.0)	Sex	1.198	1, 290	0.2745
	Female	0.5 (0.09)	78.4 (5.1) ˠ	74.43 (2.9) ˠⱠ	44.23 (2.3) ˠ	27.13 (6.3) ˠ	Time	80.88	4, 290	0.0001
							Interaction	15.74	4, 290	0.0001
Thalamus
Paraventricular nucleus *	Male	0.7 (0.1)	104.2 (10.8) ᶲⱠ	55.77 (2.8) ᶲ	85.60 (5.5) ᶲ	57.73 (6.5) ᶲ	Sex	12.38	1, 140	0.0006
	Female	0.7 (0.1)	59 (5.2) ˠ	71.67 (1.6) ˠ	66.27 (5.9) ˠ	44.27 (6.7) ˠ	Time	67.75	4, 140	0.0001
							Interaction	8.409	4, 140	0.0001

and described below.

Brain Regions Affected by Noise

In the olfactory bulb, two-way ANOVA evidenced differences due to sex (the main effect of sex, F_{(1, 290)} = 7.771, p = 0.0057) time (main effect of time, F_{(4, 290)} = 73.84, p = 0.0001), and the interaction (sex × time interaction, F_{(4, 290)} = 20.51, p = 0.0001). Šidák post hoc comparisons showed that males exposed to noise increased their expression levels at 2 h, 12 h, 7 d (p < 0.0001), and 21 d (p < 0.0154). Females exhibited higher expression levels at 2 h, 12 h, and 7 d (p < 0.001). The differences between the sexes were registered at 2 h and 7 d when males showed higher c-fos expression levels than females (p < 0.0001).

In the lateral septum, there was a significant effect of sex (F_{(1, 290)} = 18.48, p = 0.0001), time (F_{(4, 290)} = 70.97, p = 0.0001), and interaction (sex × time interaction, F_{(4, 290)} = 19.84, p = 0.0001). Post hoc comparisons showed that exposed males increased their c-fos levels at 2 h, 12 h, 7 d, and 21 d (p < 0.001). Females also registered increases at 2 h, 12 h, and 21 d (p < 0.001). Intersex comparisons showed that males overexpressed c-fos at 2 h and 7 d, compared to females (p < 0.001).

In the claustrum, the density of c-fos+ cells did not differ between males and females (main effect of sex, F_{(1, 290)} = 0.3096, p = 0.5783). Time significantly altered c-fos expression (main effect of time, F_{(4, 290)} = 141.6, p = 0.0001), and this effect differed between males and females (sex × time interaction, F_{(4, 290)} = 58.33, p = 0.0001). Increases were registered in males at 2 h, 12 h, 7 d (p < 0.001), and 21 d (p < 0.0154). Females also increased their expression levels at 2 h, 12 h, and 21 d (p < 0.001). Male vs. female comparisons revealed higher levels in males at 2 h and 7 d (p < 0.001).

In amygdala female rats had greater c-fos expression compared to males (main effect of sex, F_{(1, 290)} = 39.77, p = 0.0001). Time of exposure altered the density of c-fos+ cells (main effect of time, F_{(4, 290)} = 195.4, p = 0.0001) and this effect differed between males and females (sex × time interaction, F_{(4, 290)} = 26.64, p = 0.0001). Both males and females increased their expression levels at 2 h, 12 h, 7 d, and 21 d (p < 0.001), with respect to controls. Increases were higher in females at 12 h and 7 days (p < 0.001), in comparison to males.

In the habenula, there was no significant effect of sex (F_{(1, 290)} = 0.2757, p = 0.5999), but there was an effect of time (F_{(4, 290)} = 86.58, p = 0.0001) and interaction (sex × time interaction, F_{(4, 290)} = 15.26, p = 0.0001). Follow-up comparisons indicated that males increased their expression levels at 2 h, 12 h, 7 d (p < 0.001), and 21 d (p < 0.0154) in comparison to control, and after 21 days in comparison to females (p < 0.001). Otherwise, females showed higher levels at 2 h, 12 h (p < 0.001), and 7 d (p < 0.0038) compared to control and at 12 h (p < 0.0001), and 21 days (p < 0.0014) compared to exposed males.

In the caudate nucleus, the density of c-fos+ cells differed between the sexes (main effect of sex, F_{(1, 290)} = 15.97, p = 0.0001), was affected by time (main effect of time, F_{(4, 290)} = 69.87, p = 0.0001), and was significant for the interaction (sex × time interaction, F_{(4, 290)} = 23.15, p = 0.0001). Exposed males increased their expression levels after 2 h, 12 h, 7 d, and 21 d (p < 0.001). Exposed females also increased their c-fos levels at 2 h, 12 h, 7 d, and 21 d, compared to control (p < 0.001). These increases were higher in females at 12 h and 7 days (p < 0.001) compared to males (Figure 5).

In the dentate gyrus, there was also a significant effect of sex (F_{(1, 290)} = 19.14, p = 0.0001), time (F_{(4, 290)} = 160.4, p = 0.0001), and interaction (sex × time interaction, F_{(4, 290)} = 35.78, p = 0.0001). Males and females increased their expression levels at 2 h, 12 h, 7 d, and 21 d (p < 0.001). Males increased their numbers at 7 and 21 days (p < 0.001) compared to females (Figure 5).

In CA3 (the cornus amonis region 3) we found changes as a function of sex (main effect of sex, F_{(1, 290)} = 31.19, p = 0.0001), time (main effect of time, F_{(4, 290)} = 73.71, p = 0.0001), and interaction (sex × time interaction, F_{(4, 290)} = 38.95, p = 0.0001). Male rats showed significant increases after 2 h, 12 h, 7 d, and 21 d of exposure (p < 0.001). Additionally, females augmented their expression levels after 2 h, 12 h, and 21 d (p < 0.001). Compared to females, males increased their numbers at 2 h and 21 d (p < 0.001).

In CA2 (cornus amonis region 2) there was a significant effect of sex (F_{(1, 290)} = 54.82, p = 0.0001), time (F_{(4, 290)} = 94.84, p = 0.0001), and interaction (F_{(4, 290)} = 36.39, p = 0.0001). Males showed increases at 2 h, 12 h, 7 d (p < 0.001), and 21 d (p < 0.0154). Females also increased their c-fos levels at 2 h, 12 h (p < 0.001), and 7 d (p < 0.0038), with respect to controls. The increases were greater in males at 2 h and 7 d (p < 0.001), compared to females.

In CA1 (cornus Amonis region 1), there was a significant effect of sex (F_{(1, 290)} = 6.362, p = 0.0001), time (F_{(4, 290)} = 19.02, p = 0.0001), and interaction (F_{(4, 290)} = 14.83, p = 0.0001). Increases in male levels were significant at 2 h, 7 d, and 21 d (p < 0.001). Females registered increases only at 12 h (p < 0.0129). Changes were higher in males at 2 h and 21 days (p < 0.001), with respect to females.

In the prelimbic cortex, there was no significant effect of sex (F_{(1, 290)} = 0.2748, p = 0.6005), but time (F_{(4, 290)} = 51.18, p = 0.0001) and interaction (F_{(4, 290)} = 23.18, p = 0.0001) were significant. Males registered increases at 2 h, 7 d, and 21 d (p < 0.001). Female changes were significant at 2 h, 12 h, and 21 d (p < 0.001). Intersex differences were observed at 12 h and 7 d (p < 0.0001).

In the infralimbic cortex, the density of c-fos+ cells did not differ between males and females (F_{(1, 290)} = 0.1482, p = 0.7005), but time (F_{(4, 290)} = 78.01, p = 0.0001) and interaction (F_{(4, 290)} = 27.04, p = 0.0001) were significant. Male increases in c-fos levels were significant at 2 h, 12 h, 7 d, and 21 d (p < 0.001). Females registered increases at 2 h, 12 h, and 21 d (p < 0.001). Intersex differences were observed at 2 h, 12 h, and 7 d (p < 0.0001).

In the cingulate cortex, the density of c-fos+ cells did not differ due to sex (F_{(1, 290)} = 58.64, p = 0.4445), but was altered by time (F_{(4, 290)} = 42.87, p = 0.0001) and interaction (F_{(4, 290)} = 20.00, p = 0.0001). Exposed males increased their expression levels at 2 h, 7 d, and 21 d (p < 0.001). Exposed females also increased their c-fos levels at 2 h, 12 h (p < 0.001), 7 d (p < 0.0026), and 21 d (p < 0.0041). Changes were significantly elevated in males at 12 h (0.0001) and 7 days (p < 0.008), compared to females.

In the insular cortex, we found significant effects of sex (F_{(1, 290)} = 5.034, p = 0.0256), time (F_{(4, 290)} = 81.16, p = 0.0001), and interaction (F_{(4, 290)} = 29.07, p = 0.0001). Males and females increased their expression levels after 2 h, 12 h, 7 d, and 21 d of exposure (p < 0.001). Differences between the sexes were registered at 12 h and 7 days (p < 0.0001) when males outpointed females.

In the piriform cortex, the number of c-fos+ cells did not differ between the males and females (F_{(1, 290)} = 2.027, p = 0.1556), but was affected by time (F_{(4, 290)} = 101.4, p = 0.0001) and interaction (F_{(4, 290)} = 28.90, p = 0.0001). Males augmented their c-fos expression after 2 h, 12 h, 7 d, and 21 d (p < 0.001). Females also increased their c-fos levels at 2 h, 12 h (p < 0.001), and 7 d (p < 0.0185). These increases were greater in females at 12 h (p < 0.001).

In the auditory primary cortex, exposed males increased their expression levels after 2 h, 12 h, 7 d, and 21 d (p < 0.001). Exposed females also registered increases at 2 h, 12 h, 7 d (p < 0.001), and 21 d (p < 0.0324). The effects were higher in males at 2 h and 21 d (p < 0.001) than females.

In the auditory secondary cortex, we found the significant effect of sex (F_{(1, 290)} = 0.2242, p = 0.6362), time (F_{(4, 290)} = 164.2, p = 0.0001), and sex × time interaction (F_{(4, 290)} = 24.55, p = 0.0001). Exposed males increased their expression levels after 2 h, 12 h, 7 d, and 21 d (p < 0.001). Female increases were significant at 2 h, 12 h, 7 d (p < 0.001), and 21 d (p < 0.0058). Males were more affected than females at 2 h, 12 h(p < 0.001), and 21 d (p < 0.0003).

In the hypothalamus paraventricular nucleus, male increases were significant after 2 h, 12 h, 7 d, and 21 d (p < 0.001), compared to controls. Female increases were significant at 2 h, 12 h, 7 d (p < 0.001), and 21 d (p < 0.0147). Males exhibited higher levels than females at 21 days (p < 0.001) (Figure 5).

In the hypothalamus ventromedial nucleus, there was no significant effect of sex (F_{(1, 290)} = 1.198, p = 0.2745), but time (F_{(4, 290)} = 80.88, p = 0.0001) and sex × time interaction (F_{(4, 290)} = 15.74, p = 0.0001) were both significant. Follow-up comparisons evidenced that exposed males increased their expression levels after 2 h, 12 h, 7 d (p < 0.001), and 21 d (p < 0.0403). Females also increased their c-fos levels at 2 h, 12 h, 7 d (p < 0.001), and 21 d (p < 0.0065). These increases were statistically significant in females at 2 h (p < 0.0004) and 12 h (p < 0.0001), compared to males.

Finally, in the thalamus paraventricular nucleus, we found the significant effect of sex (F_{(1, 140)} = 12.38, p = 0.0006), time (F_{(4, 140)} = 67.75, p = 0.0001), and sex × time interaction (F_{(4, 140)} = 8.409, p = 0.0001). Exposed males increased their expression after 2 h, 12 h, 7 d, and 21 d (p < 0.001). Exposed females also increased their c-fos levels at 2 h (p < 0.0138), 12 h (p < 0.0012), and 7 d (p < 0.0036). These increases were significantly higher in males at 2 h (p < 0.001), compared to females.

4. Discussion

Our results evidence a robust increase in the cerebral expression of c-fos as a consequence of environmental noise. Remarkably, noise produced strong increases in many brain areas proposed to regulate stress response: the hypothalamus, amygdala, hippocampus, habenula, insula, lateral septum, prefrontal cortex, cingulate cortex, claustrum, and caudate-putamen. These patterns of activation confirmed environmental noise as a potent stressor [16,17,18]. Measures showing that exposed rats elevated their serum CORT levels, confirmed that noise was able to activate the neuroendocrine stress regulatory system (Figure 2). Our results are supported by experiments demonstrating that artificial noise (i.e., white noise at 60 to 105 dBA) increases the c-fos expression in the hippocampus, hypothalamus, septal area, prefrontal cortex, and thalamus [16,19]. Then, we agree with the line of thought proposing that stressful audiogenic stimulus arriving at the auditory system may also use alternative pathways (for instance, the arcuate fasciculus, anterior cingulate, and prefrontal cortices) to activate the hypothalamic PVN and operate the complex stress regulatory system [18]. Then, it is possible that noise synergizes with conditions that are difficult to separate from the audiogenic experience (i.e., sleep deprivation and handling) to increase the c-fos/CORT response. Nonetheless, it is clear that in addition to the well-known effects of noise on audition, we should also be aware of the less-investigated effects of the concomitant stress.

Apart from confirming noise as a potent stressor, our results also evidence that c-fos fluctuates as a function of time. Similar to the other experiments, the rats evaluated in our experiment increased their expression levels at acute exposures, but diminished their counts at chronic exposures [19]. As expected, rapid increases (2 h) were registered in most of the analyzed areas, confirming the relevance of these regions in the processing of noxious sounds. However, and less expected, reduced levels were found at chronic exposures (21 days). This phenomenon attracts our attention, since it may suggest that subjects habituate over time and that the involved areas could be crucial for the adaptation/habituation to this stressor [20,21,22,23]. Fluctuations can also suggest that c-fos expression might presage/accompany the down-regulatory effect of stress on dendritic retraction, learning impairment, reduced neurogenesis, or impaired plasticity, commonly associated with the deterioration of these areas [24,25,26,27,28]. Then, chronic-fos expression changes could be interpreted as an indicator of a new functional state of a given structure [29]. In our experiment, the hippocampus adjusts to this idea since c-fos expression changes were initiated later, and maintained even when other structures dropped. Additional support for these time-dependent fluctuations can be extracted from reports showing that the fos family members may express protein variants at different time points. The c-fos protein used to be expressed during the first 2 h after an acute stimulus, while fosB used to be expressed for more extended periods. Instead, members of the fos family, fra-1 and fra-2, are expressed in response to chronic/repeated stimuli [8]. Consequently, differences could be justified at acute and chronic/repeated exposures. Additional experiments should be planned to fully elucidate the complex time-dependent dynamics of c-fos activation across the many brain regions identified in the present study.

Next, this experiment revealed that males and females were differentially affected by environmental noise. It seems that males exhibited the most pronounced effects. Differences between males and females were reported previously for sound processing [30,31] and stress response [32]. While, in some reports, females exhibited higher releases of CORT after stress [33,34], other experiments presented less effects on dendritic remodeling [35] or cognitive alterations [25,36,37]. In accordance with experiments showing fewer effects, our results suggest that females are more resilient to noise, since CORT rises and c-fos changes were significantly less intense. Available evidence on stress research sustains that rodents could be more or less sensitive to noisy conditions as a function of sex. Our results align with the data showing more intense effects on males [38]. Accordingly, other experiments reported less c-fos expression in females that faced novel stressors [39]. However, differing from models using foot and tail shock [33], neonatal handling [40], restraint, and immune challenge [41], where effects on males were less intense, our results suggest a stronger noise-specific effect for males. Coincidentally, there is an elegant series of experiments elucidating this. Babb and colleagues found that, contrary to males, females increased their CORT and c-fos expression levels after restraint stress, but not after noise [42]. Therefore, females could be more resistant than males to the adverse effects of noisy environments, but not to other stressors. Complementary experiments should elucidate this, keeping in mind that some structures are sexually dimorphic. According to our results, structures that include the hippocampus, amygdala, and habenula may represent key candidates to this outcome, since sex effects were noticeable for c-fos expression.

We conclude the following: (i) environmental noise is a potent stressor capable of activating at least 17 cerebral areas outside the classic auditory circuits; (ii) the sex (male/female) and time (acute/chronic) variables are strong modulators of these effects; and (iii) the effects of noise appear more pronounced and extended in males, at least in relation to rodents.