Noise stress changes mRNA expressions of corticotropin-releasing hormone, its receptors in amygdala, and anxiety-related behaviors.

Noise is a psychological, environmental stressor that activates limbic sites in the brain. Limbic sites such as the amygdala and the amygdaloid corticotropin-releasing hormone (CRH) system play an important role in integrating stress response. We investigated the association between noise exposures, CRH-related molecules in the amygdala, and behavioral alterations. In total 54 Sprague-Dawley rats were divided into the following three groups: Control (CON), acute noise exposure (ANE), and chronic noise exposure (CNE). The ANE group was exposed to 100 dB white noise only once in 4 h and the CNE group was exposed to the same for 4 h per day for 30 days. Expression profiles of CRH and its receptors CRH-R1 and CRH-R2 were analyzed by quantitative real-time polymerase chain reaction (qPCR). The same stress procedure was applied to the ANE and CNE groups for behavior testing. The anxiety responses of the animals after acute and chronic stress exposure were measured in the defensive withdrawal test. CNE upregulated CRH and CRH-R1 mRNA levels but downregulated CRH-R2 mRNA levels. ANE led to a decrease in both CRH-R1 and CRH-R2 expression. In the defensive withdrawal test, while the ANE increased, CNE reduced anxiety-like behaviors. The present study shows that the exposure of rats to white noise (100 dB) leads to behavioral alterations and molecule-specific changes in the CRH system. Behavioral alterations can be related to these molecular changes in the amygdala.

Introduction

Noise is becoming a widespread stress source in living environments in modern societies. The World Health Organization (WHO) documented that noise has effects on mental health, decreases working capacity, and induces sleep disturbances, and may be a risk factor for cardiovascular diseases.[1] However, the impact of noise stress on cognition, mood disorders, and underlying neurobiological mechanisms have been investigated in only a few studies. Noise has been reported to alter stress hormones,[234] learning and memory,[567] and anxiety-related behaviors.[48] Neuropsychiatric disorders including anxiety have been reported to be stress-related, and they are becoming widespread in modern societies with the increasing stress levels in everyday life.[9] However, the effects of stressors on anxiety responses may be adaptive or maladaptive.[10111213] Noise stress has been reported to reduce anxiety behaviors in a few studies.[48] However, the relationship of this effect with stress-responsive molecules in the brain has not been investigated.

The corticotrophin-releasing hormone (CRH) system plays a prominent role in the coordination of neuroendocrine and behavioral responses to stress. The CRH family comprises CRH, CRH1, and CRH2 receptors, and urocortins (Ucn). CRH family members found in the amygdala and the dysregulation of the CRH system in this limbic site cause alterations in the anxiety response of animals.[14151617] Although the involvement of CRH and its receptors CRH-R1 and CRH-R2 in anxiety responses have been widely documented, the role of these molecules in the direction of the responses is less clear.[1819202122] In addition, evidence for the activation of amygdala CRH-related molecules under different stressors is still limited and the results of these studies are controversial.[232425] It seems that the direction of alterations observed in behavioral responses, either adaptive or maladaptive, and the underlying neurobiology are stressor-specific.

Anxiety responses after noise stress have been studied only in a limited number of studies, and related alterations in the CRH system are not known. To test the impact of psychological stressor noise on the amygdala CRH-related molecules and the direction of behavioral responses, we assessed CRH, CRH-R1, and CRH-R2 mRNA expressions and anxiety-related behaviors of rats.

Methods

Animals and grouping

In total, 54 adult (weighting 250 ± 10 g) Sprague-Dawley male rats were used in this study (24 for mRNA expression analyses, 30 for the defensive withdrawal test). The rats used for mRNA expression analyses were randomized into the control (CON), acute noise exposure (ANE), and chronic noise exposure (CNE) groups, each consisting of 8 animals. For determining the effects of noise stress in defensive withdrawal test, the rats were randomly placed in CON, ANE, and CNE groups, each consisting of 10 animals.

The animals were housed in polycarbonate cages in groups of 4 per cage with wood chip bedding. Food and water were provided ad libitum. The animals were maintained in standard lighting (12 h/12 h light/dark cycle) and temperature conditions (22 ± 3°C). All experimental procedures were approved by the local Ethics Committee of Istanbul University.

Noise exposure

The white noise was generated using a noise generator (Type 1390, General Radio Company, MA, USA), amplified electronically, and emitted by loudspeakers installed into a sound-isolated cabinet. The loudspeakers (one speaker per cage) were fixed directly above the cages to the shelves of the cabinet. Noise levels were measured with a sound meter (CEM DT-8820, Shenzhen Everbest Machinery Industry Co., Ltd, Shenzhen, China) in the bottom of the cages and adjusted to the 100 dB (±1 dB) sound pressure level (SPL). Another cabinet with the same specifications with unplugged loudspeakers served as the control cabinet. The background noise level in the cabinets was 50 (±5) dB SPL.

Animals from the CNE group were exposed to white noise in the stress cabinet for 4 h per day for 30 days, while the ANE group animals were exposed to the same level of stress only once in 4 h. Continuous white noise stress was applied to the animals between 8 AM and 12 PM during the stress application period. During the stress sessions, the CON group animals were kept in the control cabinet.

All animals were sacrificed by rapid decapitation always between 12:30 PM and 1:00 PM. Animals from the CON group were sacrificed without stress application. Animals in the chronically stressed groups were always sacrificed 24 h following the end of the stress procedure to avoid the acute influence of the last stress session.[2627]

Tissue dissection and RNA preparation

Brains were immediately removed and kept at –80°C. Frozen brains were placed into a 1-mm rodent brain matrix (Electron Microscopy Sciences, Hatfield, PA, USA, catalog no. 69026-C) and cut into 2-mm thick slices (−2.76 to −4.76 mm from the bregma) using brain matrix razor blades (Electron Microscopy Sciences, Hatfield, PA, USA, catalog no. 70933-70) on ice. From these slices, entire amygdala samples were removed with a 2-mm diameter punch tool according to the brain atlas of Paxinos and Watson.[28] The same procedure was applied on all animals for sampling of amygdala tissue.

Total RNA was isolated using PureLink™ RNA Mini Kit (Invitrogen, Carlsbad, CA, US) in accordance with manufacturer's instructions. The concentration of total RNA was measured fluorimetrically (Qubit fluorimeter, Invitrogen, Carlsbad, California, USA) using the Quant-iT™ RNA Assay Kit (Invitrogen, Eugene, OR, USA).

Quantification of mRNA by real-time reverse transcription polymerase chain reaction (PCR)

Total RNA (1 μg) was reverse-transcribed into cDNA using random primers (high-capacity RNA-to-cDNA Master Mix Kit, Applied Biosystems, Foster City, CA, USA). The concentration of the resulting cDNA was measured using Quant-iT™ DNA Br Assay Kit (Invitrogen, Eugene, OR, USA). Quantitative real-time PCR (qPCR) was performed with 1 ng cDNA using TaqMan Gene Expression Master Mix and TaqMan Gene Expression Assays reagents (Applied Biosystems, Foster City, CA, USA) for CRH (Rn01462137_m1), CRHR1 (Rn00578611_m1), and CRHR2 (Rn00575617_m1). As a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (4352338E_0906011) was amplified in the same experiment. The real-time reaction was carried out on ABI 7500 Real-Time PCR Systems (Applied Biosystems, Foster City, California, USA).

The results of the real-time reactions are represented as the threshold cycle (Ct) values. We controlled whether the procedure used in the study affected housekeeping gene expression by comparing GAPDH Ct values across the different groups. The absence of statistical difference between these values indicated that GAPDH expression was not affected by the procedure. The mRNA expression levels of tested genes were normalized to those of GAPDH, and analyses of the data were performed using ΔΔ Ct method.[29] Fold changes of genes were calculated using the expression 2-ΔΔCt with respect to the mean value of delta Ct in the control group.

Defensive withdrawal test

Animals from the ANE (n = 10) and CNE groups (n = 10) were tested to measure the effects of stress on anxiety-related behaviors in the defensive withdrawal test on the first and 30th of the experimental days, respectively. CON (n = 10) group animals were tested without being exposed to stress.

The test was conducted on a transparent open arena (60 × 60 × 35 cm high) with a black bottom and adjacent closed side compartment (15 × 20 × 15 cm high). Light intensity in the center of the open field was 100 lx and less than 1 lx in the closed compartment. One day prior to the start of the test, the rats were habituated to the unfamiliar test environment. They were placed into the open field and allowed to explore freely for 15 min, while access to the dark compartment was prevented. The following day, the rats were placed in the dark compartment via the upper side lid and given access to the open field. The test lasted for 10 min and was videotaped. The test apparatus was cleaned with 1% glacial acetic acid to prevent olfactory cues after the behavioral testing of each animal.

We measured the following behaviors from video records using the computer software EthoLog 2.2 (Ottoni EB, University of Sao Paulo, Sao Paulo, Brazil):[30] latency to first exit the dark compartment with all four paws, number of exits from the dark compartment, the total time spent in the open field, and locomotor activity (line crossings per min in the open field).

Statistical analyses

Statistical analysis was carried out on the SPSS software package (ver. 11.5.2.1, SPSS Inc., Chicago, IL, USA). All results are expressed as means ± SEM. Data were first tested for normality using the Shapiro-Wilk test. Normally distributed data were compared using one-way analysis of variance (ANOVA). If the normality assumption was violated, the nonparametric Kruskal-Wallis test was applied. For pairwise comparisons, Duncan and Mann-Whitney U tests were used following parametric and nonparametric tests, respectively.

Results

CRH, CRH-R1, and CRH-R2 mRNA expressions in the amygdala after noise stress

Chronic noise stress significantly increased CRH mRNA expression, while ANE did not change it compared to the control condition [fold change, acute noise 1.7 ±. 8, chronic noise 5.6 ± 1.2, control 1 ±. 1, mean ± SEM, Mann-Whitney U, z = -3.36, P = . 001 after Kruskal-Wallis, χ2 = (2) = 11.79, P = .003]. While CRH-R1 gene expression was downregulated in the ANE group, it was upregulated in the CNE group, compared to the CON group (acute noise.6 ±.0, chronic noise 4.9 ±.7, control 1 ±.1, mean ± SEM, post hoc P ˂.05 after ANOVA, F (2, 21) = 31.86, P ˂.001). Both acute and chronic noise exposure decreased mRNA expression of CRH-R2 (acute noise.5 ±.0, chronic noise.5 ±.1, control 1 ±.1, mean ± SEM post hoc P ˂.05 after ANOVA, F (2, 21) = 13.46, P ˂.001) [Figure 1].

Figure 1

Changes in the expression of mRNA for CRH, CRH-R1, and CRH-R2 in the amygdala after noise stress exposure

There was a significant effect of noise stress on anxiety-like behaviors measured in the defensive withdrawal test considering latency F (2, 27) = 4.79, P = .009 and total time spent in the open field F (2, 27) = 5.70, P = .009. The total time spent in the open field significantly decreased in the ANE group compared to the other groups, indicating increased anxiety-like behaviors in ANE animals (acute noise 49.5 ± 10.0, chronic noise 120.3 ± 12.8, control 97.4 ± 20.6, mean ± SEM, P ˂.05). The chronic stress-exposed group showed significantly shorter latencies to exit the dark chamber comparing to the ANE group, indicating decreased anxiety responses after CNE, P ˂.05. Also, the CNE group exhibited tendencies towards decreased anxiety-like behaviors compared to controls, considering latency. But it was not significant due to the high variation between animals, P = .06 (acute noise 18.5 ± 3.7, chronic noise 7.5 ±.8, control 13.2 ± 2.1, mean ± SEM, Figure 2). Locomotor activity as measured by line crossings per min in the open field was not affected by acute or chronic stress treatments (acute noise 3.0 ±.4, chronic noise 5.1 ±.7, control 3.7 ±.5, mean ± SEM).

Figure 2

Effects of noise stress on anxiety-related behaviors in the defensive withdrawal test

Discussion

In the present study, we investigated the effects of stress on the mRNA levels of genes involved in the stress response in amygdala and related behavioral consequences in rats. In this context, we studied mRNA levels of genes related to CRHergic system and anxiety-related behaviors. Our results revealed that noise stress causes behavioral alterations and induces changes in CRH-related molecules in amygdala.

The present results show that noise stress causes a molecule-specific change in the mRNA expression of CRH and its receptors CRH-R1 and CRH-R2 in the amygdala. Although the effect of noise stress on stress hormones, some neurotransmitters, oxidative status markers, and neuronal activity in different brain regions had been studied previously,[568313233] alterations in CRH-related molecules are not known, to the best of our knowledge. Noise has been defined as a psychological, emotional, or processive stressor[32] that interacts with limbic system pathways to exert its effects.[34] Therefore, changes due to noise stress could be expected in stress-related gene expressions in the amygdala, which is a limbic site.

In the present study, acute noise stress did not change CRH mRNA levels. It had been suggested that gene expression may not change after exposure to acute stress because of a long half-life and the large pool of mRNA already contained in the neuron.[3536] Furthermore, CRH gene transcription had been reported to only occur after a certain stimulus intensity threshold is reached.[37] Therefore, with the support of these reports, we could suggest that acute noise stress was not sufficient to upregulate CRH mRNA levels. However, CNE led to an increase in CRH mRNA levels. It has been previously reported that psychological stressors caused an increase in CRH mRNA levels in the amygdala and thus could activate the amygdaloid CRH system.[383940] Our results for chronic noise-induced CRH mRNA expression in the amygdala are in accordance with the results of these studies. Moreover, it had been suggested that changes in secretogogue gene expression in the neuron are required to sustain future secretory activity.[35] Therefore, mRNA levels might have been increased to restore the likely depletion of CRH levels in the face of a sustained stressor.

A number of studies have shown that CRH receptor expression in the amygdala is stressor-specific, and the regulation of CRH receptors in limbic structures is not well understood.[23414243] Amygdala CRH-R1 mRNA levels decreased via acute stress application in the present study. For most of the receptor types, receptor biosynthesis and availability decreases after increased ligand binding and vice versa.[44] CRH has been reported to act as an endogenous ligand for CRH receptors, especially for CRH-R1 in the limbic system.[15434546] As CRH levels in the amygdala increase through stress,[384748] the downregulation of the CRH-R1 mRNA expression that we observed after acute stress may be mediated by stress-induced endogenous CRH release and binding to these receptors. On the other hand, CRH-R1 mRNA expression was biphasic, with a marked increase in the chronic phase of stress following the initial decline mentioned above, suggesting an altered receptor turnover. Because the activation of postsynaptic signal transduction is required for the modulation of CRH1 receptors,[49] neural stimulation induced by chronic noise stress could lead to an increase in receptor expression.

Acute and chronic noise exposure downregulated CRH-R2 mRNA levels in the amygdala. CRH-R2 mRNA alterations after stress exposure in this region have remained relatively unexplored. A few studies reported that different stressors either did not affect or decreased expressions of CRH-R2.[24414250] These studies mainly focused on the effects of acute stressors and reported stressor-specific results. Downregulation of CRH-R2 mRNA in our study may indicate the occupancy of these receptors by the ligand. It has been reported that CRH is a more potent activator of CRH-R1 than R2.[51] Rather than CRH, Ucn II and III selectively bind to CRH2 receptors.[5253] As CRH family neuropeptides including Ucn are considered to be important regulators of the stress response,[16] a likely increase in Ucn II and III levels induced by noise stress could have been related to downregulation of CRH-R2 mRNA in the present study.

It has been long known that stress causes diverse behavioral responses, including anxiety-related behaviors. The direction of the alterations in behavioral response varies depending on the type, duration, and intensity of the stress.[4101123] We found that total time spent in the open field in the defensive withdrawal test decreased in the ANE group, suggesting increased anxiety-like behavior. On the other hand, latency to exit the dark chamber significantly decreased in the CNE group compared to that of the ANE group, and there was also a tendency to decrease in the CNE group compared to the CON group, suggesting reduced anxiety-like behavior after CNE. Although acute stressors have been previously reported to cause increased anxiety response,[245455] to our knowledge, the anxiogenic effect of acute noises stress has not been previously reported. On the other hand, the decrease in anxiety responses in CNE animals supports the hypothesis of habituation to a long-term stressor.[56] The reduced anxiety levels of CNE animals may also indicate that chronic stress favors behavioral coping in a conflict situation. Considering the illuminated open area as a stressful environment, the decrease in latency to enter this area shows that previous exposure to chronic noise might predispose the animals to better cope with the stressful environment. Although we found acute anxiogenic effects of noise stress, animals may show adaptation to long-term stressful experiences, and favorable effects of moderate chronic stress, such as better coping with stressful situations, can be observed.[5758]

The effects of noise exposure on anxiety-related behaviors have been reported in a few studies previously.[48] However, the underlying molecular mechanisms, especially the involvement of CRH-related molecules, had not been studied so far. CRH-related molecules in the amygdala are widely believed to regulate anxiety-related behaviors.[151617] It has been suggested that CRH plays a prominent role via acting CRH-R1 receptors in mediating, especially, increasing anxiety-responses.[141559] Anxiogenic effect observed after ANE in our study may be related to a decrease in mRNA expression of CRH-R1 due to possible increased ligand binding. Similar to our results, a study[23] reported that while prenatal stress increased anxiety, CRH-R1 and R2 mRNA were reduced in male rats. On the other hand, different types of stressors were reported to have anxiogenic effects but no changes in the mRNA expression levels of CRH-R1 or R2 were observed. Differences between the results may be due to the type of the stressors. The relationship between the mRNA expressions of these receptors and observed behavioral effects is still not clear.

Our findings that indicate a reduction in CRH-R2 mRNA in the amygdala and the reduced anxiety levels of animals after chronic stress may be related to each other, supporting the claim that these receptors may have an anxiogenic effect.[212260] Controversial results involving the role of CRH-R2 receptors in anxiety-related behaviors have been reported. A prominent role for CRH-R2 in the regulation of anxiety-like behaviors after stress had been suggested, but the reported direction of behavioral alterations is not consistent across studies.[1820212261] On the other hand, it is difficult to associate reduced anxious behaviors with mRNA upregulation of CRH and CRH-R1 after CNE in the present study, considering that these molecules had been reported to have a role in increased anxiety.[144362] However, controversial results had been also reported,[196364] and the exact role of CRH and CRH-R1 receptors on anxiety responses are still not well understood. Furthermore, mRNA expression is only the initial component of gene expression and biosynthesis. The availability and functionality of these receptors are required in order for the relevant effect to be observed. Therefore, further studies focusing on possible changes in receptor synthesis, functional receptors, and levels of CRH and Ucn ligands are required to reveal the mechanisms responsible for the observed behavioral alterations.

The chronic noise stress-induced upregulation of CRH and CRH-R1 mRNA levels in our study may represent a sensitization process that could provide subsequent responses to future stressors. Changes in the mRNA levels of CRH-R1 and R2 may be associated with observed alterations in the anxiety responses of animals. The present study shows that the exposure of rats to chronic white noise (100 dB) leads to adaptive behavioral alterations that can be related to molecular changes in the CRH system. Further detailed research on the association between the components of the CRH system, stress, and behaviors is required. Understanding the neurobiology of the altered ability to respond to the stressor will contribute to the maintenance of mental soundness and prevention of health problems.