Edited by: Deborah Baro, Georgia State University, USA
Reviewed by: Stephen M. Johnson, University of Wisconsin-Madison, USA; Laura M. Hurley, Indiana University Bloomington, USA
*Correspondence: Alexies Dagnino-Subiabre
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Chronic stress impairs auditory attention in rats and monoamines regulate neurotransmission in the primary auditory cortex (A1), a brain area that modulates auditory attention. In this context, we hypothesized that norepinephrine (NE) levels in A1 correlate with the auditory attention performance of chronically stressed rats. The first objective of this research was to evaluate whether chronic stress affects monoamines levels in A1. Male
Stress is a non-specific biological response of an organism to environment demands that affect their homeostasis. Consequently, stress allows them to restore homeostasis and adapt to environmental threats (Selye,
Stress can be positive (eustress) when the stressors are mild, brief and controllable (Tafet and Bernardini,
In agreement with evidence obtained in rats, psychosocial stress and stress-related disorders (e.g., depression) in humans produce alterations in cognitive functions, such as attention (Gotlib and McCann,
Attention is a cognitive function that allows animals to select relevant stimulus in the environment and ignore irrelevant stimuli (Raz,
On the other hand, norepinephrine (NE) is released from the
Together, these studies show that there is a relationship between LC-NE system, GABAergic transmission in A1 and auditory attention, which raise the question of whether chronic stress affects monoamine levels in the A1. Thus, the objectives of this study were to test whether repeated restraint stress affects: (1) NE, 5-HT and dopamine (DA) levels in the auditory cortex; and (2) the behavioral performance in an auditory attention task. We found that chronically stressed rats had lower levels of NE in A1 than controls, while 5-HT and DA levels were similar. We evaluated the effects of reboxetine on NE levels and on a performance in an auditory attention task. The main results were that control and chronically stressed rats treated with reboxetine had similar NE levels in the auditory cortex and comparable performance in the auditory attention task.
All procedures related to animal maintenance and experimentation were approved by the Institutional Animal Ethics Committee of the Faculty of Sciences Universidad de Valparaíso (Chile) and were in strict accordance with animal care standards outlined in National Institutes of Health (USA) guidelines. Efforts were made to minimize the number of animals used and their suffering. The antidepressant drug reboxetine at doses of 0.65 mg/kg (Pfizer, Pharmacia and Upjohn) or saline (vehicle, 0.9% NaCl) were intraperitoneal injected one time daily at 9:00–10:00 am for 21 days (Scheme
Hundred sixty male
After weaning (PND 23), 40 rats were trained for 3 weeks in a 2-ACT task, a behavioral task used to evaluate auditory attention in rodents (Scheme
To determine auditory attention, the animals were trained to discriminate tones in a 2-ACT paradigm (Jaramillo and Zador,
Three days after weaning, male
The 2-ACT training has three steps; in each of which it is possible to independently analyze learning, memory consolidation and auditory attention (Scheme
Scheme
Experiment | Description | Experimental group | Total | |||
---|---|---|---|---|---|---|
1 | Monoamine levels in Al | Control | Stress | |||
2 | Stress markers | |||||
Body weight and locomotor activity | ||||||
Corticosterone levels in unstimulated rats | ||||||
Corticosterone levels after acute swim | ||||||
3 | Auditory attention | |||||
4 | NE levels in Al | |||||
After completion of the chronic stress protocol each rat was decapitated under deep anesthesia with isoflurane. The brain was removed quickly and the auditory cortex was microdissected at 4°C according to Hébert et al. (
To analyze the effects of the stress procedure, we measured the percentage gain in body weight at the beginning and end of the experimental period (net change in weight after experiment ×100/weight at the beginning of the experiment).
We used a separate set of rats to determine the corticosterone concentration in plasma to avoid the stress of blood collection affecting behavioral or HPLC experiments. A set of rats (control,
Locomotor activity was evaluated in the open field test. All animals were naive to the test situations. The behavioral test was carried out from 10:00 h to 14:00 h. in the test room. The activity of each rat was recorded by IP cameras fixed above the behavioral apparatus and connected to a computer in another room. Videos were acquired by Nuuo software (Nuuo, Taipei, Taiwan). Behavioral tests were conducted in a soundproof and temperature-controlled (21 ± 1°C) room. Each rat was placed in the center of a black Plexiglass cage (70 cm × 70 cm × 40 cm) for 5 min. The background noise level in the open field was 40 dB SPL (Precision sound level meter, Model # 1100, Quest Technologies, Oconomowoc, WI, USA) and the arena was illuminated to 300 lux (measured by a digital lux meter, Model # LX-1010B, Weafo Instrument Co., Shanghai, China). Total distance traveled was analyzed from video recordings using ANY-maze video tracking system (Stoelting Co., Wood Dale, IL, USA). All mazes were wiped thoroughly clean with a 5% ethanol solution after each trial. In all experiments, animals from control and stress experimental groups were evaluated at the same time.
Trained rats were subjected to 50 trials of 2-ACT 1 day before and after the restraint-stress protocol (Scheme
After completion of the restraint-stress protocol each rat treated with vehicle or reboxetine was decapitated under deep anesthesia with isoflurane. NE content in the auditory cortex was determined as shown in the description of Experiment 1.
NE concentration in A1 was analyzed using the non-parametric Mann-Whitney U test. The results shown in Figures
Results were analyzed by a two-way ANOVA. The independent variables were treatments (vehicle and reboxetine) and stress (control and stress). The dependent variables were body weight (body weight gain), total distance traveled (locomotor activity), and pg/mg of tissue (NE content).
Corticosterone levels were analyzed by a 3 × 2 factorial ANOVA. The dependent variable was serum corticosterone levels and the independent variables were restraint stress (control and stress), treatments (vehicle and reboxetine), and acute stress (without stimulation and acute swim).
The performance of the animals in the 50 trials were analyzed by a two-way repeated-measure ANOVA (DS [groups (vehicle, reboxetine) × trials (1–50)]).
Bonferroni
Data are represented by mean ± SEM. A probability level of 0.05 or less was accepted as significant. The asterisk (*) indicates significant differences relative to control animals.
Chronically stressed rats had lower levels of NE in the auditory cortex than did control animals (
To determine whether our chronic stress protocol was effective in triggering stress response, we measured locomotor activity and stress markers, including the corticosterone level and body weight gain. There was a significant effect of the treatment-stress interaction on body weight gain (
Figure
Neither restraint stress nor reboxetine affected locomotor activity. There was no main effect of restraint stress (
The aim of this experiment was to evaluate the effect of the reboxetine on the auditory attention behavior of control and chronically stressed rats that were trained in the attentional task, an auditory frequency discrimination task used to study attention in rats (Figure
Chronically stressed rats treated with vehicle had lower levels of NE in the auditory cortex than did control animals (
In this study we found that chronic stress specifically decreased NE concentrations in A1, which could be explained in part by the relationship between the HPA axis and LC-NE system. It is possible that in our experiments chronic stress had decreased LC activity and NE release in the brain. This idea is supported by the study of Isingrini et al. (
Having demonstrated that chronic stress decreases NE levels in A1, we determined if reboxetine affects auditory attention in chronically stressed rats. Firstly, we determined if the applied chronic stress procedure was effective in triggering stress responses in the animals treated with vehicle or reboxetine. The rats subjected to restraint stress and treated with vehicle had lower body weight gain than did controls injected with vehicle (Figure
Having established that our chronic stress protocol was effective, we evaluated the effects of reboxetine on auditory attention and NE concentrations in A1. An unexpected result was that vehicle-treated control rats and vehicle-treated stressed rats (restraint) had similar performances in the attentional task (Figure
On the other hand, rats that were exposed to vehicle injections and subjected to repeated restraint stress had significantly higher corticosterone levels than rats that were not disturbed. This result suggests that repeated restraint stress was more stressful for the rats than daily injections and the HPA axis of restraint-treated rats did not adapt to restraint and exacerbated their HPA axis response to a new stressor. In this context of chronic stress, we have reported that A1 shows dendritic atrophy and decreases of GABAergic efficacy, a brain area that also regulates auditory attention (Bose et al.,
An alternative hypothesis is that the observed effects along trials in vehicle-treated animals were due to sustained attention. It is known that NE brain levels are important for maintaining sustained attention in time, and it has been shown that low dose of nortryptiline (NE reuptake blocker) can improve the performance of rats in an auditory sustained attention tasks (Roychowdhury et al.,
The effects of reboxetine did not correlate with alterations in locomotor activity because reboxetine and restraint stress did not affect the distance traveled in the open field task (Figure
An interesting fact was that the treatment with reboxetine had a different effect on each of the control and stress experimental groups, not only at a behavioral level, but also on the NE concentration in A1. In rats that were exposed to repeated restraint stress, vehicle- and reboxetine-treated stressed rats showed similar levels of attention in the first 20 trials of the 2-ACT test, after which reboxetine-treated rats had higher attention levels and NE concentration in A1 than those vehicle-treated rats (Figures
With the results obtained in this research we cannot explain how NE affected in particular the auditory attention, so that further experiments are necessary. Despite this, we can speculate a possible mechanism and suggest it in this discussion (Scheme
Interneurons exert a strong control over balance and synchronization of the brain circuit (Buzsaki and Chrobak,
On the other hand, it is possible that the effects of chronic stress and reboxetine on auditory attention could be in part induced indirectly. For example, the mPFC plays a key role in attention and NE modulates their neuronal activity (Dalley et al.,
Taken together, these evidences suggest that in our experiments NE could modulate the cholinergic system in the mPFC and/or A1, which in turn affect sustained auditory attention. For example, chronic stress could decrease NE and cholinergic activities, while reboxetine increase both NE and cholinergic activities improving sustained auditory attention as shown in Figure
In conclusion, our results suggest that chronic stress decrease the NE concentration in A1. On the other hand, reboxetine improved auditory attention and increased NE in A1 of chronically stressed rats. In addition, reboxetine had a strong anti-stress effect. We propose that NE plays a key role in the stress-induced inhibition of GABA release in A1 and by this way affects auditory attention of stressed rats.
AD-S designed research; CP-V and MFG-P performed research; CP-V, MFG-P, RS-Z and PHD analyzed data. AD-S wrote the article.
This study was primarily funded by Anillo de Ciencia y Tecnología N° ACT1403 grant, Programa PIA of Comisión Nacional de Investigación Científica y Tecnológica (CONICYT; AD-S). We also received funding from FONDECYT 111-21205 (RS-Z).
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
microlitres
two-alternative choice task
5-Hydroxyindoleacetic acid
serotonin
primary auditory cortex
acetonitrile
centimeters
corticotropin-releasing factor
decibel
difference score
difference score of correct trials
Ethylenediaminetetraacetic acid
grams
gamma-Aminobutyric acid
hour
hypothalamus-pituitary-adrenal
high performance liquid chromatographer
Internet Protocol address
inhibitory postsynaptic currents
kilograms
kilohertz
meter
molar
milligrams
minutes
milliliters
millimolar
millivolts
microlitres
normal
nanoampere
Sodium phosphate monobasic
norepinephrine
nanograms
nanometers
picograms
seconds.