Rapid eye movement (REM) sleep has an essential role in the process of learning and memory in the hippocampus. It has been reported that linalool, a major component of
Insomnia is a common problem and results from a high work load, shift work and various other tasks imposed by modern society. In our society, insomnia has come to be known as a cause of many chronic illnesses that can significantly impair functioning and have a negative influence on ones quality of life (Huang
Linalool, a monoterpene, is the major component of
Four-week-old male ICR mice (weighing 18–22 g) were purchased from the OrientBio (Seongnam, Korea), and housed in a conventional animal facility with free access to food and water in a temperature and relative humidity monitored and controlled environment under artificial lighting (12 h of light per day). Animals were allowed to acclimatize for at least 7 days before the experiments were performed. All animal related study protocols were conducted in accordance with the guidelines of the National Institutes of Health guide for the care and use of Laboratory animals (NIH publication No. 8023, revised 1978), and were approved by the Committee on Animal Research at Ajou Medical Center, Ajou University.
In ICR mice, REM-sleep deprivation was induced using the modified multiple platform method, as described previously (Silva
Animals were divided between a total of 10 groups, each comprising of 6–8 animals. Five groups were examined using the Y maze and forced swimming test (FST), the remaining five groups were examined using the passive avoidance test. Fig. 1 displays an overview of the experiments. Each group was either exposed to 72 h sleep deprivation using the multiple platform method or they remained in their home cages and acted as controls. After 30 min of 0.5% tween80-saline or linalool (0.3, 1 or 3 mg/kg, Sigma, St. Louis, MO, USA) administration, behavioral tests were performed on the day following the 72 h sleep deprivation. All behavioral tests were conducted between 10:00 and 17:00.
Spontaneous alternation behavior was examined using the Y maze test. This test is performed in a horizontal maze (30-cm long and 5-cm wide, with 12-cm high walls) with three arms (labeled A, B, and C). The maze floor and walls are constructed of black acrylic. Mice are initially placed within one arm, and the number of alternations (i.e., consecutive entry sequences of ABC, CAB, or BCA but not BAB) and the number of arm entries are manually recorded for each mouse over an 8-min period. In our experiment, 30 min before each test, the mice were given linalool (0.3, 1, 3 mg/kg, through intraperitoneal injection) or saline. The control group received 0.5% tween80-saline instead of linalool. The percentage alternation was calculated according to the following equation: Percentage alternation=[(Number of alternations)/(Total arm entries-2)]×100. The number of arm entries per trial was used as an indicator of locomotor activity. The Y maze arms were cleaned with 10% ethanol between tests to remove odors and residues.
The passive avoidance test was performed essentially as has been described by Silva and colleagues (Silva
In the FST, mice were individually forced to swim in an open cylindrical container (10×25 cm), containing water at a temperature of 23 ± 1°C and a depth of 15 cm so that they could not escape or touch the bottom. Each mouse was gently placed in the cylinder and the total duration of floating was recorded during a 6-min period. The immobility time was measured during the last 4 min of the test. Each mouse was judged to be immobile when it ceased struggling and maintained motionless floating in the water, making only those movements necessary to keep its head above water.
After performing the behavioral tests, mice were anaesthetized, and blood was collected from the abdominal vein using a 1 mL syringe, to include 60 μL 3.8% sodium citrate. Following this, plasma was clarified by centrifugation at 20°C for 10 min and stored at −80°C until experimentation. Plasma cortisol, adrenocorticotropic hormone (ACTH) and serotonin concentrations were analyzed using Mybiosource ELISA kits (San Diego, CA, USA). Absorbance was read using a Bio-Tek Synergy HT plate reader (Bio-Tek Instruments Inc., Winooski, VT, USA).
Following linalool administration and linalool withdrawal, mice were sacrificed by decapitation immediately after the behavioral tests, and the hippocampi were dissected from the brain and stored in liquid nitrogen for the determination of serotonin levels. Serotonin levels were measured with Enzo Life Sciences ELISA kits (Farmingdale, NY, USA). The hippocampal samples were weighed and 300 μL lysis buffer was added. The samples were homogenized for 15 sec and centrifuged at 4°C for 20 min. The supernatant was stored at −20°C until analysis. Absorbance was read using a Bio-Tek Synergy HT plate reader (Bio-Tek Instruments Inc.).
Data are expressed as mean ± standard error of at least three separate determinations in each group. Numerical data were compare using Student’s
Spontaneous alteration behavior in the Y maze is used to measure spatial and working memory (Kwon
To explore the anti-stress and antidepressant activity of linalool, we used the FST. The FST is a widely used behavioral test for the evaluation of potential anti-stress and antidepressant activity in rodents (de la Pena
As shown in Fig. 4A, the plasma cortisol levels in the sleep-deprived group (veh) were significantly higher than those of the control group (ctl). After 72 h REM-sleep deprivation and linalool treatment (1 or 3 mg/kg), the cortisol levels were significantly lower than those of the untreated group. Plasma ACTH levels in REM-sleep-deprived mice were not significantly different between the linalool treated group and the untreated group (Fig. 4B).
It has been reported that stress increases the serotonin turnover in animals and increases the serotonin levels in the hippocampus. As shown in Fig. 5A, hippocampal serotonin levels in the sleep-deprived group (veh) were significantly higher than in the control group. Following the administration of 3 mg/kg linalool, the serotonin levels were significantly higher than in the vehicle groups during REM-sleep deprivation. However, treatment with 0.3 or 1 mg/kg linalool produced no change in the serotonin levels compared to the untreated group. These results suggest that the plasma serotonin level is significantly reduced in REM-sleep-deprived mice, and that this reduction is inhibited by linalool treatment at 3 mg/kg (Fig. 5B).
In the present study, we demonstrated that linalool may protect mice from REM-sleep deprivation-induced stress, as demonstrated by a reduction in the behavioral impairment accompanied by memory loss. These anti-stress effects of linalool can be further substantiated by the observed decrease in cortisol levels and changes in serotonin levels.
REM-sleep is a mentally active phase of sleep. During REM-sleep, our bodies function in many ways that are similar to when we are awake. Our minds are active, with increased respiration and heart rate with more variability than is observed in other sleep stages. REM-sleep is thought to be essential for memory formation and storage, while most of our muscles shut down, severely restricting movement during this phase. REM-sleep deprivation is known to increase basal arousal and affect physiological and psychological processes as well as neurotransmitter levels, but the mechanism through which these effects are caused is not fully understood. During the initial stages of REM-sleep deprivation, plasma corticosterone levels increase, indicating that sleep deprivation results in stress on the process of maintaining body homeostasis (Mathangi
It may be assumed that the mechanism of action of linalool in improving the depression that is caused by stress during REM-sleep deprivation may involve the serotonergic pathway, because the levels of hippocampal serotonin appear to be altered by linalool. The relationship between stress and serotonin has been studied previously. It has been reported that stress increases serotonin turnover in the hypothalamus, the tonsils, and the hippocampus in rats. Additionally, serotonin-induced hyperglycemia is associated with a decrease in serotonin levels in stress patients (Dunn and Welch, 1991). Furthermore, the role of serotonin in mental disorders, such as depression, is well known. Notably, it has been suggested that serotonin may be directly or indirectly involved in depression-induced behavioral impairment, as SSRIs (selective serotonin reuptake inhibitors) are known to be protective against depression. As displayed in Fig. 5, we found that REM-sleep deprivation induced an increase in hippocampal serotonin levels, indicating that sleep deprivation-induced stress led to increased serotonin turnover, resulting in a temporary increase in serotonin levels. In addition, linalool treatment led to higher serotonin levels than treatment with a vehicle. Increased serotonin levels may have a positive effect in the short term, while maintaining a high level of serotonin in the hippocampus for a long period without a rapid decrease may have other effects. Furthermore, in our results, linalool attenuated the reduction of plasma serotonin levels that were induced by REM-sleep deprivation (Fig. 5B), consistent with previous reports that higher plasma serotonin levels lead to a better mood (Williams
We first reported that linalool improves the behavioral impairments caused by memory loss following REM-sleep deprivation. It is possible that linalool has the ability to ameliorate the behavioral impairments of REM-sleep deprivation via the serotonergic system. Serotonin has not only been related to the inhibition of sleep and the promotion of wakefulness, but also plays a role in the initiation and maintenance of sleep (Zhang
The authors declare that there are no conflicts of interest.
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (Grant number: 2015R1D1A1A01060069 and 2017R1D1A1B03028743) and by the Commercialization Promotion Agency for R&D Outcomes (COMPA) funded by the Ministry of Science and ICT (MSIT) (Development of sleep-improving functional food with fermented extract of Perilla frutescens Britton var. acuta Kudo).