Introduction

Insomnia or lack of sleep results in a wide range of behavioral and cognitive deficits [1, 2], out of which anxiety and memory problems are two major manifestations [3]. Patients with co-morbid anxiety and insomnia have a poor quality of life [4] and those with high anxiety have increased sleep disturbance [5]. Anxiety-like traits are also observed in animal models of sleep deprivation (SD) [6, 7]. In addition, the importance of sleep in learning and memory has been shown in several studies [8, 9]. Though it is extremely difficult to have an ideal animal model for insomnia, sleep deprivation (SD) procedure in rats can be considered as an acceptable procedure for studying experimentally the various aspects of insomnia [10, 11].

Insomnia-associated anxiety and cognitive deficits is associated with increased oxidative stress in several regions of brain [6, 12, 13]. Sleep loss results in an increased metabolism in brain which in turn facilitates oxidative stress [14]. Removal of free radicals and restoration and conservation of energy are considered as important functions of sleep [14].

Widely used hypnotic drugs like benzodiazepines, non-benzodiazepines and their analogs [15] do reduce anxiety that is associated with insomnia [16]. However, chronic usage of these hypnotics brings about a dose-dependent impairment in the acquisition of new information and impairment of long-term episodic memory [17]. Increased risk of Alzheimer’s disease and dementia in old age has been shown to be associated with chronic intake of these hypnotics [18]. In addition, hypnotic drugs produce several undesirable side effects [19] and are notable for producing amnesia and hallucinations, especially when used in large doses [20]. Consequently, efforts are on to find some sedative substances from the herbs that are commonly suggested for treatment of insomnia and associated problems in traditional medicines [21].

α-Asarone, an active principle extracted from the Acorus species, has hypnotic property at low dose (10 mg/kg), with no withdrawal effect in rats. At this dose, it improved the quality of sleep even under SD conditions [22]. α-Asarone has also been reported to have anxiolytic and cognition enhancing properties in various animal models [23,24,25,26,27,28,29]. In rodents, α-asarone reversed the cognitive deficits induced by scopolamine [24], lipopolysaccaride [25] and amyloid-β (25–35) [26] and reduced anxiety under normal conditions [27] and after corticosterone administration [28]. However, the effect of α-asarone on changes in anxiety and cognition associated with SD is not well-studied. Therefore, the present study was undertaken to investigate the effect of α-asarone on anxiety and spatial memory deficits in rats subjected to SD.

α-Asarone produces a reduction in the oxidative stress and an improvement in the antioxidant levels in the brain [24, 26, 29]. This can probably reverse anxiety and cognitive deficits produced by SD. Antioxidant levels in the cortex, subcortex (including the thalamus, hypothalamus, limbic structures and basal fore-brain) and brainstem (including the midbrain, pons and medulla) were studied to have a gross idea about the magnitude of change, and extend of areas involved in their actions [30]. Hypnotic effect of 10 mg/kg α-asarone was observed after 5 h of SD for 5 consecutive days [22]. So the antioxidant status in brain was studied after 5 h of SD for 5 consecutive days.

Materials and methods

Animals

A total of 60 adult male Wistar rats (14–16 weeks; 250–350 g) housed in polystyrene cages, kept in controlled temperature (26 ± 1 °C) and light–dark schedule of 12 h each (lights on at 6:00 h), with food and water provided ad libitum, were used in the study. All the procedures employed in this study were approved by the Institutional Animal Ethics Committee of the Sree Chitra Tirunal Institute for Medical Sciences and Technology, Trivandrum, Kerala.

Study design

Elevated plus maze (EPM) and the open field maze test (OFT) for anxiety testing

After testing baseline anxiety levels, the rats (N = 20) were randomly distributed into three groups. First group received vehicle (N = 7), second (N = 8) received 10 mg/kg α-asarone and the third group (N = 5) received 2 mg/kg midazolam (positive control) intra-peritoneally at 9:00 h for 5 days, before 5 h of SD. EPM test was conducted on days 1 and 4, and OFT was performed on days 2 and 5 from 13:30 to 14:00 h (Fig. 1a).

Fig. 1
figure 1

Diagrammatic representation of the experimental schedule. a EPM and OFT were performed on different days, before sleep deprivation. After a gap of 7–10 days rats were sleep deprived for 5 h for 5 days (D1–D5). On days D1 and D4 EPM test and on days D2 and D5 OFT were performed. b Rats were prepared for RAM test by restricting their food intake so that their body weight was maintained at 85–90% of the original. They were then trained in the radial arm maze for 2–3 weeks. On completion of training, a baseline memory test was conducted. After that they were sleep deprived for 5 h for 5 days (D1–D5). On days D1 and D5 RAM tests were performed

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Radial arm maze (RAM) test for spatial memory

Spatial memory was tested using RAM in another set of rats (N = 12) that underwent training and food restriction. On completion of training, the baseline memory levels were checked and all the animals (N = 10) were randomly divided into two groups (Fig. 1b) The animals (N = 2) which were not complying with the test requirement during the training sessions were excluded. The first group received vehicle (N = 5) and the second group received 10 mg/kg α-asarone (N = 5) intra-peritoneally at 9:00 h for 5 days before starting the SD procedure for 5 h. The RAM test was conducted from 13:30 to 14:00 h on days 1 and 5 of SD (Fig. 1b).

Antioxidant analysis

The animals were randomly distributed into five groups. Group I (N = 7) was taken as control without any treatment. Group II (N = 6) and III (N = 7) received vehicle and 10 mg/kg α-asarone, respectively, at 9:00 h for 1 day before starting the SD procedure for 5 h. Group IV (N = 5) and V (N = 5) received vehicle and 10 mg/kg α-asarone, respectively, at 9:00 h for 5 consecutive days before starting the SD procedure for 5 h. Animals in Group II and III were decapitated after 5 h of SD and animals in Group IV and V were decapitated after 5 days of SD using guillotine at 14:00 h. Different brain regions (Cortex, Subcortex and Brainstem) were dissected out in ice-cold saline and stored at − 80 °C for oxidative stress markers analysis.

Drug administration

α-Asarone (trans-1,2,4-trimethoxy-5-(1-propenyl) benzene) and Tween80 (Polyoxyethylene (20) sorbitan monooleate) were obtained from Sigma-Aldrich Co. LLC. and midazolam was procured from the Neon Laboratories Ltd. α-Asarone was freshly dissolved in the base containing normal saline and 5% Tween80. The base was taken as the vehicle.

Procedures and parameters measured

Sleep deprivation

All the animals were sleep deprived for 5 h (9:00–14:00 h), by gentle handling for 5 days [22]. The rats were kept awake by introducing new objects into the cage, tapping the cage and mildly touching the animal with soft bristled brush. The method of gentle handling used for SD does not involve forced locomotion, and it gives minimum stress to the animals [31].

Anxiety tests

The animals were tested for anxiety in EPM and OFT using ANY-maze video-tracking system (version 4.82) from Stoelting Co. (U.S.A.). The plexiglass EPM having two open arms (50 × 10 cm) and two closed arms (50 × 10 × 50 cm), arranged in plus shape, was kept at a height of 45 cm. The arms of same type faced each other and were connected through an open center zone (10 × 10 cm). Experiment was conducted for 5 min. The parameters such as entries to open arm and the closed arm, average speed, time spent in the open arm and the closed arm, total distance traveled, time spent in mobility and ethologically derived parameters like head dipping, rearing, grooming and stretch-attend posture were assessed [22].

The plexiglass OFT maze chamber of dimension 100 × 100 × 40 cm was divided into three concentric zones, namely, outer, middle and inner zone. Experiment was conducted for 5 min. Entries and time spent in the inner and outer zones was noted. Parameters including average speed, time spent in moving around, total distance traveled and ethologically derived parameters like rearing and grooming were also measured.

Spatial memory test

RAM apparatus had eight black painted radial arms (45 × 10 × 10 cm), placed at a height of 35 cm above the floor, in a room with fixed extra-maze visual cues. Food was placed at the end of each arm. The signal acquisition was done using ANY-Maze video-tracking system (version 4.82) from Stoelting Co. (USA) and scored manually.

Rats were subjected to food restriction (10 g/day/rat) to maintain their body weights at 85–90% of free-feeding values for the first 10 days. Food restriction continued throughout the training period with free access to water. Animals were trained to consume the food rewards (Kellogg’s Chocos flakes, ~ 20 mg) from four arms randomly chosen as baited arms within a stipulated time of 10 min. The baited arms remained the same for a given rat, but varied between rats. RAM training took place between 10:00 and 16:00 h daily and was continued till all the animals learned to respond to the food rewards with minimal errors. The rats underwent test for their spatial memory skills on days 1 and 5 immediately after SD.

Entries into un-baited arms (reference memory errors), re-entries into baited arms (working memory errors) and percentage of correct choices were recorded on every trial [32].

Biochemical estimations

Tissue homogenates (10% w/v) were prepared in ice-cold 0.1 M phosphate buffer (pH 7.4) in a motor homogenizer and centrifuged at 10,000 rpm for 10 min at 4 °C. The supernatants were used for the biochemical analysis. The level of lipid peroxidation was measured by quantifying malondialdehyde (MDA), a thiobarbituric acid (TBA) reacting substance (TBARs), according to the method of Buege and Aust [33]. CAT activity was assayed by measuring the decomposition of H2O2 [34]. GSH-R activity was determined following the procedures of Smith et al. [35] by measuring the disappearance of NADPH. SOD activity was estimated by the method of Marklund and Marklund [36]. The degree of inhibition of autoxidation of pyrogallol at an alkaline pH by SOD was used as a measure of the enzyme activity. GSH-Px activity was determined according to the method of Rotruck et al. [37]. Protein content in the tissue was determined according to the method of Lowry et al. [38].

Statistical analysis

Assuming a 5% significance level and a two-sided test, a sample size of seven per group was obtained with an S/N ratio of 1.6 and a power of 80%. In the RAM test, based on their poor performance during training, four animals were excluded from the study. Shapiro–Wilk test was done to check the normality of the data. All values expressed as mean ± SEM. p ≤ 0.05 were considered as statistically significant. All statistical analyses were done in SPSS (version 16.0). For EPM and OFT parameters, Kruskal–Wallis test was done to compare among the groups and Wilcoxon signed rank test was used for comparison with the baseline. For RAM test parameters, Mann–Whitney test was used to make comparison between the groups and Wilcoxon signed rank test was used for comparison with the baseline. Various parameters were analyzed using one-way ANOVA with Tukey’s post hoc test.

Results

Effects of administration of α-asarone on the anxiety levels in EPM test

The data from the vehicle-treated group showed that the time spent by the rats in the open arm was significantly reduced (p = 0.02) after SD. The entries into the open arms were also markedly reduced (p = 0.01) after 4 days of SD in those rats that received vehicle treatment. The total distance traveled and the average speed on the maze was also reduced (p = 0.03) in these sleep-deprived rats (Fig. 2; Table 1).

Fig. 2
figure 2

Changes in EPM parameters after administration of vehicle, α-asarone and midazolam in sleep-deprived rats. Changes in the parameters in EPM test show decreased levels of anxiety in those rats that received vehicle, α-asarone and midazolam before SD. The box plot shows the a total distance traveled, b average speed, c the entries into the open arms and d the time spent in the open arms on day 4 of SD as compared to the baseline values (taken as 100%) taken before SD. *Indicates significant change from vehicle group and # indicates significance from the baseline. Levels of significance *, #p ≤ 0.05 and **p ≤ 0.01. N = 7 for vehicle group, N = 8 for α-asarone group and N = 5 for midazolam group

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Table 1 Values of all parameters of EPM after administration of α-asarone, midazolam and vehicle in SD rats
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On the other hand, the time spent in the open arm after SD was increased by day 4 (p = 0.008) in the α-asarone-treated rats (Fig. 2; Table 1). Entry in to the open arm was also improved (p = 0.04) by day 4 (Fig. 2; Table 1). Furthermore, the distance traveled on EPM and the average speed of mobility was also improved (p = 0.01) in α-asarone group in comparison to the vehicle group (Fig. 2; Table 1). Though there was difference in the magnitude of change, effects of α-asarone on most of the parameters were similar to that of midazolam in the sleep-deprived rats (Fig. 2; Table 1).

Effect of α-asarone on the anxiety levels in OFT

After 5 days of SD, vehicle-treated rats made significantly decreased entries (p = 0.04) and time spent (p = 0.03) into the inner zone (Fig. 3; Table 2). Along with decreased time in the inner zone, the vehicle-treated rats had spent increased (p = 0.03) time in the outer zone after 5 days of SD. But the α-asarone-treated rats entered the open arm more frequently (p = 0.04) even after 5 days of SD (Fig. 3). After 5 days of SD, there was reduction in mobility on maze in both vehicle- and α-asarone-treated rats (Table 2). Even though not statistically significant, α-asarone group spent more time in the inner zone than the midazolam group (Table 2).

Fig. 3
figure 3

Changes in OFT parameters after administration of vehicle, α-asarone and midazolam in sleep-deprived rats. Changes in the parameters in OFT shows decreased levels of anxiety in those rats that received vehicle, α-asarone and midazolam before SD. The box plot shows a the time spent in the outer zone, b entries in to inner zone and c the time spent in the inner zone on day 5 of SD. Effect of 5 days SD is compared to the baseline values (taken as 100%) before SD. *Indicates significant change from vehicle group and #indicates significance from the baseline. Levels of significance *, #p ≤ 0.05. N = 7 for vehicle group, N = 8 for α-asarone group and N = 5 for midazolam group

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Table 2 Values of all parameters of OFT after administration of α-Asarone, midazolam and vehicle in SD rats
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Effects of administration of α-asarone on spatial memory in RAM test

As indicated in Fig. 4 and Table 3, the percentage of correct choices was significantly higher and working memory and reference memory errors were significantly lower in the α-asarone-treated rats when compared to the vehicle-treated group. The correct choices made by vehicle group were reduced (p = 0.02) after 5 days of SD (Fig. 4a; Table 3). Number of entries into the un-baited arm (reference memory errors) was significantly higher (p = 0.03) in the vehicle group when compared to the α-asarone group by day 5 of SD (Fig. 4b; Table 3). The number of re-entries into the baited arm, indicating working memory error, was also significantly higher in the vehicle-treated group, as compared to the drug-treated group, by day 5 of SD (Fig. 4b; Table 3).

Fig. 4
figure 4

Changes in RAM parameters after administration of α-asarone and vehicle in sleep-deprived rats. Changes in the parameters in RAM test show increased spatial memory in those rats that received α-asarone before SD. The graph shows a the percentage of correct choices made and b reference memory (RME) and working memory (WME) errors on day 5 of SD compared to the baseline values (taken as 100%) taken before SD. *Indicates significance of the differences between vehicle and α-asarone group and #indicates significance from the baseline. Levels of significance *, #p < 0.05 and **p < 0.01. N = 5 for each group

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Table 3 Values of all parameters of RAM after administration of α-asarone and vehicle in SD rats
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Effects of administration of α-asarone on the antioxidant levels in cortex, subcortex and brainstem

Table 4 summarizes the changes in antioxidant levels in vehicle- and α-asarone-treated SD rats. The MDA levels in the brainstem and subcortex lowered after 5 days of SD (Table 4). CAT activity was also significantly decreased in the cortex and subcortex in vehicle group after 5 days of SD in comparison to the control group (Table 4). Increase in the activities of SOD, GSH-Px and GSH-R was also observed after 5 days of SD (Table 4).

Table 4 Effect of acute SD by gentle handling on antioxidant levels after administration of vehicle and α-Asarone
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In comparison to the vehicle group, α-asarone reduced (p = 0.02) the MDA levels in the subcortex and brainstem after 5 days of SD (Table 4). CAT activity in the cortex and subcortex increased (p = 0.03) on day 5 in SD rats when treated with α-asarone (Table 4). Subcortical region also showed increased GSH-R activity (p = 0.05) in the α-asarone-treated rats after 5 days of SD.

Discussion

Increased anxiety levels and errors in reference and working memory in sleep-deprived rats were alleviated by α-asarone administration. This was associated with decreased MDA level and increased activities of CAT and GSH-R.

Changes in anxiety and cognition produced by SD

Increased anxiety after SD observed in the present study is supported by an earlier report and contradicted by few other reports [39,40,41]. Cohen et al. [39] reported that SD by gentle handling for 6 h induced anxiety in rats. On the contrary, the same method was found to reduce anxiety and increase risk-taking behavior in EPM test [40] and produced no anxiety in the OFT [41]. It is noted that this is the first report on the effect of repeated SD by gentle handling on anxiety. Repeated SD by other methods, however, has shown to increase anxiety [7, 13].

Cognitive decline, observed in the present study, as a result of SD is supported by earlier reports [42, 43]. Impairment in the spatial reference memory was observed in rats subjected to 6 h of SD by gentle handling [42]. Furthermore, mice subjected to 3 h of SD for 30 days by gentle handling, showed significantly impaired spatial learning and spatial memory (reference and working) retention [43].

Anxiolytic and cognitive enhancing effect of α-asarone

The animals treated with 10 mg/kg of α-asarone were less anxious when compared to their vehicle counterpart after being subjected to SD. Moreover, the anxiolysis of α-asarone was on par with the commonly used anxiolytic midazolam [44, 45]. This indicates that α-asarone at a dose of 10 mg/kg is not just an effective hypnotic [22], but also a potent anxiolytic to alleviate anxiety associated with SD.

In the present study, α-asarone improved reference as well as working memory and enhanced the performance of rats on RAM. This signifies the efficacy of α-asarone in alleviating the cognitive decline associated with SD and thus, may be considered as a better alternative for the management of insomnia since the commonly used hypnotics produces cognitive deficits [17, 18].

Effects of administration of α-asarone on the antioxidant levels

In the present study, oxidative stress level in cortex, subcortex and brainstem was altered in the rats subjected to SD. Even though SD for 5 h produced moderate oxidative stress, by day 5 of SD, the lipid peroxidation was considerably lowered along with an increase in the GSH-R and GSH-Px activities. This might be a cellular adaptive response to recoup from the acute stress associated with SD as previously reported [46, 47]. It is noted that the brain is capable of responding to the stress associated with acute sleep loss and thereby preventing oxidative stress [48].

On the other hand, the cellular adaptive response after 5 days of SD was not evident in the behavior of the animals. They showed an increased anxiety and reduced spatial memory after 5 days of SD. This might be due to the reduction in the activity of CAT especially in the subcortical region along with increase in the SOD activity. Catalase over-expression is reported to be sufficient to enhance cognition and reduce anxiety even in the absence of alteration in levels of oxidative stress [49]. Furthermore, in the present study, both subcortex and cortex were affected the most by SD associated oxidative stress which in turn would have lead to cognitive decline and anxiety. Previously, a systematic review reported that the brain areas, namely, cortex, hypothalamus, hippocampus, thalamus and amygdala are found to be more vulnerable to oxidative stress [50].

The anxiolytic and cognitive enhancing effect of α-asarone in the present study may be partly due to its antioxidant property. The present study indicated that the oxidative cellular damage induced by sleep loss was alleviated moderately by α-asarone treatment which reduced the MDA levels and increased the activity of the antioxidants CAT and GSH-R in the subcortex and the brainstem regions. It is further noted that the effect of α-asarone in the present study was more drastic in the subcortical region which probably reflected in the anxiety and the cognitive testing in the rats subjected to SD. In various other models, improvement in the cognitive functions after α-asarone administration was associated with an increased antioxidant status, mainly in the subcortical regions like hippocampus and striatum, and to a lesser extent in the cortex [24, 26, 29, 51]. It is also emphasized that the effect of α-asarone on the antioxidant status of brain may be dose dependent and other doses might probably showcase an entirely different pattern of antioxidant activity.

Improvement in sleep quality by α-asarone treatment is also associated with lowering of hyperthermia caused by 5 h SD for 5 days [22]. Hyperthermia is a leading cause to anxiety and cognitive impairment [52, 53] and is linked to increased oxidative stress [54]. So the antioxidant effect observed in the present study, along with mild temperature lowering effect of α-asarone might have facilitated sleep, anxiolysis and enhancement of cognition.

It is not possible to have an ideal animal model for insomnia, but the SD procedure done in this study can be considered acceptable for studying insomnia [10, 11]. All the SD-induced changes in sleep like increased latency to sleep (difficulty in falling asleep), sleep fragmentation/ increased arousal index (frequent awakenings) and reduced sleep quality [22], satisfy the clinical guidelines laid down as criteria for chronic insomnia [10]. Moreover, the SD procedure used in the present study (gentle handling) is reported to be a valid animal model of insomnia with many overlapping pathologies [11]. So it is reasonable to expect similar results in patients with insomnia.

Conclusion

α-Asarone 10 mg/kg reduced the SD-induced anxiety and memory deficit with reduced oxidative stress in the subcortex. This study provided evidences to indicate that the hypnotic dose of α-asarone (10 mg/kg) can effectively manage anxiety associated with sleep loss and could also assist in counteracting the associative cognitive deficits under such conditions. Improved antioxidant level especially CAT activity might have facilitated the improvement in the cognitive functioning. This is the first report to show that the hypnotic α-asarone may be a potential therapeutic agent in the management of insomnia-associated changes in anxiety and memory.