Effects of the 5-HT_1A Receptor Agonist Tandospirone on ACTH-Induced Sleep Disturbance in Rats

Abstract

The aim of this study was to compare the effect of the serotonin (5-HT)_1A receptor agonist tandospirone versus that of the benzodiazepine hypnotic flunitrazepam in a rat model of long-term adrenocorticotropic hormone (ACTH)-induced sleep disturbance. Rats implanted with electrodes for recording electroencephalogram and electromyogram were injected with ACTH once daily at a dose of 100 µg/rat. Administration of ACTH for 10 d caused a significant increase in sleep latency, decrease in non-rapid eye movement (non-REM) sleep time, and increase in wake time. Tandospirone caused a significant decrease in sleep latency and increase in non-REM sleep time in rats treated with ACTH. The effect of tandospirone on sleep patterns was antagonized by the 5-HT_1A receptor antagonist WAY-100635. In contrast, flunitrazepam had no significant effect on sleep parameters in ACTH-treated rats. These results clearly indicate that long-term administration of ACTH causes sleep disturbance, and stimulating the 5-HT_1A receptor by tandospirone may be efficacious for improving sleep in cases in which benzodiazepine hypnotics are ineffective.

Eighty to ninety percent of patients with major depression have disturbed sleep patterns,¹⁾ and it is widely accepted that sleep disturbance may be a risk factor for and/or a prodromal symptom of depression.^2–4) Furthermore, disturbed sleep is characteristic of individuals undergoing treatment for depression, as well as those exhibiting a relapse of depressive symptoms.³⁾ Therefore the treatment of sleep disturbance is likely important in preventing the development of depression.

Benzodiazepines and their analogues are commonly effective in the clinical treatment of sleep disturbances via their effects on the gamma-aminobutyric acid (GABA)-ergic system. The GABAergic system plays an important role in the control of the sleep–wake cycle; however, sensitivity to benzodiazepines has been shown decreased in depressed patients with disturbed sleep.⁵⁾ Therefore it is conceivable that sleep patterns in these individuals are influenced by non-GABAergic systems. For example, abnormal functioning of the serotonergic system was proposed to be closely associated with depression and sleep disturbances.^6,7) Furthermore, serotonin (5-HT)_1A receptor agonists have been reported efficacious against depression; however, only a limited number of studies have supported the efficacy of 5-HT_1A receptor agonists for improving sleep disturbance in depression in humans and animals.⁸⁾

The hypothalamic-pituitary-adrenocortical (HPA) axis, which is activated in response to stress, may be hyperfunctional in patients with depression.⁹⁾ Kitamura et al.¹⁰⁾ reported an overactive HPA axis in rats chronically administered adrenocorticotropic hormone (ACTH) and indicated that this model may be useful for studying depression and tricyclic antidepressant treatment-resistant conditions. Although these rats have been demonstrated valuable as tricyclic antidepressant treatment-resistant animals in the forced swim test, whether they are also useful as a model of sleep disturbance has not yet been investigated.

In the present study, we looked at sleep parameters in rats administered ACTH. We also compared the effect of the 5-HT_1A receptor agonist tandospirone with that of the benzodiazepine hypnotic flunitrazepam on sleep patterns.

MATERIALS AND METHODS

Animals

Male Wistar rats weighing 240–260 g (Japan SLC, Shizuoka, Japan) were maintained in an air-conditioned room with controlled temperature (24±2°C) and humidity (55±15%) under a light–dark cycle (lights on from 07 : 00 to 19:00). The rats were housed in plastic cages with sawdust and allowed free access to food and water. All procedures involving these animals were conducted in accordance with the Guidelines for Animal Experiments at Okayama University Advanced Science Research Center.

Surgery

Rats were anesthetized with pentobarbital sodium (Nembutal, 35 mg/kg intraperitoneally (i.p.); Abbott Laboratories, North Chicago, IL, U.S.A.) and fixed to a stereotaxic apparatus (SR-5N; Narishige, Tokyo, Japan). To record electroencephalogram (EEG), a stainless steel screw electrode (diameter, 0.5 mm; depth, 1.7 mm) was chronically implanted into the cortex (A, −4.5; L, +2.5), according to coordinates from the atlas of Paxinos and Watson.¹¹⁾ A stainless steel screw fixed in the left frontal bone served as a reference electrode. To record electromyogram (EMG), stainless steel wire electrodes (0.2 mm) were implanted into the dorsal neck muscle. These electrodes were connected to a miniature receptacle, and the entire assembly was fixed to the skull with dental cement. At least 7 d was allowed for recovery from the surgery.

Animal Model Development

ACTH (Cortrosyn-Z; Daiichi-Sankyo & Co., Ltd., Japan) was subcutaneously administered once daily for 1, 5, or 10 d, at a dose of 100 µg/rat in a 0.2-mL injection volume.

EEG and EMG Recordings

EEG and EMG were recorded with an electroencephalograph (Model EEG 4314; Nihon Kohden, Tokyo, Japan) from 7:00 to 19:00. Recordings were conducted as described previously.^12,13) The signals were amplified and filtered (EEG, 0.5–30 Hz; EMG, 16–60 Hz), then digitized at a sampling rate of 128 Hz, and recorded by SleepSign ver. 2.0 (Kissei Comtec, Nagano, Japan) data acquisition program. Recordings were made in a cylindrical plastic cage (diameter, 26 cm; height, 31 cm) placed on sawdust with food and water. The observation cage was placed in a soundproof and electrically shielded box (70×60×60 cm).

Sleep–Wake State Analysis

Sleep–wake states were automatically classified in 10-s epochs as wake, non-rapid eye movement (non-REM) sleep, or rapid eye movement (REM) sleep by SleepSign ver. 2.0, according to criteria described previously.^14,15) As a final step, defined sleep–wake stages were examined visually and corrected, if necessary. Each state was characterized as follows: wake, low-amplitude EEG and high-voltage EMG activities; non-REM sleep, high-amplitude slow or spindle EEG and low-EMG activities; and REM sleep, low-voltage EEG and EMG activities. Sleep latency was defined as the time from drug administration until the first 12 consecutive 10-s epochs of sleep.

Drugs

The following drugs were used: tandospirone citrate (Sediel; Dainippon Sumitomo Pharma Co., Ltd., Osaka, Japan); flunitrazepam (Rohypnol; Chugai Pharmaceutical Co., Ltd., Tokyo, Japan); and WAY-100635 (Sigma, St. Louis, MO, U.S.A.). Tandospirone and flunitrazepam were suspended in a 0.5% carboxymethyl cellulose solution and administered orally. WAY-100635 was dissolved in saline and administered intraperitoneally. All drugs were administered at 7:00, and EEG and EMG were measured for 12 h after drug administration. Eight rats were used in each group, and a counterbalanced design was used for drug dosing.

Data Analysis and Statistics

Values shown are mean±S.E.M. A one-way ANOVA with Dunnett’s test was used to estimate the effects of each drug. Probability values <0.05 were considered significant.

RESULTS

Changes of Sleep Patterns in ACTH-Treated Rats

Rats were administered ACTH for 10 d, and EEG and EMG were measured prior to and on days 1, 5, and 10 following treatment. Administration of ACTH for 10 d resulted in a significantly longer sleep latency and wake time (Figs. 1A, C), and shorter non-REM sleep time (Fig. 1B) compared with pretreatment values. ACTH decreased REM sleep time, but not significantly (Fig. 1D). We also examined the hourly effects of ACTH on non-REM sleep time over 12 h. ACTH caused significant decreases in non-REM sleep time on day 10 for 7 to 10 h and 16 to 18 h (Fig. 1E).

Fig. 1. Variations in Sleep–Wake Cycles in ACTH-Treated Rats

Sleep latency (A), non-REM sleep time (B), wake time (C), and REM sleep time (D) were measured in rats before (open column) and 1 d (light gray column), 5 d (dark gray column), and 10 d (filled column) after administration of ACTH. Columns and vertical bars represent mean±S.E.M. (n=8). **Significantly different versus before administration of ACTH (p<0.01). (E) Effects of ACTH on the time–course of non-REM sleep time in rats measured before (open circles) and on day 10 (filled circles) following administration of ACTH. Circles and vertical bars represent mean±S.E.M. (n=8). Significantly different versus before administration of ACTH: * p<0.05; ** p<0.01.

Effect of Tandospirone on Sleep Parameters in Rats Treated with ACTH

We used rats treated with ACTH to study the effect of tandospirone on sleep parameters. Tandospirone caused significant decreases in sleep latency at 10, 20, and 50 mg/kg (Fig. 2A). Moreover, this agent caused a significant increase in non-REM sleep time at 50 mg/kg (Fig. 2B). Further analysis revealed that tandospirone caused an increase in non-REM sleep time (20 mg/kg, 7 to 9 h; 50 mg/kg, 7 to 11 h) (Fig. 2C).

Fig. 2. Effect of Orally Administered Tandospirone on Sleep Parameters in ACTH-Treated Rats

The effects of tandospirone on sleep latency (A) and non-REM sleep time (B) were measured in rats treated with ACTH for 10 d: control (open column); 10 mg/kg (light gray column); 20 mg/kg (dark gray column); and 50 mg/kg (filled column). Columns and vertical bars represent mean±S.E.M. (n=8). Significantly different versus control: * p<0.05; ** p<0.01. (C) Effects of tandospirone on the time–course of non-REM sleep time in rats treated with ACTH for 10 d: control (open circles); 10 mg/kg (closed circles); 20 mg/kg (open triangles); and 50 mg/kg (closed triangles). Circles and vertical bars represent mean±S.E.M. (n=8). Significantly different versus control: * p<0.05; ** p<0.01.

Effect of Flunitrazepam on Sleep Parameters in Rats Treated with ACTH

We also investigated the effects of flunitrazepam on sleep parameters in rats treated with ACTH. Flunitrazepam (0.1-3 mg/kg per os (p.o.)) had no significant effect on sleep latency (Fig. 3A) and non-REM sleep time (Fig. 3B). Moreover, flunitrazepam had no significant effect on non-REM sleep time at any time-point (Fig. 3C).

Fig. 3. Effect of Orally Administered Flunitrazepam on Sleep Parameters in ACTH-Treated Rats

The effects of flunitrazepam on sleep latency (A) and non-REM sleep time (B) were measured in rats treated with ACTH for 10 d: control (open column); 0.3 mg/kg (light gray column); 1 mg/kg (dark gray column); and 3 mg/kg (filled column). Columns and vertical bars represent mean±S.E.M. (n=8). (C) Effects of flunitrazepam on the time course of non-REM sleep time in rats treated with ACTH for 10 d: control (open circles); 0.3 mg/kg (closed circles); 1 mg/kg (open triangles); and 3 mg/kg (closed triangles). Circles and vertical bars represent mean±S.E.M. (n=8).

Effect of WAY-100635 on Tandospirone-Induced Improvements in Sleep Disturbance in Rats Treated with ACTH

To investigate the relation between the effect of tandospirone and the 5-HT_1A receptor, we examined the effects of WAY-100635. WAY-100635, at doses of 0.3 and 1 mg/kg i.p., antagonized the hypnotic effect of tandospirone on sleep latency (Fig. 4A) and non-REM sleep time (Fig. 4B). Further investigation revealed that WAY-100635 antagonized the hypnotic effect of tandospirone on hourly non-REM sleep times (0.1 mg/kg, 9 to 11 h; 0.3 mg/kg, 9 to 11 h and 13 to 14 h; 1 mg/kg, 9 to 12 h and 13 to 15 h) (Fig. 4C).

Fig. 4. Effects of WAY-100635 on Tandospirone-Induced Improvements in Sleep Disturbance Induced by ACTH

The effects of WAY-100635 (i.p.) on sleep latency (A) and non-REM sleep time (B) were measured in rats treated with ACTH for 10 d and a single dose of tandospirone (50 mg/kg p.o.): control (open column); 0.1 mg/kg (light gray column); 0.3 mg/kg (dark gray column); and 1 mg/kg (filled column). Columns and vertical bars represent mean±S.E.M. (n=8). Significantly different versus control: * p<0.05; ** p<0.01. (C) Effects of WAY-100635 (i.p.) on the time–course of non-REM sleep time in rats treated with ACTH for 10 d and a single dose of tandospirone (50 mg/kg p.o.): control (open circles); 0.1 mg/kg (closed circles); 0.3 mg/kg (open triangles); and 1 mg/kg (closed triangles). Circles and vertical bars represent mean±S.E.M. (n=8). Significantly different versus control: * p<0.05; ** p<0.01.

Effect of Tandospirone, Flunitrazepam, and WAY-100635 on Sleep Parameters in Normal Rats

We also investigated the effects of tandospirone, flunitrazepam, and WAY-100635 on sleep parameters in normal rats. Tandospirone (50 mg/kg p.o.), flunitrazepam (3 mg/kg p.o.), and WAY-100635 (1 mg/kg i.p.) had no significant effect on sleep latency, wake time, non-REM sleep time, or REM sleep time (data not shown).

DISCUSSION

It has been recognized that sleep disturbance in depressive patients is characterized by an increase in sleep latency and a decrease in non-REM sleep time.^1,16,17) It is also well known that an increase in immobilized time after chronic administration of ACTH in rat swim test is useful as a depression model in human beings.¹⁰⁾ In the present study, a significant increase in sleep latency and decrease in non-REM sleep time were observed following chronic administration of ACTH in rats. From the above findings, it may be hypothesized that ACTH-induced sleep disturbance in rats is similar to depression-related sleep disturbance in human beings.

Next, we examined the effects of flunitrazepam and tandospirone on sleep disturbance in rats treated with ACTH. Flunitrazepam had no significant effect on any of the parameters examined, even at the highest dose tested (3 mg/kg). Contrariwise, Shinomiya et al.¹⁸⁾ reported that flunitrazepam at 3 mg/kg caused a decrease in sleep latency, a decrease in wake time, and an increase in non-REM sleep time in rats whose sleep was disturbed by both water- and altitude-induced stress. Flunitrazepam is a benzodiazepine hypnotic that binds to the GABA_A receptor and enhances the effect of GABA. In recent years, patients with sleep disturbances in conjunction with psychiatric illness, especially depression, have been prescribed multiple and/or high doses of benzodiazepine hypnotics.^19,20) This indicates that sleep disturbance associated with psychiatric diseases exhibits tolerance or resistance to benzodiazepine hypnotics and is related to abnormalities not only with GABAergic neurotransmission, but also with 5-HT, noradrenaline, and dopamine.

Why flunitrazepam had no hypnotic effect on sleep disturbance induced by the long-term administration of ACTH remains unclear; however, we believe that the mechanism may be closely related to upregulation of alpha5-GABA_A receptors. Flunitrazepam has been shown to produce a hypnotic effect by binding to GABA_A receptors, which comprise various alpha, beta, and gamma subunits. It has been reported that the hypnotic effects of benzodiazepines are mediated by the alpha1 subunit. Conversely, alpha5-GABA_A receptors were insensitive and developed tolerance to the sedative actions of benzodiazepines.^21–23) Verkuyl et al.²⁴⁾ reported that expression of alpha5, not alpha1-GABA_A receptors was increased in patients with hyperfunctional HPA axes. Therefore the absence of a flunitrazepam-induced hypnotic effect may be the result of tolerance developed by upregulation of alpha5-GABA_A receptors in ACTH-treated rats.

The 5-HT_1A receptor is related to depression, and 5-HT_1A receptor agonists have been shown to exhibit antidepressant-like effects. Kitamura et al.²⁵⁾ reported that the 5-HT_1A agonist 8-hydroxy-2-di-n-propylamino tetralin (8-OH-DPAT) decreased immobility time for ACTH-treated rats in the forced swim test. Our results suggest that tandospirone caused a significant decrease in sleep latency and an increase in total non-REM sleep time in rats administered ACTH, which were different to those observed with flunitrazepam. This effect was significantly inhibited by the 5-HT_1A receptor antagonist WAY-100635. Therefore tandospirone had a clear, 5-HT_1A-mediated hypnotic effect in this sleep-disturbance model.

Previous studies reported that stimulation of 5-HT_1A receptors produced a sleep-inducing effect in animal models of sleep disturbance whereas tandospirone had no significant effect on non-REM sleep time.^26,27) As shown in the present data, however, tandospirone increased non-REM sleep time in rats treated with ACTH. This difference may be due to upregulation of 5-HT_2A receptors in ACTH-treated rats. We previously reported that chronic treatment with ACTH increased expression of 5-HT_2A receptor mRNA in the frontal cortex, and enhanced the function of 5-HT_2A receptors in rats.^28,29) Some studies in rats and humans have shown that 5-HT_2A receptor agonists increase wakefulness and inhibit slow-wave sleep (SWS), whereas 5-HT_2A receptor antagonists enhance duration of SWS and EEG low-frequency activity in non-REM sleep.^30–32) On the other hand, 5-HT_1A receptors were reported to regulate the activity of the serotonin nervous system, which inhibited the function of 5-HT_2A receptors.^33,34) Kitamura et al.²⁸⁾ demonstrated that 8-OH-DPAT, a potent agonist of 5-HT_1A receptors, inhibited ACTH-induced 5-HT_2A receptor hyperfunction. This led us to conclude that long-term administration of ACTH upregulates 5-HT_2A receptors, and the effect of the partial 5-HT_1A agonist tandospirone on sleep disturbance in ACTH-treated rats may be attributable to 5-HT_1A-receptor-mediated inhibition of 5-HT_2A receptors.

In summary, sleep was disturbed in rats repeatedly administered ACTH. The sleep disturbance observed in the present model was markedly improved in rats treated with tandospirone, but not flunitrazepam, which suggests a close association between disturbed sleep and the serotonin nervous system, especially the 5-HT_1A receptor. We also confirmed that a 5-HT_1A agonist was useful for treating sleep disturbance in cases in which benzodiazepine hypnotics were ineffective.

Conflict of Interest

The authors declare no conflict of interest.

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