INTRODUCTION

Benzodiazepine (Bz) is known to have sedative/hypnotic effects on the brain function represented by the induction of sleepiness and decline in psychomotor performance. Bz-binding site ligands cause allosteric modulation of GABA_A–Cl^- channel complex, which subsequently induces an opening of ion channel pore and Cl^- flows across the neuronal membrane (Barnard et al, 1998; McKernan and Whiting, 1996; Polc, 1988). The principal characteristic of BZP action has been assumed to be the inhibition of other neuronal systems, including the monoaminergic and/or glutaminergic projections, which could account in part for its sedative effects and side effects related to attention and balance, for example. However, the 'physiological process' underlying Bz's sedative/hypnotic effects remains poorly understood.

Growing evidence suggests that thermoregulation, especially the process of heat loss, is a key pathway generating sleepiness and impaired psychomotor performance in humans. The circadian profiles of both alertness and psychomotor performance are mirror images of the endogenous core body temperature (core BT) rhythm, with a trough of alertness and psychomotor performance levels occurring during the several hours around the minimum core BT (Daurat et al, 1993; Drummond et al, 2001; Gillberg et al, 1994; Leproult et al, 1997; Monk et al, 1997). Recent studies have shown that the sleep-producing mechanism in humans is preceded by increased heat loss (Campbell and Broughton, 1994; Gilbert et al, 1999; Krauchi et al, 1999,1997) and that various sleep-producing manipulations act, at least in part, by enhancing the heat loss mechanism (Deacon et al, 1994; Dorsey et al, 1999; Gilbert et al, 1999; Krauchi et al, 1999). Furthermore, physiological sleepiness has been shown to cause a decrease in psychomotor per-formance (De Gennaro et al, 2001; De Valck and Cluydts, 2001; Hood and Bruck, 1996; Kinnari et al, 2000; Nakano et al, 2000; Nave et al, 2002; Porcu et al, 1998; Schulz et al, 1997; Thomas et al, 2000; Wesensten et al, 2002).

We report here that a standard Bz, diazepam (DZP), induced a sedative effect in humans via the thermoregulatory system such that the DZP-related sleepiness and impaired psychomotor performance progressed in parallel with the magnitude of heat loss. The goal of our study was to evaluate the effects of DZP administered at clinical doses on thermoregulation, sleepiness, and psychomotor performance using high time resolution in young healthy volunteers under a modified constant routine condition (Mills et al, 1978), in which various masking effects produced by physical movement, posture, calorie intake, clothing, ambient temperature, and environmental light intensity (Campbell and Dawson, 1990; Rietveld et al, 1993) are strictly controlled.

METHODS AND SUBJECTS

Subjects

Before enrollment in the present study, healthy volunteers under 25 years of age were screened by three physicians who conducted rigorous physical and psychological evaluations of each subject. Volunteers were surveyed to exclude the following: irregularity in the sleep–wake pattern according to a self-registering sleep diary kept for 2 weeks, a history of physical disease that could affect sleep states suggested by the International Classification of Sleep Disorders (ICSD, 1990), for example, chronic headaches, asthma, gastoroesophageal reflux, peptic ulcer disease, etc, history of psychiatric disease identified by the Mini-International Neuropsychiatric Interview (MINI) (Sheehan et al, 1998) or a structured diagnostic psychiatric interview for DSM-IV disorders (including depression and anxiety disorders), use of medications that might modify sleep states during the prior 3 months, any airline travel that could cause jet lag during the prior 6 months, and any abnormal hematology or urinalysis findings. Eight healthy male volunteers with a mean age of 19.75 years (range 18–23) who gave written informed consent participated in the study.

Experimental Procedures

General setting

The study protocol is illustrated in Figure 1. All subjects abstained from drugs, tobacco, alcohol, heavy exercise, and medications for at least 7 days before the study (prestudy period). During this prestudy period, all subjects were asked to keep their daily routine at home but to make sure the light intensity during the time in bed was kept under 10 lux. Sleep quality and regularity were assessed by means of an actigraph (AMI Inc., Ardsley, NY) fitted to the nondominant wrist of each subject; the data were analyzed for computer-calculated sleep–wake determinations by Cole's algorithm (Cole et al, 1992). For each subject, the sleep onset times calculated by actigraph during the prestudy period were averaged, and the average onset time was defined as 0000 h. Each subject participated in three experiments (two drug experiments and one placebo experiment) that were conducted at 2-week intervals. On the day before each experiment, the subject entered the sleep laboratory at 8 h before 0000 h (-0800 h). In the period before -0600 h, the subject donned cotton pajamas provided by the laboratory to control for the influence of clothing on thermoregulation. At -0600 h, a 750-kcal meal and as much water as desired was given to each subject. From the time after the meal and for over 22 h until the end of the study at 1630 h the next day, subjects rested in the supine position on a reclining seat during the wake time. Standing and walking were prohibited, and movement of the limbs was discouraged. An adjoining room was fitted with a portable toilet, movement to the toilet was assisted, and sitting was permitted while the toilet was being used. Subjects were allowed to recline and sleep on a bed in the laboratory only at 0000–0800 h. During the sleep time, each subject underwent polysomnography and was confirmed to have no sleep disorder as defined by the ICSD (1990). Sleeping was forbidden outside the sleep period. Participants were subjected to continuous EEG monitoring, and two or more laboratory workers were always present to observe the subjects and provide assistance. Laboratory lighting was maintained at less than 10 lux during the sleep period and at 100 lux near the subjects' eyes at other times to avoid the alerting and alleviating effects of bright light on sleepiness and psychological performance (Campbell and Dawson, 1990). The room temperature was maintained at 231°C throughout the study period.

Figure 1.

Experimental protocol. PSG: Polysomnography, VAS: Visual Analog Scale, SSS: Stanford Sleepiness Scale, CRT: Choice Reaction Time. Vertical arrows indicate the time of either DZP or placebo administration, which occurred 12 h after each subject's average sleep onset time (1200 h).

Full figure and legend (60K)

Drug administration

On the day of the experiment, the subject ingested a 100-kcal meal and 100 ml of orange juice at 0830 h to ensure alimentary canal absorption of DZP and to suppress the influence of calorie intake on temperature and psychomotor performance. The experiments tested the subject's responses to placebo (lactose), 5 mg of DZP, or 10 mg of DZP, all of which were packed in indistinguishable gelatin capsules and were administered with 100 ml of water at 1200 h. The study was conducted in a randomized, single-blind, crossover fashion.

Evaluation of plasma concentration of DZP and desmethyldiazepam

Blood samples were used to monitor plasma DZP and its active metabolite desmethyldiazepam (dmDZP). Samples were drawn every 20 min from 1200 to 1500 h and again at 1600 h via intravenous catheter placed in a forearm vein at 0900 h. Blood samples were immediately centrifuged at 3000 rpm for 5 min, and the plasma was collected and frozen at -20°C for later assay. Plasma concentrations of DZP and dmDZP were determined according to a high-performance liquid chromatography (HPLC) method previously described (Miura et al, 2002; Nagasaki et al, 1999) but with some modifications. The apparatus used for HPLC was the EP-300 chromatography pump (EICOM, Kyoto, Japan) equipped with an EICOM NOD-10 NOx detector. The wavelength was set at 230 nm. Test samples were introduced with a 234 autoinjector (Gilson Inc., Wisconsin) at an effective volume of 75 l. The HPLC column used was a Grand pack ODS-5 NK stationary phase (5 m, Masis Inc. Owani, Japan). The column temperature was maintained at 25.0°C in an EICOM ATC-300 column oven. The mobile phase consisted of 0.5% KH₂PO₄ (pH 4.5)–acetonitrile (60 : 40, v/v), which was degassed in an EICOM DG-300 degasser prior to use. The flow rate was 1.0 ml/min. All solvents used were of HPLC grade (Wako Pure Chemical Industries, Osaka, Japan). After flunitrazepam (320 ng) in methanol (10 l) was added to plasma samples (1 ml) as an internal standard, the plasma samples were diluted with 5 ml water, and the solution was mixed briefly. The mixture was applied to a Sep-Pak CN cartridge (Waters, Bedford, MA) that had been activated previously with 10 ml acetonitrile and water. The cartridge was then washed with 10 ml water and 5 ml 20% acetonitrile in water. The desired fraction was eluted with 5 ml 70% acetonitrile in water. The elute was evaporated in a vacuum at 40°C by rotary evaporator (Tokyo Rikakikai, Tokyo, Japan). The residue was dissolved in 50 l methanol, and 100 ml mobile phase was added. The samples were injected into the HPLC apparatus. The ratios of drug to internal standard were calculated from the recorded peaks. The results obtained from spiked plasma samples containing known amounts of drug were calculated on the basis of linear regression analysis.

Evaluation of heat loss after DZP administration

In the period prior to -0600 h, skin-temperature thermistors were attached to the left and right wrist area and the instep of both feet. A rectal thermistor (polyethylene-covered thermoprobe, accuracy within 0.01°C) was inserted 10 cm into the subject's rectum. The thermistors were connected to an ambulatory temperature monitoring system (Kohden Medical Inc., Tokyo, Japan), and sampling occurred at 1-min intervals. All temperature recordings taken on the day of the experiment were later collapsed into 20-min bins from 1100 to 1600 h. The data at 1200 h were averaged for 10 min just before drug administration to control for the acute drug effects. After that, bin data from each subject were expressed relative to the data at 1200 h. Proximal body temperature (p-BT), that is, rectal temperature, and distal body temperature (d-BT), that is, average skin temperature from four sites were used to calculate distal-proximal BT gradient (DPG, difference between d-BT and p-BT) as an indicator of blood flow in distal skin regions (Krauchi et al, 1999; Rubinstein and Sessler, 1990). For further analysis, the change in DPG (DPG) induced by DZP was defined as the difference in the corresponding values between the DZP and placebo experiments at each point of measure.

Evaluation of subjective sleepiness and psychomotor performance

Subjective sleepiness and psychomotor performance were monitored every 20 min from 1000 to 1600 h. During the first 5 min of each 20 min epoch, the Visual Analog Scale (VAS) (Folstein and Luria, 1973), Stanford Sleepiness Scale (SSS) (Hoddes et al, 1973), and Choice Reaction Time (CRT) (Sekine et al, 2001) were applied consecutively.

The VAS technique, self-registering on a line 100 mm in length ranging from 'very alert' to 'very sleepy,' and the SSS technique, self-registering by a seven-grade scale ranging from 'feeling active, vital, alert, wide awake' to 'almost in reverie, cannot stay awake, sleep onset appears imminent,' have been used widely to evaluate the subjective sleepiness.

CRT was used to assess objective psychomotor performance, as reported previously (Sekine et al, 2001). Two different brief tones (2000 and 1000 Hz), each with a duration of 100 ms, were generated by an automatic stimulator (Nihon Kohden SS-1449, Nihon Kohden, Tokyo, Japan) and presented at 60 dB (normal hearing level). The 1000 Hz tone was presented 80% of the time and the target 2000 Hz was presented 20% of the time. The order of presentation was randomized. The interstimulus interval was also randomized within the range of 1.0–3.0 s (mean 2.0 s). The subjects were instructed to respond only to the target stimuli (2000 Hz tone) by pressing a button attached to the palm of their right hand with their thumb ('oddball' paradigm). The button-press response was converted to an electrical signal that was monitored and recorded on a personal computer. Reaction time latencies from the target stimuli to button press were averaged for a total of 33 times as the CRT data at each point of measure. VAS and CRT were performed at 1200 h for 5 min just before drug administration. All other VAS and CRT data were expressed relative to the data obtained at 1200 h. For further analysis, the change in either VAS (VAS), SSS (SSS), or CRT (CRT) induced by DZP was defined as the difference in the corresponding values between the DZP and placebo experiments at each point of measure.

Evaluation of heart rate

Heart rate (HR) was monitored continuously from 1000 to 1630 h on the day of the experiment by means of an ambulatory HR monitoring system (AC-301 Activetracer, GMS Corp., Tokyo, Japan), and values were later collapsed into 20-min bins from 1100 to 1600 h. The data at 1200 h were averaged for 10 min just before drug administration. Bin data were expressed relative to the data at 1200 h. For further analysis, the change in HR (HR) induced by DZP was defined as the difference in the corresponding values between the DZP and placebo experiments at each point of measure.

Data Analysis

One- and two-way analyses of variance (factors: time and dose) followed by the Bonferroni's post hoc analysis were used to examine differences in BT, VAS, SSS, CRT, and HR values obtained for the placebo and for each dose of DZP, appropriately. Relations between DPG and plasma DZP and dmDZP concentrations, subjective sleepiness, and psychomotor performance were determined by Pearson's correlation analysis. Results are shown as meanSEM. p<0.05 was considered statistically significant.

RESULTS

Proximal Body Temperature

Changes in the p-BT for the placebo, 5- and 10-mg DZP experiments are shown in Figure 2a. In comparison with p-BT with administration of placebo, p-BT with administration of DZP at 10 mg was significantly decreased (F=4.14, df=15, p<0.0001) such that the maximum p-BT suppression occurred 80 min after DZP administration for 0.170.04°C followed by a gradual recovery to the placebo level, which occurred 240 min after DZP administration. A two-phase thermoregulatory effect, that is, short-term hypothermia followed by a hyperthermic phase of 180 min, was observed after administration of DZP at 5 mg; however, this change did not reach a statistically significant level compared to that with administration of placebo (F=0.90, df=15, p=0.568).

Figure 2.

Changes in (a) the proximal BT, (b) distal BT, (c) DPG, and (d) plasma DZP and dmDZP concentrations after oral administration of DZP at 5 and 10 mg. Horizontal bars indicate the time relative to 1200 h of drug administration. MeanSEM values are shown. The proximal and distal BT data are shown as open circles for the 5-mg DZP experiment, closed circles for the 10-mg DZP experiment, and open squares for the placebo experiment. In (a) and (b), SEM values for the 5-mg DZP experiments were omitted for convenience. The DPG in both the 5-mg (open circles) and 10-mg (closed circles) DZP experiments was defined as the difference in the corresponding values between the placebo and DZP experiments at each measured point. In (d), large and small circles correspond to DZP and dmDZP data, respectively. Scales on the y-axis were, respectively, modified for DZP and dmDZP data.

Full figure and legend (78K)

Distal Body Temperature

Changes in d-BT for the placebo, 5- and 10-mg DZP experiments are shown in Figure 2b. In comparison with d-BT with administration of placebo, d-BT with administration of DZP at 10 mg was significantly increased (F=2.42, df=15, p<0.01) such that the maximum d-BT elevation occurred 40 min after administration for 1.070.29°C followed by a gradual recovery to the placebo level, which occurred 120 min after DZP administration. No statistically significant change in d-BT was observed with administration of DZP at 5 mg compared to that with administration of placebo (F=0.29, df=15, p=0.996).

Distal-Proximal BT Gradient

Changes in DPG for the 5- and 10-mg DZP experiments are shown in Figure 2c. The DZP administration at 10 mg induced a significant increase in DPG (F=2.68, df=15, p<0.002) such that the maximum DPG elevation occurred 40 min after DZP administration for 1.170.30°C followed by a gradual recovery to the placebo level, which occurred 180 min after DZP administration. No statistically significant change in DPG was observed with administration of DZP at 5 mg (F=0.37, df=15, p=0.983).

Plasma DZP and dmDZP Concentrations

Changes in the plasma DZP and dmDZP concentrations for the 5- and 10-mg DZP experiments are shown in Figure 2d. Orally administered DZP was absorbed rapidly with mean t_max (time of peak concentration) values of 50.07.56 and 45.09.06 min as well as mean C_max (peak plasma DZP concentration) values of 234.623.1 and 484.6931.11 ng/ml in the 5- and 10-mg DZP experiments, respectively.

Subjective Sleepiness

Changes in VAS for the 5- and 10-mg DZP experiments are shown in Figure 3a. The DZP administration at 10 mg induced a significant increase in VAS (F=4.03, df=15, p<0.0001) and SSS (F=4.65, df=15, p<0.0001), while no statistically significant increases in VAS and SSS were observed with the administration of DZP at 5 mg (F=0.87, df=15, p=0.597 and F=1.46, df=15, p=0.133, respectively). There was a significant intergroup difference in the average VAS score from 20 to 240 min after DZP administration (F=9.38, df=2, p<0.01, Figure 3a) such that the average VAS score in the 10-mg DZP experiment was significantly larger than that in the placebo experiment (p<0.001).

Figure 3.

Changes in the VAS and CRT for the 5- and 10-mg DZP experiments. In (a) (upper) and (b) (lower) panels, horizontal bars indicate the time, relative to 1200 h, of drug administration. MeanSEM values are shown. The VAS and CRT in both the 5-mg (open circles) and 10-mg (closed circles) DZP experiments were defined as the difference in the corresponding values between the placebo and DZP experiments at each measured point. Middle panels show average values for VAS (a, left) and CRT (b, right) from 20 to 240 min after DZP administration in the placebo (pcb), and 5- and 10-mg DZP experiments.

Full figure and legend (132K)

Psychomotor Performance

Changes in CRT for the 5- and 10-mg DZP experiments are shown in Figure 3b. The DZP administration at 10 mg induced a significant increase in CRT (F=3.16, df=15, p<0.001), while no statistically significant increases in CRT were observed with the administration of DZP at 5 mg (F=1.66, df=15, p=0.071). There was a significant intergroup difference in the average CRT from 20 to 240 min after DZP administration (F=14.82, df=2, p<0.0001, Figure 3b) such that the average CRT in the 10-mg DZP experiments was significantly prolonged compared to that of the placebo experiment (p<0.0001) and the 5-mg DZP experiment (p<0.005).

Heart Rate

Changes in HR for the 5- and 10-mg DZP experiments are shown in Figure 4a. One subject was excluded from the data analysis; his data were lost because of an ECG recording disc error. There was a significant intergroup difference in the average HR from 20 to 240 min after DZP administration (F=4.01, df=2, p<0.05, Figure 4b), such that the average HR in the 10-mg DZP experiment was significantly higher than that of the placebo experiment (p<0.02).

Figure 4.

Changes in HR for the 5- and 10-mg DZP experiments. In (a) (upper panel), horizontal bars indicate the time, relative to 1200 h, of drug administration. MeanSEM values are shown. HR in both the 5-mg (open circles) and 10-mg (closed circles) DZP experiments were defined as the difference in the corresponding values between the placebo and DZP experiments at each measured point. The lower panel (b) shows average values for HR from 20 to 240 min after DZP administration in the placebo and 5- and 10-mg DZP experiments.

Full figure and legend (89K)

Relation between DPG, Plasma DZP, and dmDZP Concentrations

There was a strong positive correlation between the mean DPG and the mean plasma DZP concentration at each point of measure in the 10-mg DZP experiment (r=0.988, p<0.0001 (Figure 5a)), whereas correlation was lost in the 5-mg DZP experiment (r=0.397, p=0.25). A positive correlation was also observed between the mean DPG and the mean total plasma concentration for DZP plus dmDZP at each point of measure in the 10-mg DZP experiment (r=0.951, p<0.0001), whereas correlation was also lost in the 5-mg DZP experiment (r=0.397, p=0.26).

Figure 5.

Relation between DPG and (a) plasma DZP concentration, (b) VAS, and (c) CRT in the 10-mg DZP experiment. Pearson's correlation analysis was performed for the mean DPG (x-axis) and the mean of plasma DZP concentrations, VAS, or CRT (y-axis) at the corresponding time points in the 10-mg DZP experiment.

Full figure and legend (53K)

Relation between DPG and Subjective Sleepiness

There was a strong positive correlation between the mean DPG and the mean VAS at each point of measure in the 10-mg DZP experiment (r=0.876, p<0.0001) (Figure 5b), whereas the correlation was lost in the 5-mg DZP experiment (r=0.331, p=0.21). There was also a positive correlation between the mean DPG and the mean SSS at each point of measure in the 10-mg DZP experiment (r=0.873, p<0.0001); correlation disappeared in the 5-mg DZP experiment (r=0.452, p=0.08).

Relation between DPG and Psychomotor Performance

There was a strong positive correlation between the mean DPG and the mean CRT at each point of measure in the 10-mg DZP experiment (r=0.842, p<0.0001) (Figure 5c); the correlation was not as strong in the 5-mg DZP experiment (r=0.636, p<0.01).

COMMENT

We showed that DZP administered orally in a clinical dose of 10 mg induced a significant increase in skin temperature and decrease in rectal temperature, resulting in a significant increase in DPG. We know that these thermoregulatory changes were not induced by physical activity, muscle tone, posture changes, or sleep itself because the possible effects of these factors on thermoregulatory parameters were controlled with the use of the modified constant routine protocol (Mills et al, 1978). DPG has been shown to reflect accurately blood flow in the distal skin regions regulated by anastomosis arterio-venosa (Rubinstein and Sessler, 1990), and it provides an indirect index of distal heat loss (Krauchi et al, 1999). The present data with a high time resolution of 20 min have strongly confirmed previously reported hypo-thermic actions of Bz agonists in humans (Gilbert et al, 2000,1999; Matsukawa et al, 1997; Pleuvry et al, 1980) and animals (Jackson and Nutt, 1992; Zarrindast and Dibayan, 1989).

In this study, however, we could not detect a significant thermoregulatory effect after the administration of DZP at a dose of 5 mg in spite of its clinically established sedative/hypnotic effects. One explanation is that the present subjects might possess relatively high tolerance to Bz because 5 mg of DZP showed a weak thermoregulatory effect but no significant subjective sleepiness compared to the placebo condition in these subjects. The second possibility is that there is a threshold for the plasma concentration of DZP to cause an obvious heat loss reaction in humans. Profiles of plasma DZP concentrations showed clear dose-dependent pharmacokinetics between the 5- and 10-mg DZP experiments. As a result, the average peak concentration of DZP at 5-mg experiment was 178.111.2 ng/ml and it appeared 60 min after administration. This only slightly exceeded the lowest three measures appearing from 160 min (176.012.8 ng/ml) to 240 min (137.69.37 ng/ml) in the 10-mg experiment (Figure 2d). Given that a threshold exists around these plasma levels, 5 mg of DZP induced only a slight or no thermoregulatory effect. It is also possible that another Bz agonist has its own plasma concentration threshold for inducing the heat loss reaction.

We observed a strong positive correlation between plasma DZP concentrations and the increase in DPG, indicating that DZP promotes the heat loss from distal skin regions in a dose-dependent manner. While physiological mechanistic details of heat loss action induced by Bz remain unclear, two possibilities have been given, namely involvement of the central and/or peripheral GABA_A receptor. Previous studies based on receptor autoradiographic techniques have revealed that central GABA_A receptors are densely localized in the thermoregulatory center including the diagonal band, medial, and lateral preoptic area in the human brain (Najimi et al, 1999; Zezula et al, 1988). There is growing evidence that thermosensitive neurons in the preoptic area and anterior hypothalamic region play a critical role in physiological sleep and the thermoregulatory response (see the review by Szymusiak et al, 2001). In addition, microinjection of Bz agonist or antagonist into this region has been shown to produce sleep-modifying and thermoregulatory effects in rats (Ali et al, 1999; Jha et al, 2001; Mendelson and Martin, 1992; Osborne et al, 1994), suggesting the possible site of action of Bz agonists through the central GABA_A receptor to produce sleep-induction and hypothermic effects. Another series of studies indicated that the peripheral Bz receptor might directly cause distal heat loss. Existence of the peripheral Bz receptor in vascular smooth muscle has been shown (Cox et al, 1991). Although the precise function of this receptor is not fully understood (Casellas et al, 2002), previous in vivo and in vitro studies showed that Bz agonists can directly cause peripheral vasodilatation in animals (Barker and Clanachan, 1982; Clark and Lipton, 1981; Erne et al, 1989; Seubert et al, 2000). HR after DZP administration can inform us indirectly about this issue. Previous studies examining the Bz effect on HR, a marker of heat production in humans (Gilbert et al, 1999; Krauchi et al, 1997; Krauchi and Wirz-Justice, 1994), have yielded inconsistent findings (Ford et al, 1990; Matejcek et al, 1983 vs Gilbert et al, 1999; Pleuvry et al, 1980) related to HR after administration of temazepam, a clinically used Bz hypnotic. We observed a statistically significant dose-dependent increase in HR after DZP administration in subjects taking the 10-mg dose. These changes in HR after DZP administration seem to reflect the compensatory heat production for the downward deviation from the core BT set point (Kobayashi, 1989) because of direct peripheral vasodilatation by DZP rather than the sequence of heat loss promoted by a decreased BT set point driven by the thermoregulatory center, which is usually associated with suppressed heat production (Krauchi and Wirz-Justice, 1994).

There is growing evidence that the sleep-producing action in humans is commonly preceded by an increase in heat loss (Campbell and Broughton, 1994; Gilbert et al, 1999; Krauchi et al, 1999; Murphy and Campbell, 1997; Van den Heuvel et al, 1998). It is well established that human proximal BT declines during the night time under physiological conditions, and this is not the result of the sleep itself; it is controlled by the circadian time-keeping system (Campbell and Broughton, 1994; Gillberg and Akerstedt, 1982; Lack and Lushington, 1996; Zulley et al, 1981). Sleep challenge tests under isolated as well as entrained conditions reveal that the occurrence and continuity of human sleep is intimately related to the circadian proximal BT rhythms (Czeisler et al, 1980; Lavie, 1986; Zulley et al, 1981), sleep propensity increases on the falling limb, and decreases on the rising limb of the proximal BT curve. In addition, many previous studies have consistently shown that various drug-based, light exposure-based, or passive body heating-based techniques produce sleep work via thermoregulatory actions (Bunnell et al, 1988; Deacon et al, 1994; Dorsey et al, 1999; Gilbert et al, 1999; Jordan et al, 1990; Krauchi et al, 1999). They have also shown an intimate temporal relation between increasing sleepiness and the degree of heat loss induced by thermoregulatory manipulations (Gilbert et al, 1999; Krauchi et al, 1999). Krauchi et al, applied DPG as an indicator of distal heat loss and showed that it successfully predicts the reduction in sleep onset latency induced by various sleep-promoting modifications. Subjects with higher DPG values just before sleep had shorter sleep latencies regardless of the type of manipulation (Krauchi et al, 1999). Specifically, heat loss from distal skin regions could be a strong sleepiness-inducing process in humans. We too found a strong relation between DPG and two measures of subjective sleepiness after DZP administration. Our findings of a DZP concentration-dependent heat loss reaction and a heat loss-dependent increase in sleepiness lead us to believe that the heat loss process plays an important role in the onset of sleepiness after DZP administration. The findings that DZP at 5 mg showed only slight heat loss and not significant subjective sleepiness in young healthy individuals support this assumption.

The present study has also shown that the heat loss process is intimately related to the decline that occurs in psychomotor performance after DZP administration. That increased physiological sleepiness can cause impaired psychomotor performance has been shown in human extended-wakefulness studies (De Gennaro et al, 2001; Nakano et al, 2000; Thomas et al, 2000), shift-work model (Porcu et al, 1998), a clinicopathologic survey for disorders characterized by excessive daytime sleepiness (Cohen-Zion et al, 2001; Hood and Bruck, 1996; Kinnari et al, 2000; Schulz et al, 1997), and a clinicopharmacological survey of alertness-modifying drugs such as caffeine, modafinil, and melatonin (De Valck and Cluydts, 2001; Nave et al, 2002; Wesensten et al, 2002). Although it remains unclear whether impaired psychomotor performance after DZP administration could also be caused by increased sleepiness, the magnitude of heat loss induced by Bz agonist may be a potent indicator to predict sedative effects on brain function. Given this is the case, it is possible that Bz agonist possessing more robust hypothermic properties shows more prominent sleep-induction effects and impairment of psychomotor performances. Future study should verify that there exists not only an associative but also a causative link between heat loss and brain functions after DZP administration, in which, for example, whether prevention of the heat loss induced by Bz results in an absence or diminution of sedative/hypnotic effects should be examined.

In spite of the many studies, the physiological processes of Bz action through GABA_A receptor remain poorly understood. One reason is that the GABAergic projection is the most widespread of the inhibitory neurotransmitters and could affect many circuits that are responsive to the various effects of Bz agonists. In addition, there exist various subtypes of GABA_A receptor showing different affinity and channel functions in response to Bz agonists and possessing various combinations of plural types of alpha-, beta-, and gamma-subunits (Barnard et al, 1998; McKernan and Whiting, 1996). These subtypes of GABA_A receptor broadly distribute in various brain circuits to form an overlapping mosaic of receptor subtypes (Wisden and Stephens, 1999), making it difficult to understand the functional mechanisms of Bz agonists through the GABA_A system from the physiological point of view (Rudolph et al, 1999). The thermoregulatory effects observed in the present and previous studies offer important information about the physiological pathway of sedative/hypnotic actions induced by Bz agonists and could be useful physiological markers for future studies.

References

1. Ali M, Jha SK, Kaur S, Mallick BN (1999). Role of GABA-A receptor in the preoptic area in the regulation of sleep-wakefulness and rapid eye movement sleep. Neurosci Res 33: 245–250. | PubMed |
2. Barker PH, Clanachan AS (1982). Inhibition of adenosine accumulation into guinea pig ventricle by benzodiazepines. Eur J Pharmacol 78: 241–244. | PubMed |
3. Barnard EA, Skolnick P, Olsen RW, Mohler H, Sieghart W, Biggio G et al (1998). International Union of Pharmacology. XV. Subtypes of gamma-aminobutyric acid A receptors: classification on the basis of subunit structure and receptor function. Pharmacol Rev 50: 291–313. | PubMed | ISI | ChemPort |
4. Bunnell DE, Agnew JA, Horvath SM, Jopson L, Wills M (1988). Passive body heating and sleep: influence of proximity to sleep. Sleep 11: 210–219. | PubMed |
5. Campbell SS, Broughton RJ (1994). Rapid decline in body temperature before sleep: fluffing the physiological pillow? Chronobiol Int 11: 126–131. | PubMed | ISI | ChemPort |
6. Campbell SS, Dawson D (1990). Enhancement of nighttime alertness and performance with bright ambient light. Physiol. Behav. 48: 317–320. | PubMed |
7. Casellas P, Galiegue S, Basile AS (2002). Peripheral benzodiazepine receptors and mitochondrial function. Neurochem Int 40: 475–486. | Article | PubMed | ISI | ChemPort |
8. Clark SM, Lipton JM (1981). Effects of diazepam on body temperature of the aged squirrel monkey. Brain Res Bull 7: 5–9. | PubMed |
9. Cohen-Zion M, Stepnowsky C, Marler ST, Kripke DF, Ancoli-Israel S (2001). Changes in cognitive function associated with sleep disordered breathing in older people. J Am Geriatr Soc 49: 1622–1627. | PubMed |
10. Cole RJ, Kripke DF, Gruen W, Mullaney DJ, Gillin JC (1992). Automatic sleep/wake identification from wrist activity. Sleep 15: 461–469. | PubMed | ChemPort |
11. Cox DA, Ellinor PT, Kirley TL, Matlib MA (1991). Identification of a 17-kDa protein associated with the peripheral-type benzodiazepine receptor in vascular and other smooth muscle types. J Pharmacol Exp Ther 258: 702–709. | PubMed |
12. Czeisler CA, Weitzman ED, Moore EM, Zimmerman JC, Knauer RS (1980). Human sleep: its duration and organization depend on its circadian phase. Science 210: 1264–1267. | PubMed | ChemPort |
13. Daurat A, Aguirre A, Foret J, Gonnet P, Keromes A, Benoit O (1993). Bright light affects alertness and performance rhythms during a 24-h constant routine. Physiol Behav 53: 929–936. | PubMed |
14. De Gennaro L, Ferrara M, Curcio G, Bertini M (2001). Visual search performance across 40 h of continuous wakefulness: Measures of speed and accuracy and relation with oculomotor performance. Physiol Behav 74: 197–204. | Article | PubMed | ChemPort |
15. De Valck E, Cluydts R (2001). Slow-release caffeine as a countermeasure to driver sleepiness induced by partial sleep deprivation. J Sleep Res 10: 203–209. | PubMed |
16. Deacon S, English J, Arendt J (1994). Acute phase-shifting effects of melatonin associated with suppression of core body temperature in humans. Neurosci Lett 178: 32–34. | PubMed |
17. Dorsey CM, Teicher MH, Cohen Zion M, Stefanovic L, Satlin A, Tartarini W et al (1999). Core body temperature and sleep of older female insomniacs before and after passive body heating. Sleep 22: 891–898. | PubMed |
18. Drummond SP, Gillin JC, Brown GG (2001). Increased cerebral response during a divided attention task following sleep deprivation. J Sleep Res 10: 85–92. | Article | PubMed | ChemPort |
19. Erne P, Chiesi M, Longoni S, Fulbright J, Hermsmeyer K (1989). Relaxation of rat vascular muscle by peripheral benzodiazepine modulators. J Clin Invest 84: 493–498. | PubMed |
20. Folstein M, Luria R (1973). Reliability, validity, and clinical application of the Visual Analogue Mood Scale. Psychol Med 3: 479–486. | PubMed | ChemPort |
21. Ford GA, Hoffman BB, Blaschke TF (1990). Effect of temazepam on blood pressure regulation in healthy elderly subjects. Br J Clin Pharmacol 29: 61–67. | PubMed |
22. Gilbert SS, Burgess HJ, Kennaway DJ, Dawson D (2000). Attenuation of sleep propensity, core hypothermia, and peripheral heat loss after temazepam tolerance. Am J Physiol Regul Integr Comp Physiol 279: R1980–R1987. | PubMed |
23. Gilbert SS, Van den Heuvel CJ, Dawson D (1999). Daytime melatonin and temazepam in young adult humans: equivalent effects on sleep latency and body temperatures. J Physiol 514: 905–914. | PubMed |
24. Gillberg M, Akerstedt T (1982). Body temperature and sleep at different times of day. Sleep 5: 378–388. | PubMed |
25. Gillberg M, Kecklund G, Akerstedt T (1994). Relations between performance and subjective ratings of sleepiness during a night awake. Sleep 17: 236–241. | PubMed |
26. Hoddes E, Zarcone V, Smythe H, Phillips R, Dement WC (1973). Quantification of sleepiness: A new approach. Psychophysiology 10: 431–436. | Article | PubMed | ISI | ChemPort |
27. Hood B, Bruck D (1996). Sleepiness and performance in narcolepsy. J Sleep Res 5: 128–134. | PubMed |
28. ICSD (1990). International Classification of Sleep Disorders, diagnostic and coding manual, American Sleep Disorders Association (eds). Rochester, Minnesota.
29. Jackson HC, Nutt DJ (1992). Strain differences in sensitivity to the hypothermic effects of benzodiazepine receptor ligands in mice. Psychopharmacology (Berl) 109: 365–368. | PubMed |
30. Jha SK, Yadav V, Mallick BN (2001). GABA-A receptors in mPOAH simultaneously regulate sleep and body temperature in freely moving rats. Pharmacol Biochem Behav 70: 115–121. | PubMed |
31. Jordan J, Montgomery I, Trinder J (1990). The effect of afternoon body heating on body temperature and slow wave sleep. Psychophysiology 27: 560–566. | PubMed |
32. Kinnari K, Peter JH, Pietarinen A, Groete L, Penzel T, Varri A et al (2000). Vigilance stages and performance in OSAS patients in a monotonous reaction time task. Clin Neurophysiol 111: 1130–1136. | PubMed |
33. Kobayashi S (1989). Temperature-sensitive neurons in the hypothalamus: a new hypothesis that they act as thermostats, not as transducers. Prog Neurobiol 32: 103–135. | PubMed |
34. Krauchi K, Cajochen C, Werth E, Wirz-Justice A (1999). Warm feet promote the rapid onset of sleep. Nature 401: 36–37. | Article | PubMed |
35. Krauchi K, Cajochen C, Wirz-Justice A (1997). A relationship between heat loss and sleepiness: effects of postural change and melatonin administration. J Appl Physiol 83: 134–139. | PubMed |
36. Krauchi K, Wirz-Justice A (1994). Circadian rhythm of heat production, heart rate, and skin and core temperature under unmasking conditions in men. Am J Physiol 267, (Regulatory Integrative Comp. Physiol. 36) R819–R826. | PubMed |
37. Lack LC, Lushington K (1996). The rhythms of human sleep propensity and core body temperature. J Sleep Res 5: 1–11. | PubMed |
38. Lavie P (1986). Ultrashort sleep-waking schedule. III. 'Gates' and 'forbidden zones' for sleep. Electroencephalogr Clin Neurophysiol 63: 414–425. | Article | PubMed | ISI | ChemPort |
39. Leproult R, Van RO, Byrne MM, Sturis J, Van CE (1997). Sleepiness, performance, and neuroendocrine function during sleep deprivation: effects of exposure to bright light or exercise. J Biol Rhythms 12: 245–258. | PubMed |
40. Matejcek M, Neff G, Abt K, Wehrli W (1983). Pharmaco-EEG and Psychometric study of the effect of single doses of temazepam and nitrazepam. Neuropsychobiology 9: 52–65. | PubMed |
41. Matsukawa T, Hanagata K, Ozaki M, Iwashita H, Koshimizu M, Kumazawa T (1997). I.m. midazolam as premedication produces a concentration-dependent decrease in core temperature in male volunteers. Br J Anaesth 78: 396–399. | PubMed |
42. McKernan RM, Whiting PJ (1996). Which GABAA-receptor subtypes really occur in the brain? Trends Neurosci 19: 139–143. | Article | PubMed | ISI | ChemPort |
43. Mendelson WB, Martin JV (1992). Characterization of the hypnotic effects of triazolam microinjections into the medial preoptic area. Life Sci 50: 1117–1128. | PubMed |
44. Mills JN, Minors DS, Waterhouse JM (1978). Adaptation to abrupt time shifts of the oscillator(s) controlling human circadian rhythms. J Physiol 285: 455–470. | PubMed |
45. Miura M, Ohkubo T, Sugawara K, Okuyama N, Otani K (2002). Determination of estazolam in plasma by high-performance liquid chromatography with solid-phase extraction. Anal Sci 18: 525–528. | PubMed |
46. Monk TH, Buysse DJ, Reynolds CRIII, Berga SL, Jarrett DB, Begley AE et al (1997). Circadian rhythms in human performance and mood under constant conditions. J Sleep Res 6: 9–18. | Article | PubMed | ISI | ChemPort |
47. Murphy PJ, Campbell SS (1997). Nighttime drop in body temperature: a physiological trigger for sleep onset? Sleep 20: 505–511. | PubMed |
48. Nagasaki T, Ohkubo T, Sugawara K, Yasui N, Furukori H, Kaneko S (1999). Determination of risperidone and 9-hydroxy-risperidone in human plasma by high-performance liquid chromatography: application to therapeutic drug monitoring in Japanese patients with schizophrenia. J Pharm Biomed Anal 19: 595–601. | Article | PubMed | ISI | ChemPort |
49. Najimi M, Bennis M, Chigr F, Kopp N, Moyse E, Miachon S (1999). Benzodiazepine binding sites in the human hypothalamus. Autoradiographic study. J Hirnforsch 39: 493–502. | PubMed |
50. Nakano T, Araki K, Michimori A, Inbe H, Hagiwara H, Koyama E (2000). Temporal order of sleepiness, performance and physiological indices during 19-h sleep deprivation. Psychiatry Clin Neurosci 54: 280–282. | PubMed |
51. Nave R, Iani C, Herer P, Gopher D, Lavie P (2002). Residual effects of daytime administration of melatonin on performance relevant to flight. Behav Brain Res 131: 87–95. | PubMed |
52. Osborne PG, Onoe H, Watanabe Y (1994). GABAergic system inducing hyperthermia in the rat preoptic area: its independence of prostaglandin E2 system. Brain Res 661: 237–242. | Article | PubMed | ChemPort |
53. Pleuvry BJ, Maddison SE, Odeh RB, Dodson ME (1980). Respiratory and psychological effects of oral temazepam in volunteers. Br J Anaesth 52: 901–906. | PubMed |
54. Polc P (1988). Electrophysiology of benzodiazepine receptor ligands: multiple mechanisms and sites of action. Prog Neurobiol 31: 349–423. | PubMed |
55. Porcu S, Bellatreccia A, Ferrara M, Casagrande M (1998). Sleepiness, alertness and performance during a laboratory simulation of an acute shift of the wake-sleep cycle. Ergonomics 41: 1192–1202. | PubMed |
56. Rietveld WJ, Minors DS, Waterhouse JM (1993). Circadian rhythms and masking: an overview. Chronobiol Int 10: 306–312. | PubMed |
57. Rubinstein EH, Sessler DI (1990). Skin-surface temperature gradients correlate with fingertip blood flow in humans. Anesthesiology 73: 541–545. | PubMed | ISI | ChemPort |
58. Rudolph U, Crestani F, Benke D, Brunig I, Benson JA, Fritschy JM et al (1999). Benzodiazepine actions mediated by specific gamma-aminobutyric acid(A) receptor subtypes. Nature 401: 796–800. | Article | PubMed | ISI | ChemPort |
59. Schulz H, Wilde FJ, Grabietz KU (1997). Cognitive deficits in patients with daytime sleepiness. Acta Neurol Belg 97: 108–112. | PubMed |
60. Sekine A, Niiyama Y, Kutsuzawa O, Shimizu T (2001). A negative component superimposed on event-related potentials during light drowsiness. Psychiatry Clin Neurosci 55: 473–478. | PubMed |
61. Seubert CN, Morey TE, Martynyuk AE, Cucchiara RF, Dennis DM (2000). Midazolam selectively potentiates the A(2A) - but not A1- receptor–mediated effects of adenosine: role of nucleoside transport inhibition and clinical implications. Anesthesiology 92: 567–577. | PubMed |
62. Sheehan DV, Lecrubier Y, Sheehan KH, Amorim P, Janavs J, Weiller E et al (1998). The Mini-International Neuropsychiatric Interview (M.I.N.I.): the development and validation of a structured diagnostic psychiatric interview for DSM-IV and ICD-10. J Clin Psychiatry 59(Suppl 20): 22–33. | PubMed | ISI |
63. Szymusiak R, Steininger T, Alam N, McGinty D (2001). Preoptic area sleep-regulating mechanisms. Arch Ital Biol 139: 77–92. | PubMed |
64. Thomas M, Sing H, Belenky G, Holcomb H, Mayberg H, Dannals R et al (2000). Neural basis of alertness and cognitive performance impairments during sleepiness. I. Effects of 24 h of sleep deprivation on waking human regional brain activity. J Sleep Res 9: 335–352. | Article | PubMed | ISI | ChemPort |
65. Van den Heuvel CJ, Noone JT, Lushington K, Dawson D (1998). Changes in sleepiness and body temperature precede nocturnal sleep onset: evidence from a polysomnographic study in young men. J Sleep Res 7: 159–166.
66. Wesensten NJ, Belenky G, Kautz MA, Thorne DR, Reichardt RM, Balkin TJ (2002). Maintaining alertness and performance during sleep deprivation: modafinil versus caffeine. Psychopharmacology (Berl) 159: 238–247. | PubMed | ChemPort |
67. Wisden W, Stephens DN (1999). Towards better benzodiazepines. Nature 401: 751–752. | Article | PubMed | ISI | ChemPort |
68. Zarrindast MR, Dibayan M (1989). Involvement of GABAA receptor sites in diazepam hypothermia. Gen Pharmacol 20: 855–859.
69. Zezula J, Cortes R, Probst A, Palacios JM (1988). Benzodiazepine receptor sites in the human brain: autoradiographic mapping. Neuroscience 25: 771–795. | Article | PubMed | ChemPort |
70. Zulley J, Wever R, Aschoff J (1981). The dependence of onset and duration of sleep on the circadian rhythm of rectal temperature. Pflugers Arch 391: 314–318.

Acknowledgments

The present study was performed through Special Coordination Funds of the Ministry of Education, Culture, Sports and Technology, and a Grant-in-Aid for Cooperative Research from the Ministry of Health, Labour and Welfare of Japan.