Chemical Senses Vol. 30 No. suppl 1 © Oxford University
Press 2005; all rights reserved
Effects of Plant-derived Odors on Sleep–Wakefulness and Circadian Rhythmicity in Rats
1 Department of Regulatory Physiology, Dokkyo University School of Medicine, Tochigi 321-0293, Japan and 2 Department of Applied Biological Chemistry, Yamaguchi University, Yamaguchi 753-8511, Japan
Correspondence to be sent to: Sadao Yamaoka, e-mail: syamaoka{at}tcat.ne.jp
Key words: 
, -pinene, circadian
rhythm, green odors, paradoxical sleep, slow-wave sleep
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Introduction |
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Plant-derived odorants promote good feeling, refresh spirits and sometimes relieve various stresses in humans. Physiological and psychological effects of plant-derived volatile chemicals have long been acknowledged in folk medicine and aromatherapy. Recent evidence from animal experiments suggests that these plant-derived chemicals affect various animal behaviors via modulating neural or humoral mechanisms (Torii et al., 1988
;
Sano et al., 1998;
Ilmberger et al., 2001;
Akutsu et al., 2002;
Nakashima et al., 2004).
However, the majority of those animal experiments were performed under unusual
environmental conditions (various stresses, anesthesia). Therefore, we examined the
effects of these odorants on sleep–wakefulness and circadian rhythm in freely
moving animals. We used two volatile substances: -pinene and mixture of green odor
(n-hexenal and n-hexenol). |
Materials and methods |
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TopIntroductionMaterials and methodsResultsDiscussionReferences |
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Animals
Male Sprague–Dawley (SD) rats (CD-IGS rat, aged 5 months; Charles River Japan) were used for the sleep–wakefulness study and male microphthalmic (MP, kindly supplied by Professor S. Sugita, Utsunomiya University) rats (aged 10–18 months), which completely lack the optical nerve, for the study of aging effects on circadian locomotor rhythms. They were housed at a controlled temperature (24 ± 1.0°C) and illumination (light 05:00–19:00 h) and given food and water ad libitum. Over 7 days before the experiments, SD rats were implanted with electrodes for polygraphic recording under pentobarbital sodium anesthesia (35 mg/kg body wt i.p., nembutal; Dainippon Pharmaceutical). After the surgery, SD rats were housed individually. MP rats were also housed individually prior to the experiments.
Green odor (Soda Aromatic, Tokyo, Japan) and -pinene (Sigma-Aldrich Co.) were
diluted with triethyl citrate (TEC) on the day of the experiments. The concentration of
odorants in the present study was 0.3 % (w/w), which was 10 times higher than in
previous studies (Sano et al.,
1998;
Akutsu et al., 2002;
Nakashima et al., 2004). We
were able to apply these odorants to rats without any disturbance of free-moving
behaviors, because we used a new odorant delivery system. In this system,
0.03–0.1% odorants did not show any significant effects on
sleep–wakefulness and circadian rhythm (data not shown).
Odorant delivery system
The air, ventilated by an air pump in a flow volume of 3 l/min, was deodorized by
passage through a charcoal packed in U-shaped glass tube. A gas-washing glass bottle with
60 ml of 0.3% odorant solution or vehicle (TEC), connected with silicon tube from
the U-shaped [SANS U] glass tube, was bubbled by an air pump. The other side of
the bottle was connected to a three-way plastic connector for the EEG study or a six-way
plastic connector for the rhythm study. Each pathway was connected to a silicon tube and
opened through a glass funnel over the top of two cylindrical observation cages
(
350 x 400 mm height) or five individual plastic covered cages (300
x 350 x 250 mm) with two ventilation holes. In this system, we attempted to
avoid the effect of odorant chemicals through pathways other than the olfactory sensing
system. We have also confirmed this in other experiments (paper in preparation) in which
the olfactory tract was cut to disrupt odorant-induced changes in this system.
Experimental procedure
Long-term polygraphic records (EEG, EMG, for 7 days; 1 mm/s paper speed, total 605 m length) were obtained simultaneously from two SD rats using an EEG recorder (EEG7410; Nihon Koden Ltd). Vehicle exposures started at 18:00 on the third day and odorant exposures from 18:00 on the fifth day to 12:00 on the sixth day and were performed by changing the gas-washing bottle. The polygraph records were divided into three stages by visual inspection: alertness, slow wave sleep (SWS) and paradoxical sleep (PS). Six SD rats were used for these experiments and four rats were selected for statistical comparison. For statistical analysis, two-way ANOVA followed by the Bonferroni post-tests were applied using Prism 4 (Graphpad Software Inc.).
The circadian locomotor rhythm was measured using an area sensor (F5B; Omron, Tokyo)
and the data were stored in a personal computer by using the Chronobiology Kit (Stanford
Software Systems, CA). Vehicle exposure started at 18:00 on the fifth day and lasted for
5 days, followed by 5 days of odorant exposure. The odorant bottle was changed for a new
one on the third day of odorant exposure. We used >40 MP rats (aged 10–18
months) and selected 35 healthy and normally cyclic rats with aging signals such as
shorter period and lower amplitude in circadian rhythm (
2 periodogram).
Among them, 28 MP rats were selected for exposure to green odor (14 rats) and
-pinene (14 rats). For statistical analysis, the paired t-test was
applied. All experimental procedures followed Guidelines for the Care and Use of
Laboratory Animals, Dokkyo University School of Medicine.
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Results |
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Effects of odorants on sleep
Amounts of each sleep–wakefulness stage were calculated every 2 h by manual measuring using 7 day records. The two-way ANOVA revealed no significant differences in mean 2 h amounts of each sleep–wakefulness stage over 24 h all day (06:00–06:00) or over 12 h of daytime (06;00–18:00) between the control and odorant-treated rats. There was no significant difference in alertness and SWS during nighttime (18:00–06:00) between the vehicle and odorant-treated rats, however, the odorant-treated rats showed significant increase in the duration of PS at the 22:00–0:00 period compared with the vehicle-treated rats (Table 1).
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Effects of odorants on circadian locomortor rhythm activity
Circadian locomortor rhythms were measured using 10 day records within several-month
rhythm records in aged (10–18 months) MP rats. Five days exposure to both odors
induced a significant increase in circadian amplitude in 2 periodograms.
On the other hand, both odors slightly increased the circadian period, but there was no
significant difference (Table
2).
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Discussion |
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In the first experiment, a significant increase in the 2 h appearance of PS was observed during the night-time zone (22:00–00:00) in adult SD male rats exposed to plant-derived odorants. The rats dominantly sleep during daytime, but they have small peaks of SWS and PS during the same time zone without any treatment. We have reported similar phenomena in ovariectomized female rats, orchiectomized male rats, septally lesioned rats and aged rats which showed te marked night-time PS peaks (Yamaoka, 1978
, 1980, 1983).
MP rats, a congenital mutant rat with growth inhibition of the optic cup (Kobayashi and Otani, 1981), showed an increase in
amplitude of circadian activity in 2 periodograms during 5 days of
exposure to odors. MP rats showed regular free-running circadian rhythm without
entraining by the light–dark cycle (Shim
et al., 1997). It is well known that aging decreases circadian
amplitude and shortens the circadian period. The aging effects of MP rats also started
from 
10 months of age and the rhythmicity was almost disrupted by 24 months of
age (data not shown).
Amir et al. (1999) postulated
that olfactory stimulation by cedar wood essence enhanced light-induced phase shift of
wheel-running rhythms and suprachiasmatic Fos expression. These findings suggest that
these plant-derived odors stimulate olfactory cues and modulate circadian rhythm.
Our previous reports showed that estradiol-treated OVX rats and rats with posterior
deafferentation of the hypothalamus increased the amplitude of circadian SWS and PS
rhythms (Yamaoka, 1978, 1980, 1983).
The present studies and our previous reports may suggest that green odor and
-pinene accelerate PS and ameliorate aging effects, in which some neuroendocrine
mechanisms may be involved.
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References |
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TopIntroductionMaterials and methodsResultsDiscussionReferences |
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Akutsu, H., Kikusui, T., Takeuchi, Y., Sano, K., Hatanaka, A. and Mori, Y. (2002) Alleviating effects of plant-derived fragrances on stress-induced hyperthermia in rats. Physiol. Behav., 75, 355–360.[CrossRef][Medline]
Amir, S., Cain, S., Sullivan, J., Robinson, B. and Stewart, J. (1999) Olfactory stimulation enhances light-induced phase shifts in free-running activity rhythms and Fos expression in the suprachiasmatic nucleus. Neuroscience, 92, 1165–1170.[CrossRef][Web of Science][Medline]
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