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Day–night difference in thermoregulatory responses to olfactory stimulation
Neuroscience Letters 439 (2008) 192–197
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Day–night difference in thermoregulatory responses to olfactory stimulation Mamoru Tanida a,b,c,∗ , Jiao Shen a,b , Takuo Nakamura a , Akira Niijima d , Katsuya Nagai a,b a
Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan ANBAS Corporation, Kita-ku, Osaka 531-0072, Japan c College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan d Niigata University School of Medicine, Niigata, Niigata 951-8510, Japan b
a r t i c l e
i n f o
Article history: Received 3 March 2008 Received in revised form 2 May 2008 Accepted 7 May 2008 Keywords: Sympathetic nerve Suprachiasmatic nucleus Body temperature Circadian rhythm Brown adipose tissue
a b s t r a c t Previously, we observed that olfactory stimulation with scent of grapefruit oil (SGFO) or scent of lavender oil (SLVO) affected, elevated or lowered brown adipose tissue temperature (BAT-T) in conscious mice, respectively. In the present study, to test the day–night difference in the actions of olfactory stimulations, we examined the responses of BAT-T and body temperature (BT) measured as the abdominal temperature to SGFO or SLVO during day-time at 14:00 and night-time at 2:00 in conscious rats. In the light period, BATT and BT were suppressed after SLVO and elevated after SGFO whereas in the dark period, these parameters remained unchanged with olfactory stimulations. Bilateral lesions of the hypothalamic suprachiasmatic nucleus (SCN) eliminated the effects of olfactory stimulations with SGFO and SVLO on BAT-T and BT. Moreover, sympathetic nerve activity innervating brown adipose tissue (BAT-SNA) changes after SGFO or SLVO were abolished in SCN-lesioned rats. Thus, we concluded that there is day–night difference in the effects of SGFO or SLVO on BAT-T and BT, and that the SCN might be involved in these effects. © 2008 Elsevier Ireland Ltd. All rights reserved.
Brown adipose tissue (BAT) functions as a thermogenic organ, that produces heat in the mitochondria via the activation of uncoupling protein-1, which shunts the energy obtained from the oxidation of free fatty acids into heat; this heat is then stored in the BAT vessel and distributed throughout the body. Thermogenesis is evoked by acceleration of BAT-sympathetic nerve activity (BATSNA) secondary to the commands from the autonomic center in the hypothalamus [11]. The functions of the circadian rhythm is to maintain homeostatic or vital parameters such as blood pressure, body temperature (BT) and energy expenditure. It is known that circadian rhythm functions as a biological clock and is located in the suprachiasmatic nucleus (SCN) of the hypothalamus, which functions as a circadian oscillator and controls the rhythm of these homeostatic functions [3]. Recently, we observed evidences suggesting that the SCN is connected to peripheral tissue via the autonomic nerves [2], and that biphasic effects of l-carnosine, a di-peptide hormone of muscle origin with central nervous system activity, on BAT-SNA and BT were eliminated in SCN-lesioned rats [19], indicating that the SCN modulates autonomic nerves to achieve BT control.
∗ Corresponding author at: College of Life Sciences, Ritsumeikan University, 1-1-1 Noji-kita, Kusatsu, Shiga 525-8577, Japan. Tel.: +81 77 561 3378; fax: +81 77 561 2629. E-mail address: [email protected] (M. Tanida). 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.05.021
Our recent studies found that olfactory stimulation with the scent of grapefruit oil (SGFO) or lavender oil (SLVO) in the light period elevated or lowered, respectively, brown adipose tissue temperature (BAT-T) and BT through changes in the autonomic nerve activities [16,17]. Conversely, Granados-Fuentes et al. reported that there is day–night difference in olfactory responsiveness in the brain [5]. However, it has not been examined whether olfactory stimulations affect BAT-T and BT in the dark period as well as in the light period. Thus, in the present study, we investigated the effects of olfactory stimulation with SGFO or SLVO on thermogenesis both at day-time and night-time by measuring BAT-T and BT in conscious rats. Moreover, our previous studies observed that bilateral lesions in the SCN abolished not only suppressive actions of SLVO, but also the enhancing effects of SGFO on both renal sympathetic outflow and blood pressure [20,21]. Hence, we determined the possible role of the SCN in olfactory stimulation-induced changes of BAT-T, BT and BAT-SNA using SCN-lesioned rats. Male Wistar rats weighing 280–350 g were used (n = 100). Rats were housed in a cage and the room was maintained at 24 ± 1 ◦ C and illuminated for 12 h (08:00–20:00) daily. Food and water was provided ad libitum. Rats were made to adapt to the environment at least 1 week prior to the experiment. All animal care and handling procedures were approved by the Institutional Animal Care and Use Committee of Osaka University. To measure the BT and BAT-T, the Telemetry System (Star Medical Corp., Tokyo, Japan) was used as described previously [24]. In
M. Tanida et al. / Neuroscience Letters 439 (2008) 192–197
a subset of rats (n = 36), a capsule containing a temperature sensor, battery, and transmitter was either implanted into the abdominal cavity or placed above the BAT fat pad under the influence of intraperitoneal (IP) pentobarbital anesthesia (35 mg/kg), 10–14 days prior to olfactory stimulation. On the day of the experiment, the rats were made to fast 4 h prior to stimulation to avoid evoking diet-induced thermogenesis. With the animal being in the conscious state, baseline measurements of BAT-T or BT were recorded for 5 min prior to olfactory stimulation with water, SGFO [grapefruit oil (Citrus Paradisii, Pranarom International, Belgium) suspended in 10,000 volumes of water] or SLVO [lavender oil (Lavendula angustifolia, Pranarom International, Belgium) suspended in 100,000 volumes of water] in the light (14:00) or the dark (2:00) period. The stimulation method and effective dose were determined by our previous studies [16,17]. After stimulation, these parameters were recorded for 120 min. General preparation for electro-physiological experiment was carried out as described previously [19]. In brief, on the day of the experiment, the animals were made to fast 4–6 h prior to surgery. Anesthesia was induced by an IP injection of 1 g/kg urethane, and a polyethylene catheter was inserted into the left femoral vein for injections. After tracheal cannulation, the left sympathetic nerve innervating interscapular BAT was exposed, ligated and attached to a pair of silver wire electrodes. The rat was allowed to stabilize for
193
30–60 min after the nerve was placed on the recording electrodes. The BAT-SNA was amplified, filtered, monitored on an oscilloscope, and converted to standard pulses by a window discriminator. For olfactory stimulation, grapefruit and lavender essential oils were suspended in a 100-fold volume of water, the concentration of which was determined by our previous studies [16,17]. On completion of the experiment hexamethonium chloride was administered (10 mg/kg) intravenously to ensure that the recording was generated due to post-ganglionic efferent sympathetic nerve activity. In some rats (n = 15), 2–3 weeks prior to the olfactory stimulation, bilateral electrolytic lesions were made in the SCN by experimental methods described previously [4]. In brief, under the influence of pentobarbital anesthesia (35 mg/kg, IP), a stainless steel electrode was inserted into the SCN with coordinates [A–P, 1.2 mm posterior to the bregma; L, 0 mm from the midline; V, 9.0 mm from the skull surface] of the atlas of Paxisons and Watson [13], and a 1.0-mA anodal direct current was passed through the electrode for 20 s. Control rats (n = 17) were sham-operated using the same procedure without the current. After the operation, only SCN-lesioned rats with complete loss of circadian rhythm activity were used. Recordings of BAT-SNA, BAT-T and BT of sham-operated and SCN-lesioned rats were performed in the light period (14:00). On completion of the experiment, the brain was dissected from each rat and a histological examination was performed to verify
Fig. 1. Effects of olfactory stimulation with SGFO or SLVO on brown adipose tissue temperature (BAT-T) and body temperature (BT) in light and dark periods. Time course changes in BAT-T (A) and BT (B) after olfactory stimulation with water, SGFO or SLVO in light or in dark periods are expressed as mean ± S.E.M. of temperature change from values at 0 min. Numbers of animals used are shown in the parentheses. *Significant differences between water group and olfactory stimulation groups (P < 0.05).
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M. Tanida et al. / Neuroscience Letters 439 (2008) 192–197
Table 1 Basal levels (chain) of BAT-SNA, BAT-T and BT in experimental group Groups
BAT-SNA (spikes/5 s)
Experiment 1 Light period, water Light period. SGFO Light period, SLVO Dark period, water Dark period, SGFO Dark period, SLVO Experiment 2 Sham, SGFO Sham, SLVO SCNL, SGFO SCNL, SLVO
(n) 134.8 ± 30.2 (5) 138.3 ± 43.6 (4) 158.4 ± 15.7 (4) 126.2 ± 39.1 (3)
BAT-T (◦ C)
BT (◦ C)
(n) 36.1 ± 1.1 (3) 36.3 ± 1.0 (3) 36.2 ± 0.P (3) 36.9 ± 0.4 (3) 36.7 ± 0.2 (3) 37.2 ± 0.2(3)
(n) 37.3 ± 0.3 (3) 37.3 ± 0.9 (3) 37.3 ± 0.8 (3) 38.0 ± 0.2 (3) 38.4 ± 0.7 (3) 37.7 ± 0.3 (3)
(n) 37.6 ± 0.8 (4) 37.8 ± 0.9 (4) 37.4 ± 0.3 (4) 37.6 ± 0.5 (4)
(n) 38.4 ± 0.6 (4) 38.6 ± 0.6 (4) 38.5 ± 0.3 (4) 38.1 ± 0.6 (4)
BAT-SNA: brown adipose tissue sympathetic nerve activity; BAT-T: brown adipose tissue temperature; BT: body temperature. Data are presented as mean + S.E.M. (n) = number of rats.
adequate placement of bilateral lesions in the SCN by cresyl violet staining. Only rats with accurately placed lesions were used as SCN-lesioned animals. The BAT-SNA, BAT-T, and BT were measured every 5 min after olfactory stimulation with SGFO, SLVO or water and was evaluated by digital signal processing and statistical analyses. All data were expressed as mean ± S.E.M. Difference in basal values (0 min) was determined by the Mann–Whitney U-test. Because of the inter-individual variability in the pre-injection state, percent and temperature changes from the baseline were calculated for BAT-SNA and temperature data, respectively. To compare group responses of BAT-SNA, BAT-T and BT, analysis of variance (ANOVA) and post hoc tests were performed. When analysis reached significance, Fisher’s PLSD (when there are equal number of animals in each group) or the Tukey–Kramer (when the number of animals differs between groups) tests were used to compare the mean values among the different levels of rat stimulations and treatments. We examined whether time-dependent effects of olfactory stimulation with SGFO or SLVO on BAT-T and BT were evoked in the presence of light (14:00) and dark (2:00) periods. As shown in Fig. 1, in the light period, BAT-T and BT were significantly elevated by olfactory stimulation with SGFO (Fig. 1A and B), while they were significantly suppressed by olfactory stimulation with SLVO (Fig. 1A and B). Following SGFO, BAT-T (Fig. 1A) and BT (Fig. 1B) increased gradually, with the greatest levels of elevations occurring at 95 and 120 min, respectively; the highest levels were attained +0.85 ± 0.14 and +0.66 ± 0.13 ◦ C, respectively. These elevations were significant beginning 5 min after SGFO. Following SLVO, BAT-T (Fig. 1A) and BT (Fig. 1B) decreased gradually, with the greatest level of suppressions occurring at 90 and 70 min, respectively; the lowest levels were attained at −0.59 ± 0.02 and −0.71 ± 0.29 ◦ C, respectively. These suppressions were significant from 35 to 50 min after SLVO. In the dark period, SGFO and SLVO did not evoke significant changes of BAT-T and BT. Absolute basal (0 min) BAT-T and BT values for the experiments shown in Fig. 1 are summarized in Table 1. Differences in respective basal values were not statistically significant. Fig. 2A depicts representative photomicrographs of sections including the SCN from sham-operated and lesioned rats. The success rate of the SCN lesions was 15 of 47 rats. In some SCN-lesioned rats, a part of the optic chiasm was damaged as well. However, a pupillary reflex was induced with bilateral light stimulation of the eyes in all SCN-lesioned rats used in the study. In addition, the rhythmic locomotor activity, a constant pattern repeating action of about 12 h and rest of about 12 h, disappeared in all SCN-lesioned rats (Fig. 2B). Fig. 2 summarizes the data from both sham-operated and SCN-lesioned rats. In sham-operated rats, the levels of BAT-T
and BT were significantly elevated by SGFO, and reduced by SLVO. However, in SCN-lesioned rats, olfactory stimulation with neither SGFO nor SLVO resulted in any effect on levels of BAT-T and BT (Fig. 2C). Thus, bilateral lesions of the SCN eliminated both the elevating effects of the SGFO and the suppressing effects of the SLVO on BAT-T and BT. Absolute basal (0 min) BAT-T and BT values for the experiments are shown in Fig. 2 and summarized in Table 1. Differences in respective basal values were not statistically significant. Sample recordings of BAT-SNA before and throughout a 70-min period following olfactory stimulation with SGFO or SLVO in the light period (14:00) are presented in Fig. 3A. BAT-SNA was elevated by SGFO and was suppressed by SLVO. Fig. 3B summarizes the data from both sham-operated and SCN-lesioned rats. In sham-operated rats, the level of BAT-SNA was significantly reduced by SLVO with significant suppression beginning at 50 min after stimulation, and elevated by SGFO with significant activation beginning at 55 min after stimulation. However, in SCN-lesioned rats, neither SGFO nor SLVO caused any effect in the level of BAT-SNA (Fig. 3B). Thus, bilateral lesions of the SCN eliminated both the suppressive effects of SLVO and the elevating effects of SGFO on BAT-SNA. Absolute basal (0 min) BAT-SNA values for the experiments are shown in Fig. 3 and summarized in Table 1. Differences in respective basal values were not statistically significant. In aromatherapy, it is known that the aroma of grapefruit is useful in postpartum perineal healing [7], and the aroma of lavender is beneficial in relaxing the mind [9]. Since close relations have been demonstrated between psychology and physiological conditions such as cardiovascular and thermal responses in humans [6,15], it is possible that these vital functions in mammals are affected by odor sensation. Moreover, we obtained the data that olfactory stimulation with SGFO or SLVO elevated or suppressed, respectively, BAT-T in mice [16,17], thereby supporting this theory. However, the effects of olfactory stimulation on thermogenic function of rats have not been examined. The present study demonstrated that SGFO or SLVO elevated or suppressed, respectively, BAT-T and BT of rats (Fig. 1). Thus, it is suggested that not only mice but also rats, have regulatory actions of odor stimulation on BT and BAT-T. Since it has been recently shown that there is a day–night difference in responses of c-Fos expression in the olfactory bulb to scent stimulation with a decline in the subjective day period and elevation in the subjective night period under constant darkness condition [5], our present study investigated the effects of SGFO or SLVO on BAT-T and BT in light and dark periods. Our result that that the thermal effects of SGFO or SLVO were preserved in the light period, but not in the dark period supports the idea that the responses of BAT-T and BT to odor exposure are dependent on circadian time. Currently, we do not have a detailed mechanism to explain the circadian changes in these autonomic responses, but elevation of basal BT in the dark period [10] might reach a saturation point, resulting in decreased responsiveness to activity of the olfactory pathways. In other words, it is possible that neurons of the autonomic center in the hypothalamus containing the SCN might be nonreactive to olfactory stimulation with SGFO or SLVO in the dark period secondary to effects of the circadian cycle on thermoregulatory organs. To investigate this theory, evaluation of the circadian effects of SGFO or SLVO on c-Fos expression of the SCN needs to be evaluated in the future. To determine the mechanisms of time-dependent effects of olfactory stimulation on BAT-T and BT, we noted that the SCN is thought to be a master circadian oscillator of circadian rhythms in synchronization with the light–dark cycle, regulating the daily rhythm of locomotive activity [4,10], feeding [12], energy metabolism, and thermoregulation [10]. In fact, abnormal rhythm of BT was detected in SCN-lesioned rats [10]. Moreover, we pre-
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Fig. 2. Effects of bilateral lesions of the SCN on changes in brown adipose tissue temperature (BAT-T) and body temperature (BT) after olfactory stimulation with SGFO or SLVO. Representative photomicrographs of coronal sections including the SCN from sham-operated (Sham) rats and from rats that received SCN lesions (SCNL) (A). SCN, the suprachiasmatic nucleus (arrows show the intact bilateral SCN in the SCN–sham rats); 3 V, the third ventricle. Scale bars = 300 m. Representative locomotor activity of Shamand SCN-lesioned (SCNL) rats under constant dark conditions are presented as a double plot (B). BAT-T and BT after olfactory stimulation with SGFO or SLVO are expressed as mean ± S.E.M. of temperature change from values at 0 min (C). Data from sham-operated (sham) and SCN-lesioned (SCNL) rats are shown. *P < 0.05 vs. sham rats.
viously confirmed that bilateral lesions of the SCN eliminated cardiovascular responses to SGFO or SLVO [20,21]. Thus, we tested the effects of SGFO or SLVO on BAT-T and BT in SCN-lesioned rats, and found that bilateral lesions of the SCN completely eliminated the responses of BAT-T and BT to SGFO or SLVO (Fig. 2). These findings suggest that the SCN might mediate the olfactory modulations of BAT-T and BT. Conversely, BIT/SHPS-1, a transmembrane glycoprotein implicated in signal transduction of growth factors and cell-to-cell interaction [14], has been identified in our previous examinations exploring the brain substance associated with BT modulation [23]. BIT/SHPS-1 has tyrosine phosphorylation sites in its intracellular domain, and is associated with the protein tyrosine phosphatase SHP-2 upon phosphorylation. Hypothalamic injection
of an anti-BIT/SHPS-1 monoclonal antibody, which reacts with the extracellular domain of BIT/SHPS-1 and stimulates tyrosine phosphorylation, enhances BAT-T and BT [24]. In addition, olfactory stimulation with SGFO or SLVO enhanced or attenuated, respectively, the tyrosine phosphorylation of BIT/SHPS-1 in the SCN [18]. Consistently, we previously observed that immunoreactivity of cFos, a marker of neuronal activation, in the SCN was changed after exposure to SGFO or SLVO [22]. These findings suggested that olfactory stimulation might affect SCN neurons and change tyrosine phosphorylation level of BIT/SHPS-1 in the SCN. Therefore, it is possible that tyrosine phosphorylation of BIT/SHPS-1 in the SCN might function as a molecular regulator of the central mechanism mediating BAT-T and BT changes due to SGFO or SLVO.
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M. Tanida et al. / Neuroscience Letters 439 (2008) 192–197
Fig. 3. Effects of bilateral lesions of the SCN on changes in neural activity innervating brown adipose tissue (BAT-SNA) after olfactory stimulation with SGFO or SLVO. Representative trace data from recordings of BAT-SNA of a sham-operated (sham) rat and a SCN-lesioned (SCNL) rat before and after stimulation with SGFO or SLVO (A). Lower bars represent 20 min, vertical scale bars to the right of the recordings represent neural activity rates of 200 spikes/5 s.Time course change in BAT-SNA (B) after olfactory stimulation with water, SGFO or SLVO is expressed as mean ± S.E.M. of percentages of values at 0 min. Data from sham-operated (sham) and SCN-lesioned (SCNL) rats are shown. *P < 0.05 vs. sham rats.
Sympathetic nerves supplying BAT are an important regulator of BAT thermogenesis. We previously confirmed that SGFO or SLVO accelerated or suppressed, respectively, BAT-SNA in urethaneanesthetized rats [16,17], suggesting the existence of autonomic pathways between the SCN and peripheral organs [2]. With regard to BAT, a neural connection between the SCN and BAT was found in a pseudorabies virus study [1]. To determine whether the SCN played a role in olfactory stimulation-evoked change of BAT-SNA, our study investigated the effects of a SCN-lesion on BAT-SNA change after olfactory stimulation with SGFO or SLVO. This study provides new evidence that bilateral lesions of the SCN clearly abolished the response of BAT-SNA to SGFO or SLVO (Fig. 3) and supported the idea that the SCN is involved in the effects of olfactory stimulation with SGFO or SLVO on BAT-SNA. Moreover, ablation of the olfactory bulb of rats eliminates renal sympathetic responses to SGFO or SLVO (unpublished data). Currently, the exact mechanism through which the olfactory pathway affects the SCN and changes BAT-SNA is unclear. Previous studies confirmed that there was a neural connection between the olfactory bulb and the SCN [8], and the efferent pathway from the SCN to BAT via a sympathetic neu-
ral route was determined in an anatomical study [1]. Brain stem regions, such as the ventrolateral medulla and the raphe pallidus, contain sympathetic pre-motoneurons controlling BAT-SNA and exist as intermediate sites of the pathway [11]. It is possible that scent signals received in the olfactory bulb might be sent to the SCN via a neural pathway, and following BAT-SNA, BAT-T, and BT might be affected by a central mechanism. In conclusion, the present findings suggest that olfactory stimulation with SGFO or SLVO acts in a time-dependent manner, affecting BAT-T and BT. Furthermore, SCN lesions suppressed the effects of olfactory stimulation on BAT-T, BT, and BAT-SNA. Therefore, these data suggest that the SCN might be involved in the effects of olfactory stimulation with SGFO or SLVO on BAT-SNA and BT. However, further study is required to reveal the precise pathway responsible for these effects. References [1] M. Bamshad, C.K. Song, T.J. Bartness, CNS origins of the sympathetic nervous system outflow to brown adipose tissue, Am. J. Physiol. 276 (1999) R1569–1578.
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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Day–night difference in thermoregulatory responses to olfactory stimulation Mamoru Tanida a,b,c,∗ , Jiao Shen a,b , Takuo Nakamura a , Akira Niijima d , Katsuya Nagai a,b a
Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan ANBAS Corporation, Kita-ku, Osaka 531-0072, Japan c College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan d Niigata University School of Medicine, Niigata, Niigata 951-8510, Japan b
a r t i c l e
i n f o
Article history: Received 3 March 2008 Received in revised form 2 May 2008 Accepted 7 May 2008 Keywords: Sympathetic nerve Suprachiasmatic nucleus Body temperature Circadian rhythm Brown adipose tissue
a b s t r a c t Previously, we observed that olfactory stimulation with scent of grapefruit oil (SGFO) or scent of lavender oil (SLVO) affected, elevated or lowered brown adipose tissue temperature (BAT-T) in conscious mice, respectively. In the present study, to test the day–night difference in the actions of olfactory stimulations, we examined the responses of BAT-T and body temperature (BT) measured as the abdominal temperature to SGFO or SLVO during day-time at 14:00 and night-time at 2:00 in conscious rats. In the light period, BATT and BT were suppressed after SLVO and elevated after SGFO whereas in the dark period, these parameters remained unchanged with olfactory stimulations. Bilateral lesions of the hypothalamic suprachiasmatic nucleus (SCN) eliminated the effects of olfactory stimulations with SGFO and SVLO on BAT-T and BT. Moreover, sympathetic nerve activity innervating brown adipose tissue (BAT-SNA) changes after SGFO or SLVO were abolished in SCN-lesioned rats. Thus, we concluded that there is day–night difference in the effects of SGFO or SLVO on BAT-T and BT, and that the SCN might be involved in these effects. © 2008 Elsevier Ireland Ltd. All rights reserved.
Brown adipose tissue (BAT) functions as a thermogenic organ, that produces heat in the mitochondria via the activation of uncoupling protein-1, which shunts the energy obtained from the oxidation of free fatty acids into heat; this heat is then stored in the BAT vessel and distributed throughout the body. Thermogenesis is evoked by acceleration of BAT-sympathetic nerve activity (BATSNA) secondary to the commands from the autonomic center in the hypothalamus [11]. The functions of the circadian rhythm is to maintain homeostatic or vital parameters such as blood pressure, body temperature (BT) and energy expenditure. It is known that circadian rhythm functions as a biological clock and is located in the suprachiasmatic nucleus (SCN) of the hypothalamus, which functions as a circadian oscillator and controls the rhythm of these homeostatic functions [3]. Recently, we observed evidences suggesting that the SCN is connected to peripheral tissue via the autonomic nerves [2], and that biphasic effects of l-carnosine, a di-peptide hormone of muscle origin with central nervous system activity, on BAT-SNA and BT were eliminated in SCN-lesioned rats [19], indicating that the SCN modulates autonomic nerves to achieve BT control.
∗ Corresponding author at: College of Life Sciences, Ritsumeikan University, 1-1-1 Noji-kita, Kusatsu, Shiga 525-8577, Japan. Tel.: +81 77 561 3378; fax: +81 77 561 2629. E-mail address: [email protected] (M. Tanida). 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.05.021
Our recent studies found that olfactory stimulation with the scent of grapefruit oil (SGFO) or lavender oil (SLVO) in the light period elevated or lowered, respectively, brown adipose tissue temperature (BAT-T) and BT through changes in the autonomic nerve activities [16,17]. Conversely, Granados-Fuentes et al. reported that there is day–night difference in olfactory responsiveness in the brain [5]. However, it has not been examined whether olfactory stimulations affect BAT-T and BT in the dark period as well as in the light period. Thus, in the present study, we investigated the effects of olfactory stimulation with SGFO or SLVO on thermogenesis both at day-time and night-time by measuring BAT-T and BT in conscious rats. Moreover, our previous studies observed that bilateral lesions in the SCN abolished not only suppressive actions of SLVO, but also the enhancing effects of SGFO on both renal sympathetic outflow and blood pressure [20,21]. Hence, we determined the possible role of the SCN in olfactory stimulation-induced changes of BAT-T, BT and BAT-SNA using SCN-lesioned rats. Male Wistar rats weighing 280–350 g were used (n = 100). Rats were housed in a cage and the room was maintained at 24 ± 1 ◦ C and illuminated for 12 h (08:00–20:00) daily. Food and water was provided ad libitum. Rats were made to adapt to the environment at least 1 week prior to the experiment. All animal care and handling procedures were approved by the Institutional Animal Care and Use Committee of Osaka University. To measure the BT and BAT-T, the Telemetry System (Star Medical Corp., Tokyo, Japan) was used as described previously [24]. In
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a subset of rats (n = 36), a capsule containing a temperature sensor, battery, and transmitter was either implanted into the abdominal cavity or placed above the BAT fat pad under the influence of intraperitoneal (IP) pentobarbital anesthesia (35 mg/kg), 10–14 days prior to olfactory stimulation. On the day of the experiment, the rats were made to fast 4 h prior to stimulation to avoid evoking diet-induced thermogenesis. With the animal being in the conscious state, baseline measurements of BAT-T or BT were recorded for 5 min prior to olfactory stimulation with water, SGFO [grapefruit oil (Citrus Paradisii, Pranarom International, Belgium) suspended in 10,000 volumes of water] or SLVO [lavender oil (Lavendula angustifolia, Pranarom International, Belgium) suspended in 100,000 volumes of water] in the light (14:00) or the dark (2:00) period. The stimulation method and effective dose were determined by our previous studies [16,17]. After stimulation, these parameters were recorded for 120 min. General preparation for electro-physiological experiment was carried out as described previously [19]. In brief, on the day of the experiment, the animals were made to fast 4–6 h prior to surgery. Anesthesia was induced by an IP injection of 1 g/kg urethane, and a polyethylene catheter was inserted into the left femoral vein for injections. After tracheal cannulation, the left sympathetic nerve innervating interscapular BAT was exposed, ligated and attached to a pair of silver wire electrodes. The rat was allowed to stabilize for
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30–60 min after the nerve was placed on the recording electrodes. The BAT-SNA was amplified, filtered, monitored on an oscilloscope, and converted to standard pulses by a window discriminator. For olfactory stimulation, grapefruit and lavender essential oils were suspended in a 100-fold volume of water, the concentration of which was determined by our previous studies [16,17]. On completion of the experiment hexamethonium chloride was administered (10 mg/kg) intravenously to ensure that the recording was generated due to post-ganglionic efferent sympathetic nerve activity. In some rats (n = 15), 2–3 weeks prior to the olfactory stimulation, bilateral electrolytic lesions were made in the SCN by experimental methods described previously [4]. In brief, under the influence of pentobarbital anesthesia (35 mg/kg, IP), a stainless steel electrode was inserted into the SCN with coordinates [A–P, 1.2 mm posterior to the bregma; L, 0 mm from the midline; V, 9.0 mm from the skull surface] of the atlas of Paxisons and Watson [13], and a 1.0-mA anodal direct current was passed through the electrode for 20 s. Control rats (n = 17) were sham-operated using the same procedure without the current. After the operation, only SCN-lesioned rats with complete loss of circadian rhythm activity were used. Recordings of BAT-SNA, BAT-T and BT of sham-operated and SCN-lesioned rats were performed in the light period (14:00). On completion of the experiment, the brain was dissected from each rat and a histological examination was performed to verify
Fig. 1. Effects of olfactory stimulation with SGFO or SLVO on brown adipose tissue temperature (BAT-T) and body temperature (BT) in light and dark periods. Time course changes in BAT-T (A) and BT (B) after olfactory stimulation with water, SGFO or SLVO in light or in dark periods are expressed as mean ± S.E.M. of temperature change from values at 0 min. Numbers of animals used are shown in the parentheses. *Significant differences between water group and olfactory stimulation groups (P < 0.05).
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Table 1 Basal levels (chain) of BAT-SNA, BAT-T and BT in experimental group Groups
BAT-SNA (spikes/5 s)
Experiment 1 Light period, water Light period. SGFO Light period, SLVO Dark period, water Dark period, SGFO Dark period, SLVO Experiment 2 Sham, SGFO Sham, SLVO SCNL, SGFO SCNL, SLVO
(n) 134.8 ± 30.2 (5) 138.3 ± 43.6 (4) 158.4 ± 15.7 (4) 126.2 ± 39.1 (3)
BAT-T (◦ C)
BT (◦ C)
(n) 36.1 ± 1.1 (3) 36.3 ± 1.0 (3) 36.2 ± 0.P (3) 36.9 ± 0.4 (3) 36.7 ± 0.2 (3) 37.2 ± 0.2(3)
(n) 37.3 ± 0.3 (3) 37.3 ± 0.9 (3) 37.3 ± 0.8 (3) 38.0 ± 0.2 (3) 38.4 ± 0.7 (3) 37.7 ± 0.3 (3)
(n) 37.6 ± 0.8 (4) 37.8 ± 0.9 (4) 37.4 ± 0.3 (4) 37.6 ± 0.5 (4)
(n) 38.4 ± 0.6 (4) 38.6 ± 0.6 (4) 38.5 ± 0.3 (4) 38.1 ± 0.6 (4)
BAT-SNA: brown adipose tissue sympathetic nerve activity; BAT-T: brown adipose tissue temperature; BT: body temperature. Data are presented as mean + S.E.M. (n) = number of rats.
adequate placement of bilateral lesions in the SCN by cresyl violet staining. Only rats with accurately placed lesions were used as SCN-lesioned animals. The BAT-SNA, BAT-T, and BT were measured every 5 min after olfactory stimulation with SGFO, SLVO or water and was evaluated by digital signal processing and statistical analyses. All data were expressed as mean ± S.E.M. Difference in basal values (0 min) was determined by the Mann–Whitney U-test. Because of the inter-individual variability in the pre-injection state, percent and temperature changes from the baseline were calculated for BAT-SNA and temperature data, respectively. To compare group responses of BAT-SNA, BAT-T and BT, analysis of variance (ANOVA) and post hoc tests were performed. When analysis reached significance, Fisher’s PLSD (when there are equal number of animals in each group) or the Tukey–Kramer (when the number of animals differs between groups) tests were used to compare the mean values among the different levels of rat stimulations and treatments. We examined whether time-dependent effects of olfactory stimulation with SGFO or SLVO on BAT-T and BT were evoked in the presence of light (14:00) and dark (2:00) periods. As shown in Fig. 1, in the light period, BAT-T and BT were significantly elevated by olfactory stimulation with SGFO (Fig. 1A and B), while they were significantly suppressed by olfactory stimulation with SLVO (Fig. 1A and B). Following SGFO, BAT-T (Fig. 1A) and BT (Fig. 1B) increased gradually, with the greatest levels of elevations occurring at 95 and 120 min, respectively; the highest levels were attained +0.85 ± 0.14 and +0.66 ± 0.13 ◦ C, respectively. These elevations were significant beginning 5 min after SGFO. Following SLVO, BAT-T (Fig. 1A) and BT (Fig. 1B) decreased gradually, with the greatest level of suppressions occurring at 90 and 70 min, respectively; the lowest levels were attained at −0.59 ± 0.02 and −0.71 ± 0.29 ◦ C, respectively. These suppressions were significant from 35 to 50 min after SLVO. In the dark period, SGFO and SLVO did not evoke significant changes of BAT-T and BT. Absolute basal (0 min) BAT-T and BT values for the experiments shown in Fig. 1 are summarized in Table 1. Differences in respective basal values were not statistically significant. Fig. 2A depicts representative photomicrographs of sections including the SCN from sham-operated and lesioned rats. The success rate of the SCN lesions was 15 of 47 rats. In some SCN-lesioned rats, a part of the optic chiasm was damaged as well. However, a pupillary reflex was induced with bilateral light stimulation of the eyes in all SCN-lesioned rats used in the study. In addition, the rhythmic locomotor activity, a constant pattern repeating action of about 12 h and rest of about 12 h, disappeared in all SCN-lesioned rats (Fig. 2B). Fig. 2 summarizes the data from both sham-operated and SCN-lesioned rats. In sham-operated rats, the levels of BAT-T
and BT were significantly elevated by SGFO, and reduced by SLVO. However, in SCN-lesioned rats, olfactory stimulation with neither SGFO nor SLVO resulted in any effect on levels of BAT-T and BT (Fig. 2C). Thus, bilateral lesions of the SCN eliminated both the elevating effects of the SGFO and the suppressing effects of the SLVO on BAT-T and BT. Absolute basal (0 min) BAT-T and BT values for the experiments are shown in Fig. 2 and summarized in Table 1. Differences in respective basal values were not statistically significant. Sample recordings of BAT-SNA before and throughout a 70-min period following olfactory stimulation with SGFO or SLVO in the light period (14:00) are presented in Fig. 3A. BAT-SNA was elevated by SGFO and was suppressed by SLVO. Fig. 3B summarizes the data from both sham-operated and SCN-lesioned rats. In sham-operated rats, the level of BAT-SNA was significantly reduced by SLVO with significant suppression beginning at 50 min after stimulation, and elevated by SGFO with significant activation beginning at 55 min after stimulation. However, in SCN-lesioned rats, neither SGFO nor SLVO caused any effect in the level of BAT-SNA (Fig. 3B). Thus, bilateral lesions of the SCN eliminated both the suppressive effects of SLVO and the elevating effects of SGFO on BAT-SNA. Absolute basal (0 min) BAT-SNA values for the experiments are shown in Fig. 3 and summarized in Table 1. Differences in respective basal values were not statistically significant. In aromatherapy, it is known that the aroma of grapefruit is useful in postpartum perineal healing [7], and the aroma of lavender is beneficial in relaxing the mind [9]. Since close relations have been demonstrated between psychology and physiological conditions such as cardiovascular and thermal responses in humans [6,15], it is possible that these vital functions in mammals are affected by odor sensation. Moreover, we obtained the data that olfactory stimulation with SGFO or SLVO elevated or suppressed, respectively, BAT-T in mice [16,17], thereby supporting this theory. However, the effects of olfactory stimulation on thermogenic function of rats have not been examined. The present study demonstrated that SGFO or SLVO elevated or suppressed, respectively, BAT-T and BT of rats (Fig. 1). Thus, it is suggested that not only mice but also rats, have regulatory actions of odor stimulation on BT and BAT-T. Since it has been recently shown that there is a day–night difference in responses of c-Fos expression in the olfactory bulb to scent stimulation with a decline in the subjective day period and elevation in the subjective night period under constant darkness condition [5], our present study investigated the effects of SGFO or SLVO on BAT-T and BT in light and dark periods. Our result that that the thermal effects of SGFO or SLVO were preserved in the light period, but not in the dark period supports the idea that the responses of BAT-T and BT to odor exposure are dependent on circadian time. Currently, we do not have a detailed mechanism to explain the circadian changes in these autonomic responses, but elevation of basal BT in the dark period [10] might reach a saturation point, resulting in decreased responsiveness to activity of the olfactory pathways. In other words, it is possible that neurons of the autonomic center in the hypothalamus containing the SCN might be nonreactive to olfactory stimulation with SGFO or SLVO in the dark period secondary to effects of the circadian cycle on thermoregulatory organs. To investigate this theory, evaluation of the circadian effects of SGFO or SLVO on c-Fos expression of the SCN needs to be evaluated in the future. To determine the mechanisms of time-dependent effects of olfactory stimulation on BAT-T and BT, we noted that the SCN is thought to be a master circadian oscillator of circadian rhythms in synchronization with the light–dark cycle, regulating the daily rhythm of locomotive activity [4,10], feeding [12], energy metabolism, and thermoregulation [10]. In fact, abnormal rhythm of BT was detected in SCN-lesioned rats [10]. Moreover, we pre-
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Fig. 2. Effects of bilateral lesions of the SCN on changes in brown adipose tissue temperature (BAT-T) and body temperature (BT) after olfactory stimulation with SGFO or SLVO. Representative photomicrographs of coronal sections including the SCN from sham-operated (Sham) rats and from rats that received SCN lesions (SCNL) (A). SCN, the suprachiasmatic nucleus (arrows show the intact bilateral SCN in the SCN–sham rats); 3 V, the third ventricle. Scale bars = 300 m. Representative locomotor activity of Shamand SCN-lesioned (SCNL) rats under constant dark conditions are presented as a double plot (B). BAT-T and BT after olfactory stimulation with SGFO or SLVO are expressed as mean ± S.E.M. of temperature change from values at 0 min (C). Data from sham-operated (sham) and SCN-lesioned (SCNL) rats are shown. *P < 0.05 vs. sham rats.
viously confirmed that bilateral lesions of the SCN eliminated cardiovascular responses to SGFO or SLVO [20,21]. Thus, we tested the effects of SGFO or SLVO on BAT-T and BT in SCN-lesioned rats, and found that bilateral lesions of the SCN completely eliminated the responses of BAT-T and BT to SGFO or SLVO (Fig. 2). These findings suggest that the SCN might mediate the olfactory modulations of BAT-T and BT. Conversely, BIT/SHPS-1, a transmembrane glycoprotein implicated in signal transduction of growth factors and cell-to-cell interaction [14], has been identified in our previous examinations exploring the brain substance associated with BT modulation [23]. BIT/SHPS-1 has tyrosine phosphorylation sites in its intracellular domain, and is associated with the protein tyrosine phosphatase SHP-2 upon phosphorylation. Hypothalamic injection
of an anti-BIT/SHPS-1 monoclonal antibody, which reacts with the extracellular domain of BIT/SHPS-1 and stimulates tyrosine phosphorylation, enhances BAT-T and BT [24]. In addition, olfactory stimulation with SGFO or SLVO enhanced or attenuated, respectively, the tyrosine phosphorylation of BIT/SHPS-1 in the SCN [18]. Consistently, we previously observed that immunoreactivity of cFos, a marker of neuronal activation, in the SCN was changed after exposure to SGFO or SLVO [22]. These findings suggested that olfactory stimulation might affect SCN neurons and change tyrosine phosphorylation level of BIT/SHPS-1 in the SCN. Therefore, it is possible that tyrosine phosphorylation of BIT/SHPS-1 in the SCN might function as a molecular regulator of the central mechanism mediating BAT-T and BT changes due to SGFO or SLVO.
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Fig. 3. Effects of bilateral lesions of the SCN on changes in neural activity innervating brown adipose tissue (BAT-SNA) after olfactory stimulation with SGFO or SLVO. Representative trace data from recordings of BAT-SNA of a sham-operated (sham) rat and a SCN-lesioned (SCNL) rat before and after stimulation with SGFO or SLVO (A). Lower bars represent 20 min, vertical scale bars to the right of the recordings represent neural activity rates of 200 spikes/5 s.Time course change in BAT-SNA (B) after olfactory stimulation with water, SGFO or SLVO is expressed as mean ± S.E.M. of percentages of values at 0 min. Data from sham-operated (sham) and SCN-lesioned (SCNL) rats are shown. *P < 0.05 vs. sham rats.
Sympathetic nerves supplying BAT are an important regulator of BAT thermogenesis. We previously confirmed that SGFO or SLVO accelerated or suppressed, respectively, BAT-SNA in urethaneanesthetized rats [16,17], suggesting the existence of autonomic pathways between the SCN and peripheral organs [2]. With regard to BAT, a neural connection between the SCN and BAT was found in a pseudorabies virus study [1]. To determine whether the SCN played a role in olfactory stimulation-evoked change of BAT-SNA, our study investigated the effects of a SCN-lesion on BAT-SNA change after olfactory stimulation with SGFO or SLVO. This study provides new evidence that bilateral lesions of the SCN clearly abolished the response of BAT-SNA to SGFO or SLVO (Fig. 3) and supported the idea that the SCN is involved in the effects of olfactory stimulation with SGFO or SLVO on BAT-SNA. Moreover, ablation of the olfactory bulb of rats eliminates renal sympathetic responses to SGFO or SLVO (unpublished data). Currently, the exact mechanism through which the olfactory pathway affects the SCN and changes BAT-SNA is unclear. Previous studies confirmed that there was a neural connection between the olfactory bulb and the SCN [8], and the efferent pathway from the SCN to BAT via a sympathetic neu-
ral route was determined in an anatomical study [1]. Brain stem regions, such as the ventrolateral medulla and the raphe pallidus, contain sympathetic pre-motoneurons controlling BAT-SNA and exist as intermediate sites of the pathway [11]. It is possible that scent signals received in the olfactory bulb might be sent to the SCN via a neural pathway, and following BAT-SNA, BAT-T, and BT might be affected by a central mechanism. In conclusion, the present findings suggest that olfactory stimulation with SGFO or SLVO acts in a time-dependent manner, affecting BAT-T and BT. Furthermore, SCN lesions suppressed the effects of olfactory stimulation on BAT-T, BT, and BAT-SNA. Therefore, these data suggest that the SCN might be involved in the effects of olfactory stimulation with SGFO or SLVO on BAT-SNA and BT. However, further study is required to reveal the precise pathway responsible for these effects. References [1] M. Bamshad, C.K. Song, T.J. Bartness, CNS origins of the sympathetic nervous system outflow to brown adipose tissue, Am. J. Physiol. 276 (1999) R1569–1578.
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