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Short-term REM sleep deprivation and neuropeptide gene expression in the rat hypothalamus
International Congress Series 1278 (2005) 415 – 418
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Short-term REM sleep deprivation and neuropeptide gene expression in the rat hypothalamus Y. Ueta*, H. Fujihara Department of Physiology, School of Medicine, University of Occupational and Environmental Health, 1–1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807–8555, Japan
Abstract. In the present study, we examined the effects of short-term REM sleep deprivation (RSD) on the gene expressions of corticotorphin-releasing factor (CRF), galanin, arginine vasopressin, oxytocin and orexins in rats, using in situ hybridization histochemistry. Among them only galanin mRNA level in the hypothalamus was significantly increased after short-term RSD. The galaninergic neurons in the hypothalamus are suggested to be important sources of REM sleep induction. D 2004 Elsevier B.V. All rights reserved.
Keywords: REM sleep; Galanin; Rat; Hypothalamus; In situ hybridization histochemistry; Vasopressin; Oxytocin; Orexins
1. Introduction REM sleep is well discussed to be closely associated with higher nervous functions such as memory, learning, feeding, behavior and psychomotor function. The hypothalamus is an important site in regulating sleep, in particular balance of REM and non-REM sleep. In the hypothalamus neuropeptides are abundant and many of them are related to sleep regulation. In the present study, we examined the effects of short-term REM sleep deprivation (RSD) on the gene expressions of corticotorphin-releasing factor (CRF), galanin, arginine vasopressin (AVP), oxytocin (OXT) and orexins in rats, using in situ hybridization histochemistry. * Corresponding author. Tel.: +81 93 691 7420; fax: +81 93 692 1711. E-mail address: [email protected] (Y. Ueta). 0531-5131/ D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2004.11.094
416
Y. Ueta, H. Fujihara / International Congress Series 1278 (2005) 415–418
2. Methods 2.1. Surgery and RSD Male Wistar rats (6–7 weeks of age, 150–200 g of body weight at the starting point of experiment) were purchased from Slc (Shizuoka, Japan). The animals were anesthetized with Nembutal (50–70 mg/kg) and implanted with stainless steel miniature screw electrodes in the skull to record an electroencephalogram (EEG), and stainless steel wires in the neck muscles of both sides to record an electromyogram (EMG). A pair of rats were operated on the same day (one for RSD and the other is for a yokedcontrol). One week after surgery, for 2–3 days, the backs of the rats were gently stroked using a soft small brush for Japanese calligraphy to familiarize them with the RSD procedure. Polygraphic recording and digital data acquisition were started at 09:00 and continued throughout the experiments. When the RSD rat entered into REM sleep, both rats were gently stroked on their backs using the brush to wake them. If the yoked-control rat showed REM sleep simultaneously with the RSD rat, it was stroked after the spontaneous end of the REM sleep. RSD was performed for 3 h (3 h group; 12:00–15:00), 6 h (6 h group; 09:00–15:00), 6 h followed by 3 h of recovery period (6h+r group). After recording, rats were sacrificed by decapitation and the whole brains were removed. Those samples were immediately frozen on dry-ice and then stored at 80 8C. 2.2. In situ hybridization histochemistry Frozen, transverse sections were cut at 12 Am in a cryostat and mounted onto gelatin/chrome alum-coated slides that were kept at 80 8C until used. The in situ hybridization protocol has been described previously in detail [1]. The probes used were 35S3V end-labelled deoxyoligonucleotides complementary to transcripts coding for CRF (complementary to bases 496–543 of rat CRF nucleotides), AVP (complementary to basis 1843–1868 of rat AVP nucleotides), OXT (complementary to basis 912–941 of rat OXT nucleotides), preproorexin (complementary to bases 115–150 of rat orexin nucleotides) and galanin (complementary to bases 209–252 of rat galanin nucleotides). Hybridization sections were apposed to autoradiography (Hyperfilm, Amersham, Bucks, UK) for 8 h for AVP and OXT transcripts, for 4 days for CRF transcripts, for 1 day for preproorexin transcripts and for 7 days for galanin transcripts. The resulting images were analyzed by computerized densitometry using a MCID Image Analysis System (Imaging Research Ontario, Canada). The mean optical density (O.D.) of autoradiographs was measured by comparing it with simultaneously exposed [14C] micro-scale (Amersham). 2.3. Sleep scoring In order to estimate the effect of RSD on the vigilance states of the rats, off-line sleep scoring was done on the computer screen by visual assessment of EEG and EMG activity. A vigilance state in 5-s epochs was classified as wake, non-REM sleep and REM sleep. NonREM sleep was characterized by a continuous slow high-voltage EEG and low-level EMG activity. REM sleep was characterized by a low-voltage EEG with continuous theta waves and total suppression of the EMG.
Y. Ueta, H. Fujihara / International Congress Series 1278 (2005) 415–418
417
Fig. 1. Sleeping–waking durations for 3h, 6h, 6h+r groups and naive control. Values represent meanFS.E.M. *pb0.05, 3 h, 6 h and 6 h+r: same as in this figure. RSD: REM sleep-deprived rats, yoked: gently stroked rats on their backs using the brush when the RSD rat entered into REM, naive control: rats without any touch. W: wakefulness, N-REM: non-REM sleep. Data are from Ref. [3].
2.4. Statistical analysis All data are presented as meanFS.E.M. Student’s t test was performed between the RSD and sham-control groups on sleep duration. The data obtained from the in situ hybridization histochemistry were statistically analysed using a one-way analysis of variance (ANOVA) followed by Bonferroni-type adjustment for multiple comparison. For mRNAs of hypothalamic peptide analysis, the target regions of four sections, eight sites per rat were used to measure the density of the autoradiography. A Pb0.05 was considered to indicate statistical significance. 3. Result Fig. 1 shows the duration of each vigilance state in 3 h, 6 h, 6 h+r and naive-control group. In RSD rats, REM sleep was nearly completely inhibited in all groups during RSD period, while non-REM sleep was approximately the same as seen in the yoked-control
Fig. 2. The effects of REM sleep deprivation (RSD) on galanin transcript prevalence in the preoptic area (POA). The values are expressed as percentages of the value obtained from naive-control rats. Values represent meanFS.E.M. *pb0.05. Data are modified from Ref. [3].
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Y. Ueta, H. Fujihara / International Congress Series 1278 (2005) 415–418
rats. It is considered that the almost complete and selective RSD was carried out in this study. The expression of the galanin gene was observed in the preoptic area (POA) (Fig. 2). The levels of galanin mRNA were significantly increased in the RSD rats in the 6 h group compared with the yoked-control in the 6 h group and naive-control groups. The levels of CRF, AVP, OXT and preproorexin mRNAs did not show significant difference among the groups. 4. Discussion The RSD procedure used in this study did not increase the expression of the CRF gene in the paravocellular divisions of the PVN, indicating that our RSD method did not activate the hypothalamo-pituitary-adrenal stress axis. A study has reported that external AVP reduces REM sleep in humans [2]. However, in the rat, the short-term RSD deprivation used in this study had no effect on the levels of AVP and OXT mRNA in the SON and the PVN. Using this presumably non-stressful RSD procedure, only galanin mRNA in the POA significantly responded to RSD. The galanin is known to be an inhibitory peptide in the central nervous system. It is considered that the inhibition of the monoaminergic regions, such as noradrenergic in the locus coeruleus or histaminergic in the tuberomammillary nucleus, plays a critical role for REM sleep induction. And the galaninergic neurons in the ventrolateral POA are suggested to be important sources of that inhibition. The expression of the orexin gene was observed in the lateral hypothalamic area (LHA) and the surrounding areas. The levels of preproorexin mRNA in the LHA did not show any significant difference among RSD, yoked and naive-control rats, and among the groups. The control of orexin protein and/or orexin receptor may be more important in the REM sleep regulation. References [1] A. Terao, et al., Prepro-hypocretin (prepro-orexin) expression is unaffected by short-term sleep deprivation in rats and mice, Sleep 23 (2000) 867 – 874. [2] J. Born, et al., Vasopressin regulates human sleep by reducing rapid-eye-movement sleep, Am. J. Physiol. 262 (1992) E295 – E300. [3] H. Fujihara, et al., Six-hour selective REM sleep deprivation increases the expression of the galanin gene in the hypothalamus of rats, Mol. Brain Res. 119 (2) (2003) 152 – 159.
www.ics-elsevier.com
Short-term REM sleep deprivation and neuropeptide gene expression in the rat hypothalamus Y. Ueta*, H. Fujihara Department of Physiology, School of Medicine, University of Occupational and Environmental Health, 1–1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807–8555, Japan
Abstract. In the present study, we examined the effects of short-term REM sleep deprivation (RSD) on the gene expressions of corticotorphin-releasing factor (CRF), galanin, arginine vasopressin, oxytocin and orexins in rats, using in situ hybridization histochemistry. Among them only galanin mRNA level in the hypothalamus was significantly increased after short-term RSD. The galaninergic neurons in the hypothalamus are suggested to be important sources of REM sleep induction. D 2004 Elsevier B.V. All rights reserved.
Keywords: REM sleep; Galanin; Rat; Hypothalamus; In situ hybridization histochemistry; Vasopressin; Oxytocin; Orexins
1. Introduction REM sleep is well discussed to be closely associated with higher nervous functions such as memory, learning, feeding, behavior and psychomotor function. The hypothalamus is an important site in regulating sleep, in particular balance of REM and non-REM sleep. In the hypothalamus neuropeptides are abundant and many of them are related to sleep regulation. In the present study, we examined the effects of short-term REM sleep deprivation (RSD) on the gene expressions of corticotorphin-releasing factor (CRF), galanin, arginine vasopressin (AVP), oxytocin (OXT) and orexins in rats, using in situ hybridization histochemistry. * Corresponding author. Tel.: +81 93 691 7420; fax: +81 93 692 1711. E-mail address: [email protected] (Y. Ueta). 0531-5131/ D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2004.11.094
416
Y. Ueta, H. Fujihara / International Congress Series 1278 (2005) 415–418
2. Methods 2.1. Surgery and RSD Male Wistar rats (6–7 weeks of age, 150–200 g of body weight at the starting point of experiment) were purchased from Slc (Shizuoka, Japan). The animals were anesthetized with Nembutal (50–70 mg/kg) and implanted with stainless steel miniature screw electrodes in the skull to record an electroencephalogram (EEG), and stainless steel wires in the neck muscles of both sides to record an electromyogram (EMG). A pair of rats were operated on the same day (one for RSD and the other is for a yokedcontrol). One week after surgery, for 2–3 days, the backs of the rats were gently stroked using a soft small brush for Japanese calligraphy to familiarize them with the RSD procedure. Polygraphic recording and digital data acquisition were started at 09:00 and continued throughout the experiments. When the RSD rat entered into REM sleep, both rats were gently stroked on their backs using the brush to wake them. If the yoked-control rat showed REM sleep simultaneously with the RSD rat, it was stroked after the spontaneous end of the REM sleep. RSD was performed for 3 h (3 h group; 12:00–15:00), 6 h (6 h group; 09:00–15:00), 6 h followed by 3 h of recovery period (6h+r group). After recording, rats were sacrificed by decapitation and the whole brains were removed. Those samples were immediately frozen on dry-ice and then stored at 80 8C. 2.2. In situ hybridization histochemistry Frozen, transverse sections were cut at 12 Am in a cryostat and mounted onto gelatin/chrome alum-coated slides that were kept at 80 8C until used. The in situ hybridization protocol has been described previously in detail [1]. The probes used were 35S3V end-labelled deoxyoligonucleotides complementary to transcripts coding for CRF (complementary to bases 496–543 of rat CRF nucleotides), AVP (complementary to basis 1843–1868 of rat AVP nucleotides), OXT (complementary to basis 912–941 of rat OXT nucleotides), preproorexin (complementary to bases 115–150 of rat orexin nucleotides) and galanin (complementary to bases 209–252 of rat galanin nucleotides). Hybridization sections were apposed to autoradiography (Hyperfilm, Amersham, Bucks, UK) for 8 h for AVP and OXT transcripts, for 4 days for CRF transcripts, for 1 day for preproorexin transcripts and for 7 days for galanin transcripts. The resulting images were analyzed by computerized densitometry using a MCID Image Analysis System (Imaging Research Ontario, Canada). The mean optical density (O.D.) of autoradiographs was measured by comparing it with simultaneously exposed [14C] micro-scale (Amersham). 2.3. Sleep scoring In order to estimate the effect of RSD on the vigilance states of the rats, off-line sleep scoring was done on the computer screen by visual assessment of EEG and EMG activity. A vigilance state in 5-s epochs was classified as wake, non-REM sleep and REM sleep. NonREM sleep was characterized by a continuous slow high-voltage EEG and low-level EMG activity. REM sleep was characterized by a low-voltage EEG with continuous theta waves and total suppression of the EMG.
Y. Ueta, H. Fujihara / International Congress Series 1278 (2005) 415–418
417
Fig. 1. Sleeping–waking durations for 3h, 6h, 6h+r groups and naive control. Values represent meanFS.E.M. *pb0.05, 3 h, 6 h and 6 h+r: same as in this figure. RSD: REM sleep-deprived rats, yoked: gently stroked rats on their backs using the brush when the RSD rat entered into REM, naive control: rats without any touch. W: wakefulness, N-REM: non-REM sleep. Data are from Ref. [3].
2.4. Statistical analysis All data are presented as meanFS.E.M. Student’s t test was performed between the RSD and sham-control groups on sleep duration. The data obtained from the in situ hybridization histochemistry were statistically analysed using a one-way analysis of variance (ANOVA) followed by Bonferroni-type adjustment for multiple comparison. For mRNAs of hypothalamic peptide analysis, the target regions of four sections, eight sites per rat were used to measure the density of the autoradiography. A Pb0.05 was considered to indicate statistical significance. 3. Result Fig. 1 shows the duration of each vigilance state in 3 h, 6 h, 6 h+r and naive-control group. In RSD rats, REM sleep was nearly completely inhibited in all groups during RSD period, while non-REM sleep was approximately the same as seen in the yoked-control
Fig. 2. The effects of REM sleep deprivation (RSD) on galanin transcript prevalence in the preoptic area (POA). The values are expressed as percentages of the value obtained from naive-control rats. Values represent meanFS.E.M. *pb0.05. Data are modified from Ref. [3].
418
Y. Ueta, H. Fujihara / International Congress Series 1278 (2005) 415–418
rats. It is considered that the almost complete and selective RSD was carried out in this study. The expression of the galanin gene was observed in the preoptic area (POA) (Fig. 2). The levels of galanin mRNA were significantly increased in the RSD rats in the 6 h group compared with the yoked-control in the 6 h group and naive-control groups. The levels of CRF, AVP, OXT and preproorexin mRNAs did not show significant difference among the groups. 4. Discussion The RSD procedure used in this study did not increase the expression of the CRF gene in the paravocellular divisions of the PVN, indicating that our RSD method did not activate the hypothalamo-pituitary-adrenal stress axis. A study has reported that external AVP reduces REM sleep in humans [2]. However, in the rat, the short-term RSD deprivation used in this study had no effect on the levels of AVP and OXT mRNA in the SON and the PVN. Using this presumably non-stressful RSD procedure, only galanin mRNA in the POA significantly responded to RSD. The galanin is known to be an inhibitory peptide in the central nervous system. It is considered that the inhibition of the monoaminergic regions, such as noradrenergic in the locus coeruleus or histaminergic in the tuberomammillary nucleus, plays a critical role for REM sleep induction. And the galaninergic neurons in the ventrolateral POA are suggested to be important sources of that inhibition. The expression of the orexin gene was observed in the lateral hypothalamic area (LHA) and the surrounding areas. The levels of preproorexin mRNA in the LHA did not show any significant difference among RSD, yoked and naive-control rats, and among the groups. The control of orexin protein and/or orexin receptor may be more important in the REM sleep regulation. References [1] A. Terao, et al., Prepro-hypocretin (prepro-orexin) expression is unaffected by short-term sleep deprivation in rats and mice, Sleep 23 (2000) 867 – 874. [2] J. Born, et al., Vasopressin regulates human sleep by reducing rapid-eye-movement sleep, Am. J. Physiol. 262 (1992) E295 – E300. [3] H. Fujihara, et al., Six-hour selective REM sleep deprivation increases the expression of the galanin gene in the hypothalamus of rats, Mol. Brain Res. 119 (2) (2003) 152 – 159.