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Sleep in transgenic and gene-knockout mice for lipocalin-type prostaglandin D synthase
International Congress Series 1233 (2002) 429 – 433
Sleep in transgenic and gene-knockout mice for lipocalin-type prostaglandin D synthase Naomi Eguchi a, Elena Pinzar a, Yuko Kuwahata a,b, Takashi Inui a,b, Takatoshi Mochizuki a, Yoshihiro Urade a,b,*, Osamu Hayaishi a a
Department of Molecular Behavioral Biology, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874, Japan b CREST, Japan Science and Technology Corporation, c/o Osaka Bioscience Institute, Suita, Osaka 565-0874, Japan
Abstract Prostaglandin (PG) D2 is a potent sleep-inducing substance in mammals. We investigated the effects of overproduction and deficiency of endogenous PGD2 on sleep regulation by using transgenic (TG) mice overexpressing human lipocalin-type PGD synthase (L-PGDS) gene and geneknockout (KO) mice produced by a null mutation of the L-PGDS gene, respectively. The circadian rhythm of non-rapid eye movement (NREM) and REM sleep was almost unchanged among wildtype (WT), TG, and KO mice. However, we found that noxious stimulation by tail clipping (Tc) of TG, but not of WT, mice transiently ( f 5 h) increased the amount of NREM sleep without affecting REM sleep. This increase was coupled with elevation of the PGD2 content in the brain, suggesting that the overproduction of PGD2 in the brain by Tc induced NREM sleep in TG mice. We also found that sleep deprivation (SD) for 6 h induced rebound of NREM sleep in WT, but not in KO, mice, and of REM sleep in both of these mice, suggesting that the L-PGDS gene is involved in the homeostasis of NREM sleep after SD. Therefore, PGD2 endogenously produced by L-PGDS in the brain is considered to regulate NREM sleep after noxious stimulation or SD. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Prostaglandin D2; Lipocalin-type PGD synthase; Sleep; Gene-knockout; Transgenic mice
Abbreviations: PG, prostaglandin; TG, transgenic; L-PGDS, lipocalin-type PGD synthase; KO, geneknockout; NREM, non-rapid eye movement; WT, wild-type; Tc, tail clipping; SD, sleep deprivation; EEG, electroencephalogram. * Corresponding author. Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874, Japan. Tel.: +816-6872-4851; fax: +81-6-6872-2841. E-mail address: [email protected] (Y. Urade). 0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 2 ) 0 0 5 2 9 - 0
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N. Eguchi et al. / International Congress Series 1233 (2002) 429–433
1. Introduction Prostaglandin (PG) D2 is a potent endogenous sleep-promoting substance which is produced by the action of lipocalin-type PGD synthase (L-PGDS) in the brain of mammals [1]. L-PGDS is mainly produced in the leptomeninges of the entire brain and choroid plexus, and is then secreted into the cerebrospinal fluid to become htrace, a major cerebrospinal fluid protein [2]. Since infusion of Se4 + compounds, relatively selective inhibitors of L-PGDS, into the third ventricle of rats inhibited sleep in a dose-dependent manner, L-PGDS has been proposed as the key enzyme in the regulation of physiological sleep [3]. In this study, to clarify the role of PGD2 endogenously produced by L-PGDS in sleep regulation in vivo, we investigated the sleep pattern in transgenic (TG) mice and in gene-knockout (KO) mice for the LPGDS gene.
2. Materials and methods 2.1. Generation of L-PGDS-TG and L-PGDS-KO mice Human L-PGDS-overexpressing TG mice (inbred FVB/N strain) and L-PGDS-null mutation KO mice (inbred 129/Sv strain) were generated as described [4,5]. Both TG and KO mice appeared healthy and grew normally. 2.2. Sleep monitoring and analysis The electroencephalogram (EEG) and electromyogram in adult male mice (14 – 15 weeks, n = 6) were recorded for two consecutive 24-h periods, baseline and experimental days, respectively, in sound-attenuated shielded chambers (light from 8 a.m. to 8 p.m.; 23 jC; 55% humidity) as described [6]. The three vigilance states, wakefulness, non-rapid eye movement (NREM), and REM sleep, were classified by 4-s epochs by the computer software SleepSign (Kissei Comtec, Nagano, Japan) [6]. The tails of conscious TG and wild-type (WT) control mice were clipped at a point approximately 7 mm from their end with scissors at 8 p.m. (start of dark period) on the second (experimental) day, and their EEG and electromyogram were further recorded [4]. For sleep deprivation (SD) treatment, conscious KO and WT control mice were kept awake by gentle handling with cotton for 6 h in the early phase of the light period from 8 a.m. to 2 p.m. or in the late phase of the light period from 2 p.m. to 8 p.m. on the second day. 2.3. Enzyme immunoassay PGD2 in the brain was purified by HPLC, and its content was determined with a PGD2MOX enzyme immunoassay kit (Cayman Chemicals, Ann Arbor, MI, USA) as described [4].
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3. Results and discussion 3.1. Circadian profiles of sleep – wake pattern in L-PGDS-TG and L-PGDS-KO mice The circadian profiles of NREM and REM sleep in both TG and KO mice were almost identical to those in the corresponding WT control mice. Furthermore, the duration and the episode number of each vigilance state in both TG and KO mice were almost the same as those in the WT controls. These results indicate that the overexpression and deficiency of the L-PGDS gene does not affect basal sleep. 3.2. Effect of tail clipping (Tc) on sleep in L-PGDS-TG mice We accidentally found that TG mice, but not WT mice, were often sedate after Tc with scissors for DNA sampling used for genotype determination [4]. In fact, when tails of TG mice were clipped with scissors at 8 p.m., the amount of NREM sleep was significantly increased and reached a peak 3 h later (approx. 2.5-fold higher than that before Tc), and then returned to the baseline level within 6 h. However, the amount of REM sleep was unchanged by the Tc. In WT mice, the amounts of NREM and REM sleep were not significantly changed after Tc, as compared with those before it (Fig. 1A). The quality of NREM and REM sleep in TG mice after Tc was almost the same as that before Tc and as that in WT mice, as judged by analyzing the EEG power spectra of each type of sleep. To examine whether the induction of NREM sleep in TG mice after Tc was coupled to a change in the PG production, we measured the amount of PGD2 in the brain by enzyme immunoassay at different time points after Tc. Tc caused a significant change in the PGD2 content in the brain of TG mice, with a timecourse similar to that of NREM sleep induction described above. The PGD2 content in the brain at 3 h after Tc was approx. 17fold higher than that before Tc. Moreover, we confirmed in WT mice with Tc that neither the PGD2 content in the brain nor the amount of NREM sleep was changed. Therefore, these results suggest that the overproduction of PGD2 endogenously produced by L-PGDS in the TG mouse brain after Tc induced NREM sleep. 3.3. Effect of SD on sleep in L-PGDS-KO mice Since sleep is regulated as a function of prior wakefulness and sleep pressure increases during waking or SD [7], SD treatment is generally used for sleep regulation. Therefore, we investigated the effects of SD for 6 h in the early phase of the light period between 8 a.m. and 2 p.m. or in the late phase between 2 p.m. and 8 p.m. on NREM and REM sleep in WT and KO mice, and calculated the cumulative amount of each sleep during the recovery period (Fig. 1B). In WT mice (Fig. 1B, left-hand figures), the cumulative amounts of NREM and REM sleep after SD in the early phase (22% and 121% increases, respectively) were significantly higher than those before the SD. A stronger rebound of NREM sleep was observed after SD in the late phase (NREM, 43% increase), whereas REM sleep showed a 16% decrease. These data indicate that both NREM and REM sleep rebounds were induced in WT mice after SD. However, in KO mice, the cumulative amount of NREM sleep was only slightly increased after SD in the early phase (10%
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Fig. 1. Effects of Tc and SD on sleep in L-PGDS-TG and L-PGDS-KO mice, respectively. (A) The cumulative amounts of NREM (upper) and REM (lower) sleep were calculated for 6 h after Tc in TG (right) and WT control (left) mice, and compared with those on the baseline day. (B) The cumulative amounts of NREM (upper) and REM (lower) sleep were calculated during the recovery period (8 p.m. to 2 a.m.) after 6-h SD in KO (right) and WT control (left) mice, and compared with those on the baseline day. The values represent the mean F SEM (n = 6). * P < 0.05, ** P < 0.01, compared with the baseline day by the paired t test.
increase) and in the late phase (13% increase), whereas that of REM sleep was significantly increased after SD treatment at either the early (81% increase) or late (91% increase) phase (Fig. 1B, right-hand figures). In terms of the EEG power spectrum of each sleep type during the recovery period, the delta power during the NREM sleep of WT mice significantly increased after SD, whereas that during the NREM sleep of KO mice after SD was almost the same as that before SD, suggesting that NREM sleep rebound in KO mice after SD was selectively suppressed and that the deficiency of LPGDS decreased the sleep pressure for NREM sleep during SD. Therefore, these results lead us to conclude that endogenous PGD2 produced by LPGDS is involved in the homeostasis of NREM sleep after SD.
4. Conclusions We demonstrated that human L-PGDS-overexpressing TG mice exhibited excessive amounts of NREM sleep, but not of REM sleep, in response to the noxious stimulus of Tc, and that this phenomenon was coupled with a significant increase in the PGD2 content of the brain. We also showed that L-PGDS-deficient mice exhibited a remarkably weak
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rebound of NREM sleep, but normal rebound of REM sleep, after SD, when compared with the WT mice. Therefore, we consider endogenous PGD2 produced by L-PGDS in the brain to be involved in the regulation of NREM sleep. Furthermore, these genemanipulated mice should prove useful as a novel animal model to study sleep disorders.
Acknowledgements We thank N. Uodome, Y. Hoshikawa, K. Masuda, and S. Matsumoto of Osaka Bioscience Institute for technical assistance. We are also grateful to Prof. I. Tobler of Zurich University, Switzerland, and Dr. T. Endo of Hokkaido University, Japan, for setting up the sleep monitoring system for mice. This study was supported by research grants from the Special Coordination Funds for Promoting Science and Technology from the Science and Technology Agency of the Japanese Government (13557016), Suntory Institute for Bioorganic Research, Takeda Science Foundation, Yamanouchi Foundation for Research on Metabolic Disorders, ONO Medical Research Foundation, and Osaka City.
References [1] O. Hayaishi, Molecular mechanisms of sleep – wake regulation: a role of prostaglandin D2, Philos. Trans. R. Soc. London, Ser. B 355 (2000) 275 – 280. [2] Y. Urade, O. Hayaishi, Biochemical, structural, genetic, physiological, and pathophysiological features of lipocalin-type prostaglandin D synthase, Biochim. Biophys. Acta 1482 (2000) 259 – 271. [3] H. Matsumura, R. Takahata, O. Hayaishi, Inhibition of sleep in rats by inorganic selenium compounds, inhibitors of prostaglandin D synthase, Proc. Natl. Acad. Sci. U. S. A. 88 (1991) 9046 – 9050. [4] E. Pinzar, Y. Kanaoka, T. Inui, N. Eguchi, Y. Urade, O. Hayaishi, Prostaglandin D synthase gene is involved in the regulation of non-rapid eye movement sleep, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 4903 – 4907. [5] N. Eguchi, T. Minami, N. Shirafuji, Y. Kanaoka, T. Tanaka, A. Nagata, N. Yoshida, Y. Urade, S. Ito, O. Hayaishi, Lack of tactile pain (allodynia) in lipocalin-type prostaglandin D synthase-deficient mice, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 726 – 730. [6] Z.-L. Huang, W.-M. Qu, W.-D. Li, T. Mochizuki, N. Eguchi, T. Watanabe, Y. Urade, O. Hayaishi, Arousal effect of orexin A depends on activation of the histaminergic system, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 9965 – 9970. [7] I. Tobler, T. Deboer, Sleep regulation and circadian rest – activity rhythm in prion protein knockout mice, in: O. Hayaishi, S. Inoue (Eds.), Sleep and Sleep Disorders: From Molecule to Behavior, Academic Press, Tokyo, Japan, 1997, pp. 295 – 310.
Sleep in transgenic and gene-knockout mice for lipocalin-type prostaglandin D synthase Naomi Eguchi a, Elena Pinzar a, Yuko Kuwahata a,b, Takashi Inui a,b, Takatoshi Mochizuki a, Yoshihiro Urade a,b,*, Osamu Hayaishi a a
Department of Molecular Behavioral Biology, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874, Japan b CREST, Japan Science and Technology Corporation, c/o Osaka Bioscience Institute, Suita, Osaka 565-0874, Japan
Abstract Prostaglandin (PG) D2 is a potent sleep-inducing substance in mammals. We investigated the effects of overproduction and deficiency of endogenous PGD2 on sleep regulation by using transgenic (TG) mice overexpressing human lipocalin-type PGD synthase (L-PGDS) gene and geneknockout (KO) mice produced by a null mutation of the L-PGDS gene, respectively. The circadian rhythm of non-rapid eye movement (NREM) and REM sleep was almost unchanged among wildtype (WT), TG, and KO mice. However, we found that noxious stimulation by tail clipping (Tc) of TG, but not of WT, mice transiently ( f 5 h) increased the amount of NREM sleep without affecting REM sleep. This increase was coupled with elevation of the PGD2 content in the brain, suggesting that the overproduction of PGD2 in the brain by Tc induced NREM sleep in TG mice. We also found that sleep deprivation (SD) for 6 h induced rebound of NREM sleep in WT, but not in KO, mice, and of REM sleep in both of these mice, suggesting that the L-PGDS gene is involved in the homeostasis of NREM sleep after SD. Therefore, PGD2 endogenously produced by L-PGDS in the brain is considered to regulate NREM sleep after noxious stimulation or SD. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Prostaglandin D2; Lipocalin-type PGD synthase; Sleep; Gene-knockout; Transgenic mice
Abbreviations: PG, prostaglandin; TG, transgenic; L-PGDS, lipocalin-type PGD synthase; KO, geneknockout; NREM, non-rapid eye movement; WT, wild-type; Tc, tail clipping; SD, sleep deprivation; EEG, electroencephalogram. * Corresponding author. Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874, Japan. Tel.: +816-6872-4851; fax: +81-6-6872-2841. E-mail address: [email protected] (Y. Urade). 0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 2 ) 0 0 5 2 9 - 0
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N. Eguchi et al. / International Congress Series 1233 (2002) 429–433
1. Introduction Prostaglandin (PG) D2 is a potent endogenous sleep-promoting substance which is produced by the action of lipocalin-type PGD synthase (L-PGDS) in the brain of mammals [1]. L-PGDS is mainly produced in the leptomeninges of the entire brain and choroid plexus, and is then secreted into the cerebrospinal fluid to become htrace, a major cerebrospinal fluid protein [2]. Since infusion of Se4 + compounds, relatively selective inhibitors of L-PGDS, into the third ventricle of rats inhibited sleep in a dose-dependent manner, L-PGDS has been proposed as the key enzyme in the regulation of physiological sleep [3]. In this study, to clarify the role of PGD2 endogenously produced by L-PGDS in sleep regulation in vivo, we investigated the sleep pattern in transgenic (TG) mice and in gene-knockout (KO) mice for the LPGDS gene.
2. Materials and methods 2.1. Generation of L-PGDS-TG and L-PGDS-KO mice Human L-PGDS-overexpressing TG mice (inbred FVB/N strain) and L-PGDS-null mutation KO mice (inbred 129/Sv strain) were generated as described [4,5]. Both TG and KO mice appeared healthy and grew normally. 2.2. Sleep monitoring and analysis The electroencephalogram (EEG) and electromyogram in adult male mice (14 – 15 weeks, n = 6) were recorded for two consecutive 24-h periods, baseline and experimental days, respectively, in sound-attenuated shielded chambers (light from 8 a.m. to 8 p.m.; 23 jC; 55% humidity) as described [6]. The three vigilance states, wakefulness, non-rapid eye movement (NREM), and REM sleep, were classified by 4-s epochs by the computer software SleepSign (Kissei Comtec, Nagano, Japan) [6]. The tails of conscious TG and wild-type (WT) control mice were clipped at a point approximately 7 mm from their end with scissors at 8 p.m. (start of dark period) on the second (experimental) day, and their EEG and electromyogram were further recorded [4]. For sleep deprivation (SD) treatment, conscious KO and WT control mice were kept awake by gentle handling with cotton for 6 h in the early phase of the light period from 8 a.m. to 2 p.m. or in the late phase of the light period from 2 p.m. to 8 p.m. on the second day. 2.3. Enzyme immunoassay PGD2 in the brain was purified by HPLC, and its content was determined with a PGD2MOX enzyme immunoassay kit (Cayman Chemicals, Ann Arbor, MI, USA) as described [4].
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3. Results and discussion 3.1. Circadian profiles of sleep – wake pattern in L-PGDS-TG and L-PGDS-KO mice The circadian profiles of NREM and REM sleep in both TG and KO mice were almost identical to those in the corresponding WT control mice. Furthermore, the duration and the episode number of each vigilance state in both TG and KO mice were almost the same as those in the WT controls. These results indicate that the overexpression and deficiency of the L-PGDS gene does not affect basal sleep. 3.2. Effect of tail clipping (Tc) on sleep in L-PGDS-TG mice We accidentally found that TG mice, but not WT mice, were often sedate after Tc with scissors for DNA sampling used for genotype determination [4]. In fact, when tails of TG mice were clipped with scissors at 8 p.m., the amount of NREM sleep was significantly increased and reached a peak 3 h later (approx. 2.5-fold higher than that before Tc), and then returned to the baseline level within 6 h. However, the amount of REM sleep was unchanged by the Tc. In WT mice, the amounts of NREM and REM sleep were not significantly changed after Tc, as compared with those before it (Fig. 1A). The quality of NREM and REM sleep in TG mice after Tc was almost the same as that before Tc and as that in WT mice, as judged by analyzing the EEG power spectra of each type of sleep. To examine whether the induction of NREM sleep in TG mice after Tc was coupled to a change in the PG production, we measured the amount of PGD2 in the brain by enzyme immunoassay at different time points after Tc. Tc caused a significant change in the PGD2 content in the brain of TG mice, with a timecourse similar to that of NREM sleep induction described above. The PGD2 content in the brain at 3 h after Tc was approx. 17fold higher than that before Tc. Moreover, we confirmed in WT mice with Tc that neither the PGD2 content in the brain nor the amount of NREM sleep was changed. Therefore, these results suggest that the overproduction of PGD2 endogenously produced by L-PGDS in the TG mouse brain after Tc induced NREM sleep. 3.3. Effect of SD on sleep in L-PGDS-KO mice Since sleep is regulated as a function of prior wakefulness and sleep pressure increases during waking or SD [7], SD treatment is generally used for sleep regulation. Therefore, we investigated the effects of SD for 6 h in the early phase of the light period between 8 a.m. and 2 p.m. or in the late phase between 2 p.m. and 8 p.m. on NREM and REM sleep in WT and KO mice, and calculated the cumulative amount of each sleep during the recovery period (Fig. 1B). In WT mice (Fig. 1B, left-hand figures), the cumulative amounts of NREM and REM sleep after SD in the early phase (22% and 121% increases, respectively) were significantly higher than those before the SD. A stronger rebound of NREM sleep was observed after SD in the late phase (NREM, 43% increase), whereas REM sleep showed a 16% decrease. These data indicate that both NREM and REM sleep rebounds were induced in WT mice after SD. However, in KO mice, the cumulative amount of NREM sleep was only slightly increased after SD in the early phase (10%
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Fig. 1. Effects of Tc and SD on sleep in L-PGDS-TG and L-PGDS-KO mice, respectively. (A) The cumulative amounts of NREM (upper) and REM (lower) sleep were calculated for 6 h after Tc in TG (right) and WT control (left) mice, and compared with those on the baseline day. (B) The cumulative amounts of NREM (upper) and REM (lower) sleep were calculated during the recovery period (8 p.m. to 2 a.m.) after 6-h SD in KO (right) and WT control (left) mice, and compared with those on the baseline day. The values represent the mean F SEM (n = 6). * P < 0.05, ** P < 0.01, compared with the baseline day by the paired t test.
increase) and in the late phase (13% increase), whereas that of REM sleep was significantly increased after SD treatment at either the early (81% increase) or late (91% increase) phase (Fig. 1B, right-hand figures). In terms of the EEG power spectrum of each sleep type during the recovery period, the delta power during the NREM sleep of WT mice significantly increased after SD, whereas that during the NREM sleep of KO mice after SD was almost the same as that before SD, suggesting that NREM sleep rebound in KO mice after SD was selectively suppressed and that the deficiency of LPGDS decreased the sleep pressure for NREM sleep during SD. Therefore, these results lead us to conclude that endogenous PGD2 produced by LPGDS is involved in the homeostasis of NREM sleep after SD.
4. Conclusions We demonstrated that human L-PGDS-overexpressing TG mice exhibited excessive amounts of NREM sleep, but not of REM sleep, in response to the noxious stimulus of Tc, and that this phenomenon was coupled with a significant increase in the PGD2 content of the brain. We also showed that L-PGDS-deficient mice exhibited a remarkably weak
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rebound of NREM sleep, but normal rebound of REM sleep, after SD, when compared with the WT mice. Therefore, we consider endogenous PGD2 produced by L-PGDS in the brain to be involved in the regulation of NREM sleep. Furthermore, these genemanipulated mice should prove useful as a novel animal model to study sleep disorders.
Acknowledgements We thank N. Uodome, Y. Hoshikawa, K. Masuda, and S. Matsumoto of Osaka Bioscience Institute for technical assistance. We are also grateful to Prof. I. Tobler of Zurich University, Switzerland, and Dr. T. Endo of Hokkaido University, Japan, for setting up the sleep monitoring system for mice. This study was supported by research grants from the Special Coordination Funds for Promoting Science and Technology from the Science and Technology Agency of the Japanese Government (13557016), Suntory Institute for Bioorganic Research, Takeda Science Foundation, Yamanouchi Foundation for Research on Metabolic Disorders, ONO Medical Research Foundation, and Osaka City.
References [1] O. Hayaishi, Molecular mechanisms of sleep – wake regulation: a role of prostaglandin D2, Philos. Trans. R. Soc. London, Ser. B 355 (2000) 275 – 280. [2] Y. Urade, O. Hayaishi, Biochemical, structural, genetic, physiological, and pathophysiological features of lipocalin-type prostaglandin D synthase, Biochim. Biophys. Acta 1482 (2000) 259 – 271. [3] H. Matsumura, R. Takahata, O. Hayaishi, Inhibition of sleep in rats by inorganic selenium compounds, inhibitors of prostaglandin D synthase, Proc. Natl. Acad. Sci. U. S. A. 88 (1991) 9046 – 9050. [4] E. Pinzar, Y. Kanaoka, T. Inui, N. Eguchi, Y. Urade, O. Hayaishi, Prostaglandin D synthase gene is involved in the regulation of non-rapid eye movement sleep, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 4903 – 4907. [5] N. Eguchi, T. Minami, N. Shirafuji, Y. Kanaoka, T. Tanaka, A. Nagata, N. Yoshida, Y. Urade, S. Ito, O. Hayaishi, Lack of tactile pain (allodynia) in lipocalin-type prostaglandin D synthase-deficient mice, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 726 – 730. [6] Z.-L. Huang, W.-M. Qu, W.-D. Li, T. Mochizuki, N. Eguchi, T. Watanabe, Y. Urade, O. Hayaishi, Arousal effect of orexin A depends on activation of the histaminergic system, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 9965 – 9970. [7] I. Tobler, T. Deboer, Sleep regulation and circadian rest – activity rhythm in prion protein knockout mice, in: O. Hayaishi, S. Inoue (Eds.), Sleep and Sleep Disorders: From Molecule to Behavior, Academic Press, Tokyo, Japan, 1997, pp. 295 – 310.