Prostaglandin D synthase is involved in the regulation of non-rapid eye movement sleep

Elena Pinzar*, Yoshihide Kanaoka*†, Takashi Inui‡, Naomi Eguchi*, Yoshihiro Urade*‡, and Osamu Hayaishi*§

*Department of Molecular Behavioral Biology, Osaka Bioscience Institute, and ‡Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, 6-2-4 Furuedai, Suita, 565-0874 Osaka, Japan

Contributed by Osamu Hayaishi, March 3, 2000 To examine the function of prostaglandin (PG) D synthase (PGDS) overexpressed to study the effect of endogenously produced gene, as well as endogenously produced PGD2 in sleep regulation PGD2 on sleep. Here, we report that TG mice exhibit excess of in vivo, we generated transgenic (TG) mice that overexpress human NREM sleep in response to the noxious stimulus, coupled with PGDS gene to study their sleep behavior. Although no difference significant increase in PGD2 production in the brain. was observed in the sleep͞wake patterns between wild-type and TG mice, a striking time-dependent increase in non-rapid eye Materials and Methods movement (NREM), but not in rapid eye movement (REM), sleep Generation of Human PGDS-TG Mice. A 600-bp fragment containing was observed in two independent lines of TG mice after stimula- the coding region of human PGDS cDNA (16) was inserted into the tion by tail clipping. Concomitantly, the spontaneous locomotor pCAGGS vector under the control of the chicken ␤-actin promoter activity of TG animals was drastically decreased in response to the and the cytomegalovirus immediate early enhancer (17). This tail clip. Induction of NREM sleep in TG mice was positively construct then was digested with SalI and NotI and removed from correlated with the PGD2 production in the brain. Sleep, locomo- the vector sequence. The resultant 3.1-kb DNA fragment was tion, and PGD2 content were essentially unchanged in wild-type microinjected into pronuclei of fertilized eggs derived from an mice after tail clipping. The results with TG mice demonstrate the inbred FVB͞N mouse strain, as described (18). Microinjection was involvement of the PGDS gene in the regulation of NREM sleep. carried out at DNX (Princeton, NJ), and animals then were transferred to the animal facility of the Osaka Bioscience Institute. rostaglandin (PG) D2 is the most abundant The founders of the TG mice were identified by Southern blot Pproduced in the central nervous system (CNS) of mammals analysis with the human PGDS gene fragment as a probe. Five (1) and one of the most potent sleep-inducing substances (2, 3). independent TG lines were established. It has been shown to induce excess sleep in rats (4) and monkeys (5) after intracerebroventricular infusion. Most importantly, Northern Blot and Assay. Total RNA (10 ␮g) from different sleep induced by PGD2 is indistinguishable from physiological tissues was electrophoresed in an agarose-formaldehyde gel and sleep as judged by electroencephalogram (EEG), electromyo- transferred to the nylon membranes. The RNA was crosslinked to gram (EMG), brain temperature, locomotor activities, heart the membrane with UV light at 254 nm (CL-1000 UVP, Upland, rate, and general behavior of animals (4, 5). CA). The membrane then was hybridized with 32P-labeled probe of PGD2 is produced in the cascade from a the cDNA for human PGDS, exposed on imaging plates, and common precursor of various , PGH2, by the action further analyzed with Fujifilm fluorescent image analyzer FLA of PGD synthase (PGDS) (6). In the CNS, the PGDS was shown 2000. The blot was stripped of the probe and reprobed with cDNA to be produced mainly in the leptomeninges and choroid plexus for glyceraldehyde-3-phosphate dehydrogenase. (7) and secreted into the cerebrospinal fluid (CSF) as ␤-trace For detection of PGDS activity, the brains were quickly (8), the second most abundant in CSF after albumin (9). removed and homogenized in 3 vol of PBS (10 mM sodium PGD2 is most likely to exert somnogenic activity through PGD2 phosphate, pH 7.4͞150 mM NaCl). The homogenates were receptors, dominantly expressed in leptomeninges below the centrifuged at 4°C for 15 min at 400,000 ϫ g, and the resulting rostral basal forebrain (ref. 10; A. Mizoguchi, Y.U., and O.H., supernatant was used for PGDS assay in 50 ␮l of 100 mM ⅐ ␮ 14 unpublished data). Activation of PGD2 receptors results in Tris HCl (pH 8.0), 40 M [1- C]PGH2, and 1 mM DTT (19). subsequent excitation of ventral lateral preoptic neurons, a The rates of all enzymatic reactions were calculated after putative sleep center, which in its turn inhibits the tuberomam- subtracting the blank values without the enzyme. millary nucleus, a histaminergic arousal system (11). PGDS activity in the rat brain and the PGD2 concentration in Sleep Monitoring. Adult specific pathogen-free, male PGDS-TG the CSF of rats reportedly exhibit a circadian fluctuation coupled mice (n ϭ 10–20) and wild-type controls (n ϭ 7–15) of inbred with sleep͞wake cycle, being increased in the light period when FVB͞N strain weighing 25.9 Ϯ 0.9 g (12–15 wk old) were used rodents mainly sleep (12). Infusion of selective inhibitors of in the experiments. The animals were maintained under 12-h PGDS, e.g., tetravalent compounds, reversibly, time- light͞dark cycle (light from 8 a.m. to 8 p.m.) in sound-attenuated and dose-dependently inhibited both non-rapid eye movement temperature-controlled (22.0 Ϯ 1.0°C) chambers. Mice were (NREM) and rapid eye movement (REM) sleep during the daytime (13). These observations clearly showed that PGDS plays a crucial role in the regulation of physiological sleep. Abbreviations: PG, prostaglandin; PGDS, PGD synthase; EEG, electroencephalogram; EMG, In some sleep disorders such as mastocytosis (14) and African electromyogram; REM, rapid eye movement; NREM, non-REM; TG, transgenic; COX, cyclo- oxygenase; CNS, central nervous system; CSF, cerebrospinal fluid. sleeping sickness (15), profound lethargy in patients was pro- †Present address: Harvard Medical School, Brigham and Women’s Hospital, Smith Building, posed to be the result of the overproduction of PGD2. These One Jimmy Fund Way, Boston, MA 02115. clinical observations are consistent with the notion that excessive §To whom reprint requests should be addressed. E-mail: [email protected]. endogenous production of PGD2 induces sleep under certain The publication costs of this article were defrayed in part by page charge payment. This pathological conditions. However, animal experiments to verify article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. this conclusion have not been done so far. To provide experi- §1734 solely to indicate this fact.

mental evidence to substantiate this hypothesis, we generated Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073͞pnas.090093997. NEUROBIOLOGY transgenic (TG) mice, in which the human PGDS gene was Article and publication date are at www.pnas.org͞cgi͞doi͞10.1073͞pnas.090093997

PNAS ͉ April 25, 2000 ͉ vol. 97 ͉ no. 9 ͉ 4903–4907 Downloaded by guest on September 24, 2021 scissors without usage of any anesthetic; the mice then were returned to the home cage and further analysis was done.

Locomotor Activity. Horizontal locomotor activity was detected by 124 infrared photocell beams in an adjustable frame placed over the cage for every individual mouse (Biotex, Kyoto, Japan). After habituation for 1 day before recording, spontaneous locomotor activity was recorded for 48-h intervals, and day-2 recordings served as a baseline. On the third day, tail clipping was performed at 8 p.m., the beginning of the dark period, and the locomotor activity was recorded for another 48-h period.

PGD2 and PGE2 Enzyme Immunoassay. At specific time points, brains were harvested and immediately frozen in liquid nitrogen. They then were homogenized in ethanol containing 0.02% HCl at pH ϫ 3 2.0 and centrifuged at 500 g for 20 min. H-labeled PGD2 and PGE2 (60 Bq for each per assay) (New England Nuclear) were added as tracers for estimation of the recovery to the superna- tant. The PGs were extracted with ethyl acetate, which was evaporated under nitrogen; and the samples then were separated by HPLC (Gilson) (12). The quantification of PGs was per- formed with a PGD2-MOX enzyme immunoassay kit for PGD2 and a PGE2 enzyme immunoassay kit-monoclonal for PGE2 (Cayman Chemicals, Ann Arbor, MI).

Statistics. Statistic analysis was performed by two-way ANOVA followed by a post hoc Fisher’s probable least-squares difference test to establish the differences between the genotypes and different conditions; analysis of PGs production was performed by an unpaired Student’s t test; P Ͻ 0.05 was considered to be significant. Results For generation of TG mice, we used the cDNA for human PGDS, which was inserted under the control of the chicken ␤-actin promoter and cytomegalovirus enhancer (Fig. 1A). Northern blot analysis of tissue samples of TG mice showed an excessive amount of mRNA for human PGDS expressed in various organs and tissues of these mice, as expected; whereas Fig. 1. Generation of PGDS-TG mice. (A) Construct of the human PGDS cDNA mRNA for the endogenous mouse PGDS was seen only in the used for microinjection into the fertilized eggs of FVB͞N mouse strain; CMV, ␮ ͞ brain and epididymis, as reported previously (21). Five indepen- cytomegalovirus. (B) Northern blot analysis of total RNA (10 g lane) from the dent lines of TG mice were generated, and all of the animals brains of five lines of TG mice, using 32P-labeled cDNA probes for human PGDS appeared to be healthy, grew normally, and produced offspring. and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (C) PGDS activity ͞ in the brains of wild-type and two lines of TG mice (B7 and B20) measured with Usage of the inbred FVB N mouse strain for the entire process 14 [ C]PGH2 as the substrate. of transgenesis allowed us to avoid ambiguity caused by different genetic backgrounds (22). When Northern analysis was per- formed on brain samples from TG mice, the highest expression deeply anesthetized with pentobarbital (50 mg͞kg, i.p.) and of mRNA of PGDS was found in B7 and B20 lines (Fig. 1B). implanted with two EEG electrodes over the parietal cortex (2.0 Measurement of PGDS activity in the brains of these lines of TG mm lateral to the midline, 2.5 mm posterior to the bregma) and mice revealed that the B7 and B20 mice contained 5- and 2.5-fold the cerebellum (1.5 mm posterior to lambda). Two wires were greater activity than that of their wild-type counterparts, respec- inserted in the muscle of the dorsal neck as EMG electrodes. The tively (Fig. 1C). We used these TG lines to evaluate the role of electrodes were attached to the skull with dental cement. PGDS in sleep regulation. Animals were allowed at least 10 days of recovery from surgery; All mice exhibited an almost identical circadian pattern in and after habituation to experimental conditions, three consec- their sleep, and no significant differences were found between utive 24-h recordings were obtained. After baseline recordings, wild-type and TG mice (Fig. 2). Analyses of the sleep distribution in the light and dark periods showed that the amounts of NREM mice were subjected to tail clipping at the time at which the lights (light period: 35.7 Ϯ 1.6, 33.4 Ϯ 3.2, and 34.8 Ϯ 3.2 min͞h; dark were switched off (8 p.m.) and recorded for the following 2 days. period: 19.3 Ϯ 2.1, 20.3 Ϯ 1.8, and 18.4 Ϯ 2.3 min͞h for wild-type, EEG and EMG were recorded with a data acquisition program B7, and B20 TG mice, respectively) and REM (light period: (Kissei COMTEC, Matsumoto, Japan; NEC Medical Systems, 4.4 Ϯ 0.8, 4.6 Ϯ 0.9, and 4.8 Ϯ 0.8 min͞h; dark period: 3.2 Ϯ 0.7, Osaka, Japan), and subsequently analyzed by Animal Sleep 3.4 Ϯ 0.8, and 3.4 Ϯ 0.9 min͞h for wild-type, B7, and B20 TG program (Kissei COMTEC). The wake, NREM, and REM sleep mice, respectively) sleep were also similar between the geno- states were determined for 4-s epochs based on the EEG and types. When spontaneous locomotion of mice was monitored EMG recordings according to the criteria as described (20). through the dark͞light cycle, both wild-type and TG mice Tail clipping performed in the experiments is essentially displayed a circadian rhythm for locomotor activity, although the identical to the routine tail clipping procedure used for the DNA basal level of the locomotor activity of TG mice was elevated in sampling of mice. Tail ends of about 0.5 cm were clipped with comparison with that of the wild-type controls throughout the

4904 ͉ www.pnas.org Pinzar et al. Downloaded by guest on September 24, 2021 Fig. 3. Effect of tail clipping on spontaneous locomotion. (A) Tail clipping, indicated by arrow, applied in the beginning of the dark period (8 p.m.) did not alter locomotor activity of wild-type mice (n ϭ 15), yet decreased the locomotion of B7 (B)(n ϭ 20) and B20 (C)(n ϭ 20) TG mice. Data points are means Ϯ SEMper1hofrecording. E, Baseline recordings; F, after tail clipping. *, P Ͻ 0.05; **, P Ͻ 0.005.

recording time (Fig. 3). The basal locomotion of B7 mice was about 2-fold higher than that of wild-type mice (Fig. 3 A and B). Locomotor activity of B20 line appeared intermediate between B7 and wild-type mice (Fig. 3C). Thus, the higher locomotor activity of TG mice is attributable to the overexpression of the PGDS gene in almost all tissues, manifesting itself in motor Fig. 2. NREM and REM sleep in B7 and B20 TG and wild-type mice before and up-regulation. after tail clip. Tail clipping, indicated by arrow, was performed at 8 p.m., the The major difference between the genotypes was observed time at which the lights were turned off. Each value is the mean Ϯ SEM after tail clipping, performed for DNA sampling used for the estimated from1hofbaseline recording (E) or after tail clipping (F). (A) NREM genotyping. TG, but not wild-type, mice appeared sedated by the and REM sleep were not affected in wild-type mice by the tail clipping. (B)In stimulus. Therefore, we applied tail-clip stimulation to TG and the B7 mice, tail clipping increased the amount of NREM sleep without affecting the amount of REM sleep. (C) In the B20 line of TG mice, effect of tail wild-type mice and assessed their sleep by measuring EEG,

clipping was comparable to the B7 mice. n ϭ 7 for wild-type mice, n ϭ 10 for EMG, and locomotor activity. After tail clipping, the amount of NEUROBIOLOGY B7, and n ϭ 10 for B20 TG mice; *, P Ͻ 0.05; **, P Ͻ 0.005; ***, P Ͻ 0.0001. nocturnal NREM sleep of TG mice was significantly higher than

Pinzar et al. PNAS ͉ April 25, 2000 ͉ vol. 97 ͉ no. 9 ͉ 4905 Downloaded by guest on September 24, 2021 Fig. 4. Time course of PGD2 content in the brain after tail clipping. Tail clip induced production of PGD2 in B7 and B20 TG mice but not in wild-type mice. Time point ‘‘0 h’’ represents the control data, immediately after tail clip. n ϭ 6 for each genotype at each time point; *, P Ͻ 0.01; **, P Ͻ 0.001 by unpaired Student’s t test.

that of their wild-type littermate controls (Fig. 2). The effect was hand, the basal amount of PGE2 in the brain was comparable apparent in the first hour after stimulation, continued to increase between the genotypes for untreated animals (32.0 Ϯ 3.1, 30.4 Ϯ time-dependently, and dissipated within5hinboth lines of TG 5.3, and 31.3 Ϯ 3.7 pg͞brain for wild-type, B7, and B20 TG mice, mice (Fig. 2 B and C). Slight rebound of wakefulness was noted respectively). Tail-clip stimulation nonsignificantly increased in the first half of the light period. The maximal 2.5-fold production of PGE2 in both TG and wild-type mice. However, increment of NREM sleep was observed3hafterthetailhad the extent was much less than that of the PGD2 increase. Thus, been clipped. At this point, the sleep was almost saturated and the induction of NREM sleep in TG mice is consistent with this comparable to the amount of sleep during the daytime. In increase in the PGD2 production in the brain. contrast, REM sleep was unaffected by the tail clipping in either of the lines of TG mice (Fig. 2 B and C). The sleep pattern of Discussion wild-type counterparts after tail clipping was essentially similar This report shows the involvement of the endogenously pro- to the baseline recordings with only slight decreases in both duced PGD2 in the induction of physiological sleep. Previously, NREM and REM sleep, which were observed mainly in the dark the relationship between PGD2 and sleep was examined mainly period (Fig. 2A). pharmacologically by administration of exogenous PGD2 into Associated with sleep induction, tail clipping decreased loco- the brain and inhibition of PGDS in vivo by selenium compounds motor activity in the TG mice (Fig. 3 B and C). Despite the high (3, 4, 10, 13) or under certain pathological conditions as reported basal locomotor activity of these mice, tail clipping done at the in some sleep disorders (14, 15). beginning of the dark period, when mice are generally the most Although no differences were found in the baseline recordings active, caused a drastic decrease in the locomotor activity of the of vigilance states between wild-type and TG mice, the tail B7 and B20 mice, in the first5hwithsubsequent recovery, a clipping performed for DNA sampling caused a remarkable pattern similar to that seen in the case of sleep induction. increase in nocturnal sleep of TG mice consistent with drastic Wild-type mice did not change their locomotor activity in decrease in locomotor activity, whereas wild-type controls were response to the tail clipping (Fig. 3A). unaffected by the stimulus (Figs. 2 and 3). Moreover, the sleep To examine whether the induced sleep in TG mice is coupled induction in TG mice was attributed to the changes in NREM to the changes in the PGs production, we measured the amounts sleep, whereas REM sleep remained undisturbed (Fig. 2 B and of PGD2 and PGE2, which are known to induce sleep (2–5) and C). Despite the high basal locomotor activity of TG mice, tail wakefulness (23), respectively, in the brains of TG and wild-type clipping applied at the beginning of the dark period caused a mice at different time points after their tails had been clipped. dramatic decrease in the locomotion of animals in the first 4–5 Ϯ Ϯ The basal amount of PGD2 in TG mice (62.9 5.6 and 75.8 h (Fig. 3B). This observation is in good agreement with previous ͞ 21.1 pg brain for B7 and B20 mice, respectively) was 1.2- and reports indicating that i.v. injection of PGD2 produced a signif- 1.5-fold greater than that of the wild-type mice (49.8 Ϯ 6.8 icant decrease in the spontaneous locomotor activity in mice and ͞ pg brain). The PGD2 amount in the brain of TG mice increased at the same time potentiated pentobarbital-induced sleeping gradually after tail clipping, with a peak representing a 17- and time (24). The tail-clip effect on TG mice was shown to be 5-fold increase at3haftertheclipping (1,051.3 Ϯ 150.9 and consistent in two independent lines: B7 and B20 (Figs. 2 and 3). 383.9 Ϯ 46.8 pg͞brain) of B7 and B20 mice, respectively, and Thus, the increase in the amount of NREM sleep and subsequent then decreased (Fig. 4). In the case of the wild-type mice, the decrease in locomotion in TG mice in response to the tail PGD2 content was slightly increased1hafterthetailclipping clipping can be interpreted as a result of the overexpression of and then returned to the control level (Fig. 4). On the other PGDS and possibly excessive production of endogenous PGD2

4906 ͉ www.pnas.org Pinzar et al. Downloaded by guest on September 24, 2021 rather than the result of the alterations in some candidate PGD2 induced NREM sleep in clipped TG mice (Fig. 2 B and for sleep regulation caused by the insertion of the transgene. C). Although the maximal extent of PGD2 production on The interpretation of the current experiments is based on the stimulation differed between B7 and B20 mice, the NREM sleep presumption that the response of TG mice to the tail clipping is induction was comparable between these lines (Figs. 2 and 4). because of the initial overexpression of the human PGDS in This indicates that as little as 5-fold increase in the endogenous these mice, and thus increased levels of PGD2. It is well known PGD2 observed in the brains of B20 mice could induce almost that when cells are exposed to various exogenous stimuli, such as saturable amount of sleep (Figs. 2C and 4). The observation that pain, bacterial lipopolysaccharides, or ethanol, arachidonic acid NREM sleep was induced in TG mice primarily because of the is released from phospholipids in the cell membrane and is increased production of PGD2 is consistent with the findings that further metabolized by arachidonic acid cascade by the action of infusion of PGD2 in the rostral basal forebrain of rats effectively cyclooxygenase (COX) to produce PGH2, which is then con- and specifically induced NREM sleep (10). Because NREM verted to the individual PGs and thromboxane by tissue-specific sleep was selectively affected in these TG mice, it is likely that synthases, for example, PGDS or PGES (6). The rate-limiting the PGDS gene is responsible for the regulation of NREM sleep, step is generally considered to be the production of PGH2, in contrast to the recently reported orexin͞hypocretin gene, catalyzed in sequential reaction by phospholipase A2 and COX, which was identified as a gene to be involved in the pathogenesis rather than the synthase step, under normal conditions. This of narcolepsy and possibly in the regulation of REM sleep (27, suggestion is supported by the observation that, in TG mice as 28). Thus, the PGDS-TG mice proved to be a useful animal well as in wild-type ones, PGDS appeared to be constitutively model to study profound lethargy or excess sleep mediated by expressed in the brain and was not induced by tail clipping (data PGD2. not shown). On the other hand, high amounts of COX-2, an inducible form of COX, were observed in parts of the CNS We thank Profs. I. Tobler, S. Yamamoto, E. Mignot, and M. Yanagisawa involved in the processing and regulation of nociceptive, stress- for critical reading of the manuscript and valuable comments. We are ful, and inflammatory inputs, and behavioral responses (25, 26). indebted to Prof. I. Tobler for helpful advice concerning the sleep Although the transcripts of COX-2, as well as COX-1, and analyses of mice; we thank Drs. T. Mochizuki, K. Fujimori, Y. Fujitani, and C. Beuckmann for discussions pertaining to the preparation of the cytosolic phospholipase A2 were only insignificantly changed in TG and wild-type mice by the stimulation (data not shown), the manuscript, and Ms. Y. Ono, N. Uodome, and Y. Kuwahata for their technical assistance. This study was supported by grants from the Core tail clipping is likely to activate the arachidonic acid cascade at Research for Evolutional Science and Technology of the Japan Science the posttranscriptional level, including enzyme(s) activation, and Technology Agency (to Y.U.), the Japan Space Forum (to Y.U.), the finally resulting in an elevated production of PGH2. The TG mice Japan Society for the Promotion of Science (Grant 11680642, to E.P.), overexpressed PGDS, which then converted this excess PGH2 the Science and Technology Agency (to E.P.), and the Ministry of Health into an abundance of PGD2 (Fig. 4). This elevated production of and Welfare of Japan (Grant 100107, to O.H.).

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