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

Results

For generation of TG mice, we used the cDNA for human PGDS, which was inserted under the control of the chicken β-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 mRNA for the endogenous mouse PGDS was seen only in the brain and epididymis, as reported previously (21). Five independent lines of TG mice were generated, and all of the animals appeared to be healthy, grew normally, and produced offspring. Usage of the inbred FVB/N mouse strain for the entire process of transgenesis allowed us to avoid ambiguity caused by different genetic backgrounds (22). When Northern analysis was performed on brain samples from TG mice, the highest expression of mRNA of PGDS was found in B7 and B20 lines (Fig. 1B). Measurement of PGDS activity in the brains of these lines of TG mice revealed that the B7 and B20 mice contained 5- and 2.5-fold greater activity than that of their wild-type counterparts, respectively (Fig. 1C). We used these TG lines to evaluate the role of PGDS in sleep regulation.

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Figure 1

Generation of PGDS-TG mice. (A) Construct of the human PGDS cDNA used for microinjection into the fertilized eggs of FVB/N mouse strain; CMV, cytomegalovirus. (B) Northern blot analysis of total RNA (10 μg/lane) from the brains of five lines of TG mice, using ³²P-labeled cDNA probes for human PGDS 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 [¹⁴C]PGH₂ as the substrate.

All mice exhibited an almost identical circadian pattern in their sleep, and no significant differences were found between wild-type and TG mice (Fig. 2). Analyses of the sleep distribution in the light and dark periods showed that the amounts of NREM (light period: 35.7 ± 1.6, 33.4 ± 3.2, and 34.8 ± 3.2 min/h; dark period: 19.3 ± 2.1, 20.3 ± 1.8, and 18.4 ± 2.3 min/h for wild-type, B7, and B20 TG mice, respectively) and REM (light period: 4.4 ± 0.8, 4.6 ± 0.9, and 4.8 ± 0.8 min/h; dark period: 3.2 ± 0.7, 3.4 ± 0.8, and 3.4 ± 0.9 min/h for wild-type, B7, and B20 TG mice, respectively) sleep were also similar between the genotypes. When spontaneous locomotion of mice was monitored through the dark/light cycle, both wild-type and TG mice displayed a circadian rhythm for locomotor activity, although the basal level of the locomotor activity of TG mice was elevated in comparison with that of the wild-type controls throughout the 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 up-regulation.

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Figure 2

NREM and REM sleep in B7 and B20 TG and wild-type mice before and after tail clip. Tail clipping, indicated by arrow, was performed at 8 p.m., the time at which the lights were turned off. Each value is the mean ± SEM estimated from 1 h of baseline recording (○) or after tail clipping (●). (A) NREM and REM sleep were not affected in wild-type mice by the tail clipping. (B) In 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 clipping was comparable to the B7 mice. n = 7 for wild-type mice, n = 10 for B7, and n = 10 for B20 TG mice; *, P < 0.05; **, P < 0.005; ***, P < 0.0001.

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Figure 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 = 15), yet decreased the locomotion of B7 (B) (n = 20) and B20 (C) (n = 20) TG mice. Data points are means ± SEM per 1 h of recording. ○, Baseline recordings; ●, after tail clipping. *, P < 0.05; **, P < 0.005.

The major difference between the genotypes was observed after tail clipping, performed for DNA sampling used for the genotyping. TG, but not wild-type, mice appeared sedated by the stimulus. Therefore, we applied tail-clip stimulation to TG and wild-type mice and assessed their sleep by measuring EEG, EMG, and locomotor activity. After tail clipping, the amount of nocturnal NREM sleep of TG mice was significantly higher than that of their wild-type littermate controls (Fig. 2). The effect was apparent in the first hour after stimulation, continued to increase time-dependently, and dissipated within 5 h in both lines of TG mice (Fig. 2 B and C). Slight rebound of wakefulness was noted in the first half of the light period. The maximal 2.5-fold increment of NREM sleep was observed 3 h after the tail had been clipped. At this point, the sleep was almost saturated and comparable to the amount of sleep during the daytime. In 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 wild-type counterparts after tail clipping was essentially similar to the baseline recordings with only slight decreases in both NREM and REM sleep, which were observed mainly in the dark period (Fig. 2A).

Associated with sleep induction, tail clipping decreased locomotor activity in the TG mice (Fig. 3 B and C). Despite the high basal locomotor activity of these mice, tail clipping done at the beginning of the dark period, when mice are generally the most active, caused a drastic decrease in the locomotor activity of the B7 and B20 mice, in the first 5 h with subsequent recovery, a pattern similar to that seen in the case of sleep induction. Wild-type mice did not change their locomotor activity in response to the tail clipping (Fig. 3A).

To examine whether the induced sleep in TG mice is coupled to the changes in the PGs production, we measured the amounts of PGD₂ and PGE₂, which are known to induce sleep (2–5) and wakefulness (23), respectively, in the brains of TG and wild-type mice at different time points after their tails had been clipped. The basal amount of PGD₂ in TG mice (62.9 ± 5.6 and 75.8 ± 21.1 pg/brain for B7 and B20 mice, respectively) was 1.2- and 1.5-fold greater than that of the wild-type mice (49.8 ± 6.8 pg/brain). The PGD₂ amount in the brain of TG mice increased gradually after tail clipping, with a peak representing a 17- and 5-fold increase at 3 h after the clipping (1,051.3 ± 150.9 and 383.9 ± 46.8 pg/brain) of B7 and B20 mice, respectively, and then decreased (Fig. 4). In the case of the wild-type mice, the PGD₂ content was slightly increased 1 h after the tail clipping and then returned to the control level (Fig. 4). On the other hand, the basal amount of PGE₂ in the brain was comparable between the genotypes for untreated animals (32.0 ± 3.1, 30.4 ± 5.3, and 31.3 ± 3.7 pg/brain for wild-type, B7, and B20 TG mice, respectively). Tail-clip stimulation nonsignificantly increased production of PGE₂ in both TG and wild-type mice. However, the extent was much less than that of the PGD₂ increase. Thus, the induction of NREM sleep in TG mice is consistent with this increase in the PGD₂ production in the brain.

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Figure 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 = 6 for each genotype at each time point; *, P < 0.01; **, P < 0.001 by unpaired Student's t test.

Discussion

This report shows the involvement of the endogenously produced PGD₂ in the induction of physiological sleep. Previously, the relationship between PGD₂ and sleep was examined mainly pharmacologically by administration of exogenous PGD₂ into the brain and inhibition of PGDS in vivo by selenium compounds (3, 4, 10, 13) or under certain pathological conditions as reported in some sleep disorders (14, 15).

Although no differences were found in the baseline recordings of vigilance states between wild-type and TG mice, the tail clipping performed for DNA sampling caused a remarkable increase in nocturnal sleep of TG mice consistent with drastic decrease in locomotor activity, whereas wild-type controls were unaffected by the stimulus (Figs. 2 and 3). Moreover, the sleep induction in TG mice was attributed to the changes in NREM sleep, whereas REM sleep remained undisturbed (Fig. 2 B and C). Despite the high basal locomotor activity of TG mice, tail clipping applied at the beginning of the dark period caused a dramatic decrease in the locomotion of animals in the first 4–5 h (Fig. 3B). This observation is in good agreement with previous reports indicating that i.v. injection of PGD₂ produced a significant decrease in the spontaneous locomotor activity in mice and at the same time potentiated pentobarbital-induced sleeping time (24). The tail-clip effect on TG mice was shown to be consistent in two independent lines: B7 and B20 (Figs. 2 and 3). Thus, the increase in the amount of NREM sleep and subsequent decrease in locomotion in TG mice in response to the tail clipping can be interpreted as a result of the overexpression of PGDS and possibly excessive production of endogenous PGD₂ rather than the result of the alterations in some candidate genes for sleep regulation caused by the insertion of the transgene.

The interpretation of the current experiments is based on the presumption that the response of TG mice to the tail clipping is because of the initial overexpression of the human PGDS in these mice, and thus increased levels of PGD₂. It is well known that when cells are exposed to various exogenous stimuli, such as pain, bacterial lipopolysaccharides, or ethanol, arachidonic acid is released from phospholipids in the cell membrane and is further metabolized by arachidonic acid cascade by the action of cyclooxygenase (COX) to produce PGH₂, which is then converted to the individual PGs and thromboxane by tissue-specific synthases, for example, PGDS or PGES (6). The rate-limiting step is generally considered to be the production of PGH₂, catalyzed in sequential reaction by phospholipase A₂ and COX, rather than the synthase step, under normal conditions. This suggestion is supported by the observation that, in TG mice as well as in wild-type ones, PGDS appeared to be constitutively expressed in the brain and was not induced by tail clipping (data not shown). On the other hand, high amounts of COX-2, an inducible form of COX, were observed in parts of the CNS involved in the processing and regulation of nociceptive, stressful, and inflammatory inputs, and behavioral responses (25, 26). Although the transcripts of COX-2, as well as COX-1, and cytosolic phospholipase A₂ were only insignificantly changed in TG and wild-type mice by the stimulation (data not shown), the tail clipping is likely to activate the arachidonic acid cascade at the posttranscriptional level, including enzyme(s) activation, finally resulting in an elevated production of PGH₂. The TG mice overexpressed PGDS, which then converted this excess PGH₂ into an abundance of PGD₂ (Fig. 4). This elevated production of PGD₂ induced NREM sleep in clipped TG mice (Fig. 2 B and C). Although the maximal extent of PGD₂ production on stimulation differed between B7 and B20 mice, the NREM sleep induction was comparable between these lines (Figs. 2 and 4). This indicates that as little as 5-fold increase in the endogenous PGD₂ observed in the brains of B20 mice could induce almost saturable amount of sleep (Figs. 2C and 4). The observation that NREM sleep was induced in TG mice primarily because of the increased production of PGD₂ is consistent with the findings that infusion of PGD₂ in the rostral basal forebrain of rats effectively and specifically induced NREM sleep (10). Because NREM sleep was selectively affected in these TG mice, it is likely that the PGDS gene is responsible for the regulation of NREM sleep, in contrast to the recently reported orexin/hypocretin gene, which was identified as a gene to be involved in the pathogenesis of narcolepsy and possibly in the regulation of REM sleep (27, 28). Thus, the PGDS-TG mice proved to be a useful animal model to study profound lethargy or excess sleep mediated by PGD₂.