Prostaglandin D synthase in the prenatal ovine brain and effects of its inhibition with selenium chloride on fetal sleep/wake activity in utero

Abstract

It has been proposed that prostaglandin (PG) D(2) induces physiological sleep in mammals by acting on sleep centers located in the anterior hypothalamus. In fetal sheep, definitive rapid-eye-movement and non-rapid-eye-movement sleep states appear at approximately 125 d gestation (term is approximately 147 d). In adult animals, PGD synthase (PGDS) (functionally and structurally homologous to beta-trace protein) is secreted into CSF with a circadian pattern, with the highest concentrations present during sleep. In this study we show that PGDS/beta-trace protein is present in fetal sheep CSF at 125 and 135 d gestation but not at 90 d gestation. SeCl(4), a specific inhibitor of PGDS, was given to unanesthetized fetal sheep (130-140 d gestation) by intracerebroventricular infusion at a dose of 25, 100, 500, or 1000 pmol/min for 4 hr. Artificial CSF was infused in control experiments. Arousal behavior, defined as the presence of nuchal muscle electromyogram activity, electro-ocular activity, and breathing movements during low-amplitude electrocortical activity, increased from 3.8 +/- 1 min/hr to 6.6 +/- 0.5 and 7.0 +/- 0.3 min/hr at doses of 100 and 500 pmol/min, respectively (p < 0.05). SeCl(4) at 25 and 1000 pmol/min had no significant effect on arousal activity. Infusion of PGD(2) at 500 pmol/min intracerebroventricularly for 4 hr decreased the incidence of arousal from 3.8 +/- 0.5 min/hr to 0.7 +/- 0.3 min/hr (p < 0.05). When 500 pmol/min PGD(2) was infused immediately after a 4 hr infusion of SeCl(4) (500 pmol/min), the SeCl(4)-induced increase in arousal behavior was abolished. Together, the presence of PGDS/beta-trace protein in fetal CSF in late gestation and the effects of SeCl(4) in increasing the incidence of arousal-like behavior suggest that PGD(2) has a role in the induction and maintenance of prenatal sleep.

Fig. 1.

A, Western blot showing immunoreactive β-trace protein in adult human and fetal ovine CSF and its absence from fetal ovine choroid plexus (CP) hypothalamus, liver and muscle, and adult rat cortex. The molecular mass marker (27 kDa) shown by the arrow on theleft was obtained from protein standards run on the same gel (data not shown). B, Western blot of immunoreactive β-trace protein in fetal ovine CSF at 90, 125, and 135 d gestation (n = 3 for each age). β-Trace protein was undetectable at 90 d gestation. The position of the 27 kDa molecular mass marker is shown by the arrow on the left. C, Densitometric analysis of β-trace protein expression is shown in B. Results shown are means ± SEM; the asteriskindicates a significant difference between the values at 90 d compared with those at 125 and 135 d gestation (p < 0.05).

Fig. 2.

A, Polygraph recording during the pretreatment control period trace from a fetal sheep at 135 d gestation showing ECoG, EOG, integrated nuchal electromyogram (EMG_int) and fetal breathing movement (FBM) activities. REM sleep (black bar) was defined by the presence of FBM and EOG activity during low-amplitude ECoG activity. NREM sleep (open bar) was defined by the presence of varying but nearly continuous activity in the nuchal muscle during high-amplitude ECoG activity. Arousal (shaded bar) was identified by the presence of breathing movements, nuchal EMG, and EOG activities simultaneously with low-amplitude ECoG activity. B, Polygraph recording obtained from the same fetus shown in A >9 hr after the commencement of SeCl₄ at 500 pmol/min over 4 hr. Note the increase in the total amount of nuchal EMG and EOG activities and the increased incidence of the period defined as arousal (shaded bars). Periods that did not conform exactly to REM, NREM, or arousal behaviors are shown by the hatched bars at the beginning and end of the first NREM episode.

Fig. 3.

Effects of infusing aCSF (n = 5) or SeCl₄ at 500 pmol/min (n = 4) on the hourly incidence (minutes per hour) of arousal-like activity. Infusions were administered into the left ventricle of fetuses at 130–140 d gestation at 10 μl/min for 4 hr (solid bar). Administration of SeCl₄ caused a significant increase (p < 0.05, as shown byasterisks) in the incidence of arousal compared with equivalent times for infusion of aCSF. Data shown are means ± SEM.

Fig. 4.

Effect of infusing SeCl₄ at 25, 100, 500, or 1000 pmol/min on the incidence (minutes per hour) of arousal behavior in fetal sheep. Zero dose was aCSF infused at 10 μl/min for 4 hr. Data shown are means ± SEM.

Fig. 5.

Effects of infusing aCSF (n = 5) or SeCl₄ at 500 pmol/min (n = 4) on the hourly incidence of nuchal EMG activity in the presence of low-amplitude ECoG. Infusions were administered into the left cerebral ventricle of fetuses at 130–140 d gestation for 4 hr (solid bar). Administration of SeCl₄ caused a significant increase (p < 0.05, as shown byasterisks) in the incidence of nuchal EMG activity in the presence of low-voltage activity compared with equivalent times for infusion of aCSF. Data shown are means ± SEM.

Fig. 6.

Effects of infusing SeCl₄ for 4 hr into the left cerebral ventricle of fetuses at 130–140 d gestation on the hourly incidence of EOG and breathing movements. Values were calculated by accumulating all activity over the 4 hr epochs and then determining the mean incidence per hour. Administration of SeCl₄ at 25 (n = 4) and 100 (n = 4) pmol/min caused a significant increase (p < 0.05, as shown byasterisks) in the incidence of nuchal EMG activity. Infusion of aCSF or the other doses of SeCl₄ had no effects on these parameters. Data shown are means ± SEM.

Fig. 7.

Effects of infusing aCSF (n = 5) or PGD₂ at 500 pmol/min (n = 6) on the hourly incidence (minutes per hour) of arousal. Infusions were administered into the left ventricle of fetuses at 130–140 d gestation at 10 μl/min for 4 hr (solid bar). PGD₂caused a significant decrease in the incidence of arousal (p < 0.05, as shown byasterisks) compared with both the control period within the treatment group and with equivalent times for infusion of aCSF. Data shown are means ± SEM.

Fig. 8.

Effects of infusing SeCl₄ alone at 500 pmol/min (solid bars) or the same dose of SeCl₄ followed by a 4 hr infusion of PGD₂ at 500 pmol/min (shaded bars). The increased incidences of arousal and nuchal muscle EMG activities produced by the SeCl₄ treatments were abolished by the infusion of PGD₂. Data shown are means ± SEM (n = 4 fetuses) and demonstrate the incidence (minute per hour) of activity for consecutive 4 hr epochs.