Prostaglandin E2 activates the histaminergic system via the EP4 receptor to induce wakefulness in rats

Abstract

Prostaglandin (PG)E2 promotes the wakeful state when administered into the posterior hypothalamus, in which the histaminergic tuberomammillary nucleus (TMN) is located. To explore the neurotransmitter mechanisms responsible for PGE2-induced wakefulness in rats, we examined the effect of PGE2 on the activity of the histaminergic system and the involvement of PGE2 receptor subtypes in the response. PGE2 perfusion in the TMN at doses of 100, 200, and 400 pmol/min for 2 hr significantly increased histamine release from the medial preoptic area and frontal cortex in a dose-dependent manner, as measured by in vivo microdialysis. Among the agonists of the four distinct subtypes of PGE2 receptors (EP1-4) tested, only the EP4 receptor agonist (ONO-AE1-329) mimicked the excitatory effect of PGE2 on histamine release from both the medial preoptic area and frontal cortex. Perfusion of either PGE2 or the EP4 agonist into the TMN at a dose of 200 pmol/min for 1 hr increased histidine decarboxylase activity, histidine decarboxylase mRNA level, and histamine content in the hypothalamus. In situ hybridization revealed that EP4 receptor mRNA was expressed in histidine decarboxylase-immunoreactive neurons of the TMN region. Furthermore, EP4 agonist perfusion into the TMN induced wakefulness. These findings indicate that PGE2 induces wakefulness through activation of the histaminergic system via EP4 receptors.

Figure 1.

Schematic representation of the implantation sites for microdialysis probes. Coronal sections are from the stereotaxic atlas of Paxinos and Watson (1997). PGE₂ or EP agonist was administered into the TMN through a microdialysis probe with a membrane (unshaded area) of 2 mm length, and histamine was monitored by another probe implanted in the MPO (A) (membrane length, 2 mm) or the FrCx (B) (membrane length, 3 mm).

Figure 2.

Histamine release from the MPO and FrCx after perfusion of PGE₂ in the TMN for 2 hr. A, Time courses of histamine release from the MPO. B, Time courses of histamine release from the FrCx. The open circles, filled squares, filled diamonds, and filled circles stand for the groups treated with vehicle control and with PGE₂ at doses of 100, 200, and 400 pmol/min, respectively. The horizontal filled bar indicates the duration of PGE₂ perfusion. C, Dose dependency of histamine released from the MPO and FrCx during 2 hr PGE₂ perfusion. The open, light gray, medium gray, and dark gray bars stand for the groups treated with vehicle control and with PGE₂ at doses of 100, 200, and 400 pmol/min, respectively. Each value represents the mean ± SEM of five or six rats. ^*p<0.05; ^**p<0.01, significantly different from the control, as assessed by two-way (A, B) or one-way (C) ANOVA followed by the PLDS test.

Figure 3.

Amounts of histamine released from the MPO (open bars) and FrCx (filled bars) after perfusion of the TMN with four distinct EP receptor agonists or PGE₂ at the same dose of 200 pmol/min for 2 hr. Each value is expressed as the absolute amount of released histamine during these perfusions for 2 hr and as the mean ± SEM of five or six rats. ^*p < 0.05; ^**p < 0.01, significantly different from their respective control, as assessed by one-way ANOVA followed by the PLDS test.

Figure 4.

Photomicrographs showing the immunoreactivity for HDC (A, D) and the mRNA signal for EP₄(B,E) in adjacent serial sections around the TMN. C, Control section hybridized with the sense probe. The cryosection was double labeled to indicate the localization of HDC (red) (F) and EP₄ mRNA (green) (G). Both F and G are merged in H. Differential interference contrast, 4′,6′-diamidino-2-phenylindole-stained image is shown in I. Arrowheads point to HDC and EP₄ double-positive neurons. Scale bars: A–C, 200 μm; D, E, 65 μm; (in I) F–I, 25 μm.

Figure 5.

Histamine content in the hypothalamus after perfusion of the TMN with PGE₂ or EP₄ agonist at doses of 200 and 400 pmol/min for 1 hr. The control groups were perfused with ACSF containing 0.5% DMSO. Histamine content is expressed as nanomoles per gram of wet weight and as the mean ± SEM of five or six rats. The open, gray, and filled bars stand for the groups treated with vehicle control and with EP₄ agonist or PGE₂ at doses of 200 and 400 pmol/min, respectively. ^*p < 0.05; ^**p < 0.01, significantly different from the control, as assessed by one-way ANOVA followed by the PLDS test.

Figure 6.

Increase in expression of HDC mRNA and HDC activity in the hypothalamus after perfusion of the TMN with PGE₂ or EP₄ agonist at a dose of 200 pmol/min for 1 hr. A, Northern blot analysis of HDC and GAPDH mRNAs in the hypothalamus treated with PGE₂ or EP₄ agonist. Each lane contained 20 μg of total RNA. B, The mRNA for HDC was quantified as a ratio to GAPDH mRNA by Northern blotting. The HDC mRNA content is indicated as a percentage of the control. C, HDC activity was measured in the homogenate of the hypothalamus. Control rats were perfused with ACSF containing 0.5% DMSO. The open, gray, and filled bars stand for the groups treated with vehicle control, EP₄ agonist, and PGE₂, respectively. Values are expressed as the mean ± SEM of five rats. ^*p < 0.05, significantly different from the control, as assessed by one-way ANOVA followed by the PLDS test.

Figure 7.

Typical examples of polygraphic recordings and corresponding hyponograms in a rat before and after the administration of EP₄ agonist at a dose of 400 pmol/min. A, Baseline day. B, Experimental day. The horizontal filled bar indicates the duration of EP₄ agonist perfusion.

Figure 8.

Sleep-stage distribution produced by EP agonist perfusion of the rat TMN. A, Time course changes in the 400 pmol/min EP₄ agonist treatment group. Each circle represents the hourly mean ± SEM of wakefulness, NREM sleep, or REM sleep. Open and filled circles stand for the baseline and experimental day profiles, respectively. The short horizontal filled bars indicate the perfusion between 9:00 and 11:00 A.M. on the experimental day. The long horizontal open and filled bars on the x-axes indicate the 12 hr light and dark periods, respectively. B, Total time spent in wakefulness, NREM sleep, and REM sleep for 2 hr during the perfusion. Open, gray, and filled bars show the profiles of baseline days, and of experimental days treated with vehicle and EP agonists, respectively. Values are the means ± SEM (n = 5 or 6 in EP₄ agonist treatment; n = 4 in each EP₁, EP₂, and EP₃ agonist treatment group). ^*p < 0.05; ^**p < 0.01 by the paired t test. The statistical significance of amounts of each stage in EP₄ agonist-treated groups was assessed by one-way ANOVA followed by the PLDS test.