Prostaglandin E2 Attenuates Preoptic Expression of GABAA Receptors via EP3 Receptors

Takakazu Oka,Yukihiko Sugimoto,Clifford B. Saper,Kazuhiro Nakamura,Hiroyoshi Tsuchiya,Atsushi Ichikawa

doi:10.1074/jbc.m801359200

Abstract

Prostaglandin E(2) (PGE(2)) has been shown to produce fever by acting on EP3 receptors within the preoptic area of the brain. However, there is little information about the molecular events downstream of EP3 activation in preoptic neurons. As a first step toward this issue, we examined PGE(2)-induced gene expression changes at single-cell resolution in preoptic neurons expressing EP3. Brain sections of the preoptic area from PGE(2)- or saline-injected rats were stained with an anti-EP3 antibody, and the cell bodies of EP3-positive neurons were dissected and subjected to RNA amplification procedures. Microarray analysis of the amplified products demonstrated the possibility that gene expression of gamma-aminobutyric acid type A (GABA(A)) receptor subunits is decreased upon PGE(2) injection. Indeed, we found that most EP3-positive neurons in the mouse preoptic area are positive for the alpha2 or gamma2 GABA(A) receptor subunit. Moreover, PGE(2) decreased the preoptic gene expression of these GABA(A) subunits via an EP3-dependent and pertussis toxin-sensitive pathway. PGE(2) also attenuated the preoptic protein expression of the alpha2 subunit in wild-type but not in EP3-deficient mice. These results indicate that PGE(2)-EP3 signaling elicits G(i/o) activation in preoptic thermocenter neurons, and we propose the possibility that a rapid decrease in preoptic GABA(A) expression may be involved in PGE(2)-induced fever.

Highlights

Tion in body temperature by 1–2 °C and is one of the representative systemic responses against inflammation [1, 2]
The current study indicates that EP3 receptor couples to Gi/o proteins in the preoptic area (POA) neurons, leading to attenuation of gabr gene expression
If such a molecular event reflects the functional changes of the GABA type A (GABAA) channel in thermal regulation as discussed below, fever generation could be triggered by the activation of Gi/o in EP3-expressing POA neurons

Summary

EXPERIMENTAL PROCEDURES

Animals—Adult male pathogen-free Sprague-Dawley rats (250 –350 g; Taconic, Germantown, NY) were used for microarray analysis (Fig. 1 and Table 1). The brains were postfixed, saturated with a sucrose solution, and cut into coronal sections (10 ␮m), which were stained with an anti-rat EP3 rabbit antibody as described previously [11]. Single-cell microdissection and RNA amplification were performed essentially as described previously [18]. Twelve single cells derived from three rats per group (PGE2-injected and saline-injected groups) were pooled and subjected to two-round RNA amplification. Immunofluorescence Microscopy—Coronal sections (14 ␮m) of adult male EP3ϩ/Ϫ mice were fixed and incubated in phosphate-buffered saline containing a blocking reagent. Dissection of POA and Real Time-Polymerase Chain Reaction (PCR)—Wild type and EP3Ϫ/Ϫ mice anesthetized with ␣-chrolalose-HBC (40 mg/kg intraperitoneally; Sigma) were administered intracerebroventricularly (i.c.v.) with PGE2 (1 nM, 5 ␮l) or saline [20], and rectal temperatures were monitored. A comparison between two groups was performed using Student’s t test; p values Ͻ0.05 were considered significantly different

RESULTS

Gene product

GABAA subunit

DISCUSSION