Prostaglandin E₂ (PGE₂) 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 to approach this issue, we examined PGE₂-induced gene expression changes at single cell resolution in preoptic neurons expressing EP3. Brain sections of the preoptic area from PGE₂- 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 -aminobutyric acid type A receptor (GABA_A) subunits is decreased upon PGE₂ injection. Indeed, we found that most EP3-positive neurons in the mouse preoptic area are positive for the 2 or 2 GABA_A receptor subunit. Moreover, PGE₂ decreased the preoptic gene expression of these GABA_A subunits via an EP3-dependent and pertussis toxin-sensitive pathway. PGE₂ also attenuated the preoptic protein expression of the 2 subunit in wild-type but not in EP3-deficient mice. These results indicate that PGE₂-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 PGE₂-induced fever.