Different populations of prostaglandin EP3 receptor-expressing preoptic neurons project to two fever-mediating sympathoexcitatory brain regions

Abstract

The central mechanism of fever induction is triggered by an action of prostaglandin E(2) (PGE(2)) on neurons in the preoptic area (POA) through the EP3 subtype of prostaglandin E receptor. EP3 receptor (EP3R)-expressing POA neurons project directly to the dorsomedial hypothalamus (DMH) and to the rostral raphe pallidus nucleus (rRPa), key sites for the control of thermoregulatory effectors. Based on physiological findings, we hypothesize that the febrile responses in brown adipose tissue (BAT) and those in cutaneous vasoconstrictors are controlled independently by separate neuronal pathways: PGE(2) pyrogenic signaling is transmitted from EP3R-expressing POA neurons via a projection to the DMH to activate BAT thermogenesis and via another projection to the rRPa to increase cutaneous vasoconstriction. In this case, DMH-projecting and rRPa-projecting neurons would constitute segregated populations within the EP3R-expressing neuronal group in the POA. Here, we sought direct anatomical evidence to test this hypothesis with a double-tracing experiment in which two types of the retrograde tracer, cholera toxin b-subunit (CTb), conjugated with different fluorophores were injected into the DMH and the rRPa of rats and the resulting retrogradely labeled populations of EP3R-immunoreactive neurons in the POA were identified with confocal microscopy. We found substantial numbers of EP3R-immunoreactive neurons in both the DMH-projecting and the rRPa-projecting populations. However, very few EP3R-immunoreactive POA neurons were labeled with both the CTb from the DMH and that from the rRPa, although a substantial number of neurons that were not immunoreactive for EP3R were double-labeled with both CTbs. The paucity of the EP3R-expressing neurons that send collaterals to both the DMH and the rRPa suggests that pyrogenic signals are sent independently to these caudal brain regions from the POA and that such pyrogenic outputs from the POA reflect different control mechanisms for BAT thermogenesis and for cutaneous vasoconstriction by distinct sets of POA neurons.

Fig. 1

Sites of CTb injections. (A) Alexa488-conjugated CTb and Alexa594-conjugated CTb were injected into the DMH (A; bregma, −3.30 mm) and into the rRPa (B; bregma, −11.80 mm), respectively. Areas where injected CTb spread are shown on the brain maps adopted from an atlas of Paxinos and Watson (1998). Injections in the three animals (#19, #21 and #23) that were used for histochemical analyses are shown. 3V, Third ventricle; Arc, arcuate nucleus; DA, dorsal hypothalamic area; DMN, dorsomedial hypothalamic nucleus; f, fornix; Giα, alpha part of the gigantocellular reticular nucleus; ic, internal capsule; LH, lateral hypothalamic area; ml, medial lemniscus; mt, mammillothalamic tract; py, pyramidal tract; ROb, raphe obscurus nucleus; RMg, raphe magnus nucleus; VMH, ventromedial hypothalamic nucleus.

Fig. 2

Confocal images of CTb-labeled POA neurons with or without EP3R immunoreactivity. Fluorescence signals of Alexa488-conjugated CTb (transported from the DMH; green), fluorescence signals of Alexa594-conjugated CTb (transported from the rRPa; red) and immunoreactivity for EP3Rs (blue) are pseudocolored and merged. EP3R-immunoreactive neurons that were labeled with DMH-CTb (filled single arrowhead), EP3R-immunoreactive neurons that were labeled with rRPa-CTb (open arrowhead) and neurons that were labeled with both DMH-CTb and rRPa-CTb but lack EP3R immunoreactivity (arrow) were densely distributed in the MnPO and MPO. However, EP3R-immunoreactive neurons that were labeled with both DMH-CTb and rRPa-CTb (inset, double arrowhead) were hardly found. Scale bar=20 μm.

Fig. 3

Distribution of CTb-labeled neuronal cell bodies with or without EP3R immunoreactivity in the POA. Drawings of POA sections from the animal #21 are shown in a rostrocaudal order (A–D). Blue lines delineate EP3R-immunoreactive regions. Dotted lines outline the areas that were scanned with a confocal microscope. The left side is ipsilateral to the CTb injection into the DMH. ac, Anterior commissure; ox, optic chiasm. Scale bar=0.5 mm.

Fig. 4

Percentages of EP3R-immunoreactive neurons in DMH-CTb-labeled (A) and rRPa-CTb-labeled (B) populations in POA subregions. CTb-labeled cells in the EP3R-immunoreactive regions were counted in every sixth 25-mm-thick frontal section throughout the POA. The POA was divided into three subregions in (A): the MnPO and the MPO ipsilateral (Ipsi. MPO) and contralateral (Cont. MPO) to the unilateral CTb injection into the DMH, and into two subregions in (B): the MnPO and the MPO. The data represent the mean±SEM (n=3). * P<0.05, paired t-test.