Direct pyrogenic input from prostaglandin EP3 receptor-expressing preoptic neurons to the dorsomedial hypothalamus

Abstract

Fever is induced by a neuronal mechanism in the brain. Prostaglandin (PG) E2 acts as a pyrogenic mediator in the preoptic area (POA) probably through the EP3 subtype of PGE receptor expressed on GABAergic neurons, and this PGE2 action triggers neuronal pathways for sympathetic thermogenesis in peripheral effector organs including brown adipose tissue (BAT). To explore pyrogenic efferent pathways from the POA, we determined projection targets of EP3 receptor-expressing POA neurons with a special focus on rat hypothalamic regions including the dorsomedial hypothalamic nucleus (DMH), which is known as a center for autonomic responses to stress. Among injections of cholera toxin b-subunit (CTb), a retrograde tracer, into hypothalamic regions at the rostrocaudal level of the DMH, injections into the DMH, lateral hypothalamic area (LH) and dorsal hypothalamic area (DH) resulted in EP3 receptor immunolabelling in substantial populations of CTb-labeled neurons in the POA. Bilateral microinjections of muscimol, a GABA(A) receptor agonist, into the DMH and a ventral region of the DH, but not those into the LH, inhibited thermogenic (BAT sympathetic nerve activity, BAT temperature, core body temperature and expired CO2) and cardiovascular (arterial pressure and heart rate) responses to an intra-POA PGE2 microinjection. Further immunohistochemical observations revealed a close association of POA-derived GABAergic axon swellings with DMH neurons projecting to the medullary raphe regions where sympathetic premotor neurons for febrile and thermoregulatory responses are localized. These results suggest that a direct projection of EP3 receptor-expressing POA neurons to the DMH/DH region mediates febrile responses via a GABAergic mechanism.

Fig. 1

Anterograde neural tract tracing from the POA to hypothalamic regions at the rostrocaudal level of the DMH. (a) Injection of recombinant Sindbis virus into the MPO. Infected cells expressed a membrane-targeted form of EGFP; these cells formed a cluster and many neuronal fibers with EGFP fluorescence extended from the cell cluster. (b) Distribution of EGFP-immunoreactive fibers in a coronal section at the rostrocaudal level of the DMH (bregma −3.30 mm). Many axon fibers derived from infected neurons in the MPO were distributed in several hypothalamic regions. (b′) Illustration of the anatomical map of the section shown in (b). (c) EGFP immunoreactivity in the DMH. Boutons (arrowheads) as well as fibers exhibited EGFP immunoreactivity. 3V, third ventricle; Arc, arcuate nucleus; f, fornix; ox, optic chiasm; MTu, medial tuberal nucleus; VMH, ventromedial hypothalamic nucleus; ZI, zona incerta. Scale bars, 0.3 mm (a), 0.5 mm (b), 20 μm (c).

Fig. 2

EP3 receptor-expressing POA neurons directly project to the hypothalamic regions. (a) A representative view of CTb injection sites in the DMH (arrow; showing case # 26). (b) Composite drawing of CTb injection sites (gray regions) and percentages of EP3 receptor-immunoreactive neurons in CTb-labeled ones within the EP3 receptor-immunoreactive POA regions (see (d)). Injection sites that were positioned in the hypothalamic regions at around bregma −3.30 mm were drawn on a brain map taken from an atlas by Paxinos & Watson (1998). Top and bottom numbers identify injection cases and percentages for each injection case, respectively. Numbers of cells are shown in Table 1. CTb injections into the DMH, DH, and LH, which showed relatively high percentages, were colored dark gray. (c) and (c′) POA neuronal cell bodies double-labeled with EP3 receptor and CTb immunoreactivities (arrows). The photomicrographs were taken at the same site under different conditions of excitation. (d) Distribution of POA neurons labeled with CTb (open circles) and with both CTb and EP3 receptor immunoreactivities (filled circles) after a unilateral CTb injection into the DMH (#26) or into the LH (#53). Injection was made on the right side, and the injection case numbers correspond to those in (b). All CTb-labeled cells distributed in the shown regions were drawn. EP3 receptor-immunoreactive regions are colored gray. ac, anterior commissure; mt, mammillothalamic tract; SubI, subincertal nucleus. Scale bars, 0.5 mm (a and d), 30 μm (c and c′).

Fig. 3

Effects of microinjections of muscimol into hypothalamic regions on PGE₂-evoked increases in BAT SNA, T_BAT, T_rec, expired (Exp.) CO₂, AP, and HR. (a–d) Changes in the physiological variables after bilateral microinjections of saline (a and c) or muscimol (b and d) into the DMH (a and b) or LH (c and d) following thermogenic and cardiovascular responses to unilateral microinjection of PGE₂ into the POA. Horizontal scale bar represents 5 min for (a)–(d). Vertical scale bars for BAT SNA traces represent 100 μV in (a) and (d), 250 μV in (b), and 50 μV in (c). (e) Composite drawing of sites of saline and muscimol microinjections with their inhibitory effects on the increase in BAT SNA by intra-POA PGE₂. Microinjections into the hypothalamic regions were made bilaterally and their sites were positioned symmetrically. Injection sites on the right side were drawn on a brain map (bregma −3.30 mm). The inhibitory effect was expressed as inhibition percentage of PGE₂-evoked increase in BAT SNA and graded as follows: > 80%, full inhibition (filled circles); 10–80%, partial inhibition (triangles); < 10%, no inhibition (crosses). Inhibition percentages by saline injections were always less than 10% (open circles). (f) and (g) Representative views of a PGE₂ microinjection into the MPO (f) and muscimol microinjections into the DMH (g). Each injection site is clearly identified as a cluster of fluorescent beads (arrows). Scale bar, 0.5 mm (f and g).

Fig. 4

DMH neurons projecting to medullary raphe regions receive GABAergic inputs from POA neurons. (a) Site of Fluoro-Gold injection (gray region) into the rostral part of the raphe pallidus nucleus (rRPa) and the raphe magnus nucleus (RMg). (b) Distribution of Fluoro-Gold-labeled neurons in a coronal section at the rostrocaudal level of the DMH (bregma −3.30 mm) after the injection shown in (a). (c) Injection of Sindbis virus into the POA. Many infected cells, which exhibited EGFP fluorescence (green), were distributed in the region where EP3 receptor-immunoreactive neurons (red) were localized. Inset shows infection of EP3 receptor-immunoreactive neurons with the Sindbis virus; there were neurons exhibiting both EP3 receptor immunoreactivity (red) and EGFP fluorescence (green) (arrows) in the injection site. (d) Site of CTb injection (gray region) into the rRPa and RMg. (e) Confocal laser-scanning microscopy in DMH sections triple immunofluorescence-stained for EGFP (green), VGAT (red), and CTb (blue). The signals for these three labelings are shown using pseudo-colors. Axon swellings double-labeled with EGFP and VGAT immunoreactivities were closely associated with CTb-immunoreactive neuronal cells (arrowheads). Two representative cases found in the DMH are shown. Scale bars, 0.5 mm (a–d), 20 μm (inset in c), 5 μm (e).

Fig. 5

A current hypothesis on the neural pathways mediating fever on the basis of the previous and present findings. Under PGE₂-free condition (Normal), neurons in the DMH and rRPa are tonically inhibited by inputs from EP3 receptor-expressing GABAergic neurons in the POA. It is possible that the DMH neurons receive excitatory inputs from unknown regions, but cannot be activated due to stronger inhibition from the POA neurons. After infection (Infection), PGE₂, which is produced in brain vasculature in response to immune signals, suppresses the tonic firing of the POA neurons by activating the EP3 receptor, and hereby, the DMH and rRPa neurons are released from the tonic inhibition. Excitation of the DMH neurons, which can be triggered by the excitatory inputs from the unknown regions, activates sympathetic premotor neurons in the rRPa, which in turn stimulate the sympathetic output system and finally develop fever. Blue, red, and black circles denote cell bodies of activated inhibitory neurons, activated excitatory neurons, and suppressed neurons, respectively. IML, intermediolateral cell column; SPN, sympathetic preganglionic neuron.