No abstract

Sympathetic premotor neurons directly control sympathetic preganglionic neurons (SPNs) in the intermediolateral cell column (IML) of the thoracic spinal cord, and many of these premotor neurons are localized in the medulla oblongata. The rostral ventrolateral medulla contains premotor neurons controlling the cardiovascular conditions, whereas rostral medullary raphe regions are a candidate source of sympathetic premotor neurons for thermoregulatory functions. Here, we show that these medullary raphe regions contain putative glutamatergic neurons and that these neurons directly control thermoregulatory SPNs. Neurons expressing vesicular glutamate transporter 3 (VGLUT3) were distributed in the rat medullary raphe regions, including the raphe magnus and rostral raphe pallidus nuclei, and mostly lacked serotonin immunoreactivity. These VGLUT3-positive neurons expressed Fos in response to cold exposure or to central administration of prostaglandin E 2 , a pyrogenic mediator. Transneuronal retrograde labeling after inoculation of pseudorabies virus into the interscapular brown adipose tissue (BAT) or the tail indicated that those VGLUT3-expressing medullary raphe neurons innervated these thermoregulatory effector organs multisynaptically through SPNs of specific thoracic segments, and microinjection of glutamate into the IML of the BAT-controlling segments produced BAT thermogenesis. An anterograde tracing study further showed a direct projection of those VGLUT3-expressing medullary raphe neurons to the dendrites of SPNs. Furthermore, intra-IML application of glutamate receptor antagonists blocked BAT thermogenesis triggered by disinhibition of the medullary raphe regions. The present results suggest that VGLUT3-expressing neurons in the medullary raphe regions constitute excitatory neurons that could be categorized as a novel group of sympathetic premotor neurons for thermoregulatory functions, including fever.

Fever is triggered by an elevation of prostaglandin E 2 (PGE 2 ) in the brain. However, the mechanism of its elevation remains unanswered. We herein cloned the rat glutathione-dependent microsomal prostaglandin E synthase (mPGES), the terminal enzyme for PGE 2 biosynthesis, and examined its induction in the rat brain after intraperitoneal injection of pyrogen lipopolysaccharide (LPS). In Northern blot analysis, mPGES mRNA was weakly expressed in the brain under the normal conditions but was markedly induced between 2 and 4 hr after the LPS injection. In situ hybridization study revealed that LPS-induced mPGES mRNA signals were mainly associated with brain blood vessels, especially vein or venular-type ones, in the whole brain area. Immunohistochemical study demonstrated that mPGESlike immunoreactivity was expressed in the perinuclear region of brain endothelial cells, which were identified as von Willebrand factor-positive cells. Furthermore, in the perinuclear region of the endothelial cells, mPGES was colocalized with cyclooxygenase-2 (COX-2), which is the enzyme essential for the production of the mPGES substrate PGH 2 . Inhibition of cyclooxygenase-2 activity resulted in suppression of both PGE 2 level in the CSF and fever (Cao et al., 1997), suggesting that the two enzymes were functionally linked and that this link is essential for fever. These results demonstrate that brain endothelial cells play an essential role in the PGE 2 production during fever by expressing COX-2 and mPGES.

Fever is the widely known hallmark of disease and is induced by the action of the nervous system. It is generally accepted that prostaglandin (PG) E(2) is produced in response to immune signals and then acts on the preoptic area (POA), which triggers the stimulation of the sympathetic system, resulting in the production of fever. Actually, the EP3 subtype of PGE receptor, which is essential for the induction of fever, is known to be localized in POA neurons. However, the neural pathway mediating the pyrogenic transmission from the POA to the sympathetic system remains unknown. To identify the neuronal groups involved in the fever-inducing pathway, we first investigated Fos expression in medullary regions of rats after central administrations of PGE(2). PGE(2) application to the lateral ventricle or directly to the POA strikingly increased the number of Fos-positive neurons in the rostral part of the raphe pallidus nucleus (rRPa). Most of these neurons did not exhibit serotonin immunoreactivity. Microinjection of muscimol, a GABA(A) receptor agonist, into the rRPa blocked fever and thermogenesis in brown adipose tissue induced by intra-POA as well as by intracerebroventricular PGE(2) applications. Furthermore, neural tract tracing studies revealed a direct projection from EP3 receptor-expressing POA neurons to the rRPa. Our results demonstrate that the rRPa, which has never been associated with the fever mechanism, mediates the pyrogenic neurotransmission from the POA to the peripheral sympathetic effectors contributing to fever development.

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.