No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Immunocytochemical localization of prostaglandin EP3 receptor in the rat hypothalamus
Abstract
A rabbit antibody against an N-terminal portion of rat prostaglandin EP3 receptor (EP3R) was produced to examine the distribution of EP3R in the rat hypothalamus. The antibody specifically recognized EP3R proteins in rat brain extract, in membrane fractions of rat kidney, and in membrane fractions of EP3R-expressing culture cells. Intense EP3R-like immunoreactivity was observed in the median preoptic nucleus, medial preoptic area, parastrial nucleus, compact part of the dorsomedial hypothalamic nucleus, and dorsal part of the premammillary nucleus. These results suggest that prostaglandin E2 mediates various actions in the hypothalamus, such as fever induction in the preoptic area, through EP3R.
... EP3R proteins are localized in neuronal cell bodies and dendritic fibers in the median preoptic nucleus (MnPO) and medial POA (MPA) (fig. S1, A and B) (14,15) and mediate the febrile action of PGE 2 (16). However, there is no evidence that POA EP3R neurons are involved in basal thermoregulation. ...
... The tissue was cut into 30μm-thick frontal sections on a freezing microtome. The primary antibodies used are anti-EP3R rabbit antibody (1 μg/ml) (14,15), anti-Fos goat antibody (1:1000; sc-52G, Santa Cruz Biotechnology), anti-CTb goat serum (1:5000; #703, List Biological Laboratories), anti-GFP mouse antibody (1:200; A11120, Thermo Fisher Scientific), anti-GFP rabbit antibody (0.5 μg/ml) (60), anti-VGAT guinea pig serum (1:1000; 131004, Synaptic Systems), anti-VGLUT2 rabbit antibody (0.5 μg/ml) (61), anti-GAD67 mouse antibody (1:300; MAB5406, Sigma-Aldrich), anti-synaptophysin mouse antibody (1:1000; S5768, Sigma-Aldrich), and anti-monomeric red fluorescent protein (mRFP) guinea pig antibody (1 μg/ml) (62). The anti-mRFP guinea pig antibody and the anti-GFP mouse antibody showed cross-reactivity to mCherry and EYFP, respectively. ...
The bidirectional controller of the thermoregulatory center in the preoptic area (POA) is unknown. Using rats, here, we identify prostaglandin EP3 receptor–expressing POA neurons (POA EP3R neurons) as a pivotal bidirectional controller in the central thermoregulatory mechanism. POA EP3R neurons are activated in response to elevated ambient temperature but inhibited by prostaglandin E 2 , a pyrogenic mediator. Chemogenetic stimulation of POA EP3R neurons at room temperature reduces body temperature by enhancing heat dissipation, whereas inhibition of them elicits hyperthermia involving brown fat thermogenesis, mimicking fever. POA EP3R neurons innervate sympathoexcitatory neurons in the dorsomedial hypothalamus (DMH) via tonic (ceaseless) inhibitory signaling. Although many POA EP3R neuronal cell bodies express a glutamatergic messenger RNA marker, their axons in the DMH predominantly release γ-aminobutyric acid (GABA), and their GABAergic terminals are increased by chronic heat exposure. These findings demonstrate that tonic GABAergic inhibitory signaling from POA EP3R neurons is a fundamental determinant of body temperature for thermal homeostasis and fever.
... ;https://doi.org/10.1101https://doi.org/10. /2022 4 (Nakamura et al., 1999 ( Figure S1A) and mediate the febrile action of PGE2 (Lazarus et al., 2007). However, there is no evidence that POA EP3R neurons are involved in basal thermoregulation. ...
... The tissue was cut into 30-µm-thick frontal sections on a freezing microtome. The primary antibodies used are anti-EP3R rabbit antibody (1 μg/ml; Nakamura et al., 1999Nakamura et al., , 2000, anti-Fos goat antibody (1:1000; sc-52G, Santa For double immunofluoscence staining for EP3R and Fos, sections were incubated with anti-EP3R rabbit antibody and anti-Fos goat antibody overnight at 4°C and then with Alexa594-conjugated donkey antibody to goat IgG (10 μg/ml; A11058, Thermo Fisher) and biotinylated donkey antibody to rabbit IgG (1:100; AP182B, Merck Millipore) for 1 hr at room temperature. After rinsed, these sections were further (which was not certified by peer review) is the author/funder. ...
The circuit mechanism of the thermoregulatory center in the preoptic area (POA) is unknown. Using rats, here we show prostaglandin EP3 receptor-expressing POA neurons (POA EP3R neurons) as a pivotal bidirectional controller in the central thermoregulatory mechanism. POA EP3R neurons are activated in response to elevated ambient temperature, but inhibited by prostaglandin E 2 , a pyrogenic mediator. Chemogenetic stimulation of POA EP3R neurons at room temperature reduces body temperature by enhancing heat dissipation, whereas inhibition of them elicits hyperthermia involving brown fat thermogenesis, mimicking fever. POA EP3R neurons innervate sympathoexcitatory neurons in the dorsomedial hypothalamus (DMH) via tonic inhibitory signaling. Although many POA EP3R neuronal cell bodies express a glutamatergic mRNA marker, paradoxically, their axons in the DMH predominantly contain terminals with GABAergic presynaptic proteins, which are increased by chronic heat exposure. These findings indicate that tonic GABAergic inhibitory signaling from POA EP3R neurons is a fundamental determinant of body temperature for thermal homeostasis and fever.
... PGE 2 triggers febrile responses by acting on the EP3 subtype of PGE receptors (EP3R) expressed in the POA. EP3R proteins are localized in many cell bodies and dendritic fibres in the MnPO and medial preoptic area (MPA) 101,102 (Fig. 3a), consistent with its mRNA distribution 103,104 . Because the EP3R-positive profiles form a meshwork that encircles the organum vasculosum lamina terminalis (OVLT; a circumventricular organ) 101,102 , EP3R-expressing POA neurons may function to sense pyrogenic mediators and other humoral factors in the blood, which could leak out of the fenestrated vessels in the OVLT. ...
... 3V, third ventricle; ac, anterior commissure; BDNF, brain-derived neurotrophic factor; BRS3, bombesin-like receptor 3; LPO, lateral preoptic area; ox, optic chiasm; PACAP, pituitary adenylate cyclase-activating polypeptide. Photomicrographs in part a adapted with permission from reF. 101 . ...
Various environmental stressors, such as extreme temperatures (hot and cold), pathogens, predators and insufficient food, can threaten life. Remarkable progress has recently been made in understanding the central circuit mechanisms of physiological responses to such stressors. A hypothalamomedullary neural pathway from the dorsomedial hypothalamus (DMH) to the rostral medullary raphe region (rMR) regulates sympathetic outflows to effector organs for homeostasis. Thermal and infection stress inputs to the preoptic area dynamically alter the DMH → rMR transmission to elicit thermoregulatory, febrile and cardiovascular responses. Psychological stress signalling from a ventromedial prefrontal cortical area to the DMH drives sympathetic and behavioural responses for stress coping, representing a psychosomatic connection from the corticolimbic emotion circuit to the autonomic and somatic motor systems. Under starvation stress, medullary reticular neurons activated by hunger signalling from the hypothalamus suppress thermogenic drive from the rMR for energy saving and prime mastication to promote food intake. This Perspective presents a combined neural network for environmental stress responses, providing insights into the central circuit mechanism for the integrative regulation of systemic organs. Environmental stressors, including extreme ambient temperature, the presence of pathogens or predators, and a lack of food, can profoundly influence animal behaviour. In this Perspective, Nakamura, Nakamura and Kataoka present a hypothalamomedullary network model for physiological responses to various environmental stressors.
... coordinated by the hypothalamus [14]. These areas include the lateral parabrachial nucleus at the junction of the pons and medulla, inputs from spinothalamocortical relay pathways, and the preoptic area (POA) of the hypothalamus [15]. ...
... These areas include the lateral parabrachial nucleus at the junction of the pons and medulla, inputs from spinothalamocortical relay pathways, and the preoptic area (POA) of the hypothalamus [15]. The median preoptic nucleus within the POA is temperature sensitive, especially to heat [14]. In addition to the hypothalamus, the brainstem also plays a significant role in central thermoregulation through brown adipose thermogenesis (BAT) [10]. ...
Introduction . Central hyperthermia is common in patients with brain injury. It typically has a rapid onset with high temperatures and marked fluctuations and responds poorly to antibiotics and antipyretics. It is also associated with worse outcomes in the brain injured patient. Recognizing this, it is important to aggressively manage it. Case Report . We report a 34-year-old male with a right thalamic hemorrhage extending to the midbrain and into the ventricles. During his admission, he developed intractable fevers with core temperatures as high as 39.3°C. Infectious workup was unremarkable. The fever persisted despite empiric antibiotics, antipyretics, and cooling wraps. Bromocriptine was started resulting in control of the central hyperthermia. The fever spikes were reduced to minor fluctuations that significantly worsened with any attempt to wean off the bromocriptine. Conclusion . Diagnosing and managing central hyperthermia can be challenging. The use of bromocriptine can be beneficial as we have reported.
... We also identified 50 and 78 kD immunospecific bands for EP 3 protein in CAMs. The size of the EP 3 receptor has been reported as low as 33-38 kD in splice variants [28,29] while the full length protein determined by amino acid comparison is estimated at 55-63 kD. There are two sites of possible N-linked glycosylation, which can yield products of 65-75 kD that have been previously detected on Western blots [28,29]. ...
... The size of the EP 3 receptor has been reported as low as 33-38 kD in splice variants [28,29] while the full length protein determined by amino acid comparison is estimated at 55-63 kD. There are two sites of possible N-linked glycosylation, which can yield products of 65-75 kD that have been previously detected on Western blots [28,29]. Together these results suggest that constrictive effects of PGE 1 and PGE 2 are mediated at least in part by EP 3 receptors. ...
The chick chorioallantoic membrane (CAM) subserves gas exchange in the developing embryo and shell-less culture affords a unique opportunity for direct observations over time of individual blood vessels to pharmacologic interventions. We tested a number of lipids including prostaglandins PGE(1&2) for vascular effects and signaling in the CAM. Application of PGE(1&2) induced a decrease in the diameter of large blood vessels and a concentration-dependent, localized, reversible loss of blood flow through small vessels. The loss of flow was also mimicked by misoprostol, an agonist for 3 of 4 known PGE receptors, EP(2-4), and by U46619, a thromboxane mimetic. Selective receptor antagonists for EP(3) and thromboxane each partially blocked the response. This is a first report of the effects of prostaglandins on vasoreactivity in the CAM. Our model allows the unique ability to examine simultaneous responses of large and small vessels in real time and in vivo.
... Within the POA, the MPO and MnPO were found highly responsive to PGE 2 for its pyrogenic action (147). Similar POA subregions harbor many neurons that express the EP3 subtype of PGE receptor with the subcellular distribution in their somata and dendritic fibers (Fig. 5A) (99,100). PGE receptors have four subtypes, EP1, EP2, EP3, and EP4 (114), and mRNA expression for the EP1 and EP4 receptors, in addition to the EP3 receptor, is detected in the POA (120). ...
... However, neurons triplelabeled with EP3R-immunoreactivity, DMH-CTb, and rRPa-CTb were hardly found.[From Nakamura et al.(99,102,111).]. ...
Body temperature regulation is a fundamental homeostatic function that is governed by the central nervous system in homeothermic animals, including humans. The central thermoregulatory system also functions for host defense from invading pathogens by elevating body core temperature, a response known as fever. Thermoregulation and fever involve a variety of involuntary effector responses, and this review summarizes the current understandings of the central circuitry mechanisms that underlie nonshivering thermogenesis in brown adipose tissue, shivering thermogenesis in skeletal muscles, thermoregulatory cardiac regulation, heat-loss regulation through cutaneous vasomotion, and ACTH release. To defend thermal homeostasis from environmental thermal challenges, feedforward thermosensory information on environmental temperature sensed by skin thermoreceptors ascends through the spinal cord and lateral parabrachial nucleus to the preoptic area (POA). The POA also receives feedback signals from local thermosensitive neurons, as well as pyrogenic signals of prostaglandin E(2) produced in response to infection. These afferent signals are integrated and affect the activity of GABAergic inhibitory projection neurons descending from the POA to the dorsomedial hypothalamus (DMH) or to the rostral medullary raphe region (rMR). Attenuation of the descending inhibition by cooling or pyrogenic signals leads to disinhibition of thermogenic neurons in the DMH and sympathetic and somatic premotor neurons in the rMR, which then drive spinal motor output mechanisms to elicit thermogenesis, tachycardia, and cutaneous vasoconstriction. Warming signals enhance the descending inhibition from the POA to inhibit the motor outputs, resulting in cutaneous vasodilation and inhibited thermogenesis. This central thermoregulatory mechanism also functions for metabolic regulation and stress-induced hyperthermia.
... For single immunoperoxidase staining for EP 3 receptors, the brain sections (thickness, 40 m) were incubated overnight with an anti-rat EP3 receptor rabbit polyclonal antibody (1 g/ml) (27) and then for 1 h with a biotinylated donkey antibody to rabbit IgG (10 g/ml; Chemicon, Temecula, CA). The sections were further incubated for 1 h with avidin-biotinylated peroxidase complex (ABC-Elite; 1:50; Vector). ...
... Immunoperoxidase staining visualized EP 3 receptor immunoreactivity in the PVN using an anti-EP 3 receptor antibody whose specificity in rat brain tissues was confirmed in our previous studies (27)(28)(29). The PVN and the periventricular hypothalamic nucleus were clearly identified with moderate to weak EP 3 receptor immunoreactivity (Fig. 9A). ...
Prostaglandin E(2) (PGE(2)), an important mediator of the inflammatory response, acts centrally to elicit sympathetic excitation. PGE(2) acts on at least four E-class prostanoid (EP) receptors known as EP(1), EP(2), EP(3), and EP(4). Since PGE(2) production within the brain is ubiquitous, the different functions of PGE(2) depend on the expression of these prostanoid receptors in specific brain areas. The type(s) and location(s) of the EP receptors that mediate sympathetic responses to central PGE(2) remain unknown. We examined this question using PGE(2), the relatively selective EP receptor agonists misoprostol and sulprostone, and the available selective antagonists for EP(1), EP(3), and EP(4). In urethane-anesthetized rats, intracerebroventricular (ICV) administration of PGE(2), sulprostone or misoprostol increased renal sympathetic nerve activity, blood pressure, and heart rate. These responses were significantly reduced by ICV pretreatment with the EP(3) receptor antagonist; the EP(1) and EP(4) receptor antagonists had little or no effect. ICV PGE(2) or misoprostol increased the discharge of neurons in the hypothalamic paraventricular nucleus (PVN). ICV misoprostol increased the c-Fos immunoreactivity of PVN neurons, an effect that was substantially reduced by the EP(3) receptor antagonist. Real-time PCR detected EP(3) receptor mRNA in PVN, and immunohistochemical studies revealed sparsely distributed EP(3) receptors localized in GABAergic terminals and on a few PVN neurons. Direct bilateral PVN microinjections of PGE(2) or sulprostone elicited sympathoexcitatory responses that were significantly reduced by the EP(3) receptor antagonist. These data suggest that EP(3) receptors mediate the central excitatory effects of PGE(2) on PVN neurons and sympathetic discharge.
... First, specific binding sites for PGE 2 should be apparent on cells in the POA. Receptors selective for PGE 2 , more specifically the EP 3 -receptor, have been identified on the somatodendritic portion of neurons in the POA (Nakamura et al., 1999; Oka et al., 2000; Nakamura et al., 2005). Binding of PGE 2 to EP 3 -receptors is thought to produce a change in the firing rate of critical neurons in the POA. ...
... This difference in the regional expression of Fos may be a consequence of the population of neurons in the POA that are responsive to muscimol versus PGE 2 . Muscimol is thought to inhibit virtually all mammalian neurons, while PGE 2 most likely acts on a subpopulation of neurons in the POA possessing EP 3 receptors, the principal receptor for PGE 2 in the POA (Nakamura et al., 1999; Bilateral inhibition of the DMH completely abolished the increased expression of Fos in the PVN and raphe pallidus elicited by muscimol in the POA (Fig. 15). Identical injections of muscimol into the DMH have been reported to prevent stress-induced expression of Fos in the PVN (Morin et al., 2001). ...
Recent studies in anesthetized rats suggest that autonomic effects relating to thermoregulation that are evoked from the preoptic area (POA) may be mediated through activation of neurons in the dorsomedial hypothalamus (DMH). Disinhibition of neurons in the DMH produces not only cardiovascular changes but also increases in plasma adrenocorticotropic hormone (ACTH) and locomotor activity mimicking those evoked by microinjection of muscimol, a GABAA receptor agonist and neuronal inhibitor, into the POA. Therefore, I tested the hypothesis that all of these effects evoked from the POA are mediated through neurons in the DMH by assessing the effect of bilateral microinjection of muscimol into the DMH on the changes evoked by microinjection of muscimol into the POA in conscious rats. In addition, I tested the hypothesis that neurons in the DMH mediate a specific response that is thought to signal through the POA, the activation of the HPA axis evoked by systemic administration of the inflammatory cytokine IL-1β. After injection of vehicle into the DMH, injection of muscimol into the POA elicited marked increases in heart rate, arterial pressure, body temperature, plasma ACTH and locomotor activity and also increased Fos expression in the hypothalamic paraventricular nucleus (PVN), a region known to control the release of ACTH from the adenohypophysis, and the raphe pallidus, a medullary region known to mediate POA-evoked sympathetic responses. Prior microinjection of muscimol into the DMH produced a modest depression of baseline heart rate, arterial pressure, and body temperature but completely abolished all changes evoked from the POA. Microinjection of muscimol just anterior to the DMH had no effect on POA-evoked autonomic and neuroendocrine changes. Inhibition of neuronal activity in the DMH only partially attenuated the increased activity of the HPA axis following systemic injections of IL-1β. Thus, neurons in the DMH mediate a diverse array of physiological and behavioral responses elicited from the POA, suggesting that the POA represents an important source of inhibitory tone to key neurons in the DMH. However, it is clear that the inflammatory cytokine IL-1β must employ other pathways that are DMH-, and possibly POA-, independent to activate the HPA axis.
... 1998; Yamagata et al., 2001). The EP3 subtype of prostaglandin E receptor is abundantly expressed on neurons in specific subregions of the preoptic area (POA): the median preoptic nucleus (MnPO) and medial preoptic area (MPO) (Nakamura et al., 1999(Nakamura et al., , 2000, and most of these neurons contain the inhibitory neurotransmitter, GABA (Nakamura et al., 2002). The action of PGE 2 on these EP3 receptors (EP3Rs), likely an inhibition of EP3Rexpressing POA neurons (Narumiya et al., 1999), is responsible for the activation of feverproducing neuronal pathways (Lazarus et al., 2007), although EP3Rs in the POA could also be involved in other physiological functions including thermal hyperalgesia (Hosoi et al., 1997). ...
... The sections containing the POA were incubated overnight in our conventional primary antibody incubation buffer (Nakamura et al., 2000(Nakamura et al., , 2001 containing anti-rat EP3R rabbit polyclonal antibody (0.5 μg/ml; Nakamura et al., 1999Nakamura et al., , 2000. After rinses in 50 mM phosphate-buffered saline (PBS; pH 7.3) containing 0.3% Triton X-100 (PBS-T), the sections were incubated for 1 h with 10 μg/ml horseradish peroxidase-conjugated anti-rabbit IgG donkey antibody (Chemicon, Temecula, CA, USA) in azide-free PBS-T containing 10% normal goat serum. ...
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.
... Les PGE2 sont des eicosanoïdes appartenant à la famille des métabolites de l'acide arachidonique. Leur synthèse est sous la dépendance de l'action enzymatique de la cyclooxygénase 2 (COX-2) qui est induite par divers facteurs dont l'IL-1 au niveau des cellules endothéliales de la vasculature cérébrale (Matsumura & Kobayashi 2004 (Nakamura et al 1999;Rage et al 1997;Sugimoto et al 1994;Zhang & Rivest 1999). Le récepteur EP1 est associé à une protéine G de type Gq. ...
... The EP3R is densely expressed in the MnPO, the most responsive site for producing fever induced by injection of PGE2 in the brain (Scammell et al., 1996;Nakamura et al., 1999;Oka et al., 2000;Yoshida et al., 2003) and thought to be an inhibitory receptor (Vasilache et al., 2007). It was expected that fever was due to PGE2 inhibiting MnPO EP3R inhibitory neurons, and immunohistochemical studies showed that these neurons expressed glutamic acid decarboxylase 67 (GAD1; Nakamura et al., 2002). ...
Fever is a common phenomenon during infection or inflammatory conditions. This stereotypic rise in body temperature (Tb) in response to inflammatory stimuli is a result of autonomic responses triggered by prostaglandin E2 action on EP3 receptors expressed by neurons in the median preoptic nucleus (MnPO EP3R neurons). To investigate the identity of MnPO EP3R neurons, we first used in situ hybridization to show coexpression of EP3R and the VGluT2 transporter in MnPO neurons. Retrograde tracing showed extensive direct projections from MnPO VGluT2 but few from MnPO Vgat neurons to a key site for fever production, the raphe pallidus. Ablation of MnPO VGluT2 but not MnPO Vgat neurons abolished fever responses but not changes in Tb induced by behavioral stress or thermal challenges. Finally, we crossed EP3R conditional knock-out mice with either VGluT2-IRES-cre or Vgat-IRES-cre mice and used both male and female mice to confirm that the neurons that express EP3R and mediate fever are glutamatergic, not GABAergic. This finding will require rethinking current concepts concerning the central thermoregulatory pathways based on the MnPO EP3R neurons being GABAergic.
SIGNIFICANCE STATEMENT Body temperature is regulated by the CNS. The rise of the body temperature, or fever, is an important brain-orchestrated mechanism for fighting against infectious or inflammatory disease, and is tightly regulated by the neurons located in the median preoptic nucleus (MnPO). Here we demonstrate that excitatory MnPO neurons mediate fever and examine a potential central circuit underlying the development of fever responses.
... Prostaglandin E 2 (PGE 2 ) produced by endothelial cells in the POA in response to pyrogens (such as lipopolysaccharide, a component of the outer membrane of gram-negative bacteria) acts on EP3 receptors in the POA to elicit febrile responses [64,119]. Within the POA, EP3 receptors are located on neurons in the MPA and MnPO [89]. Some data have suggested that it is EP3 receptor activation in the MnPO that is necessary for febrile responses [64,136]. ...
Maintenance of mammalian core body temperature within a narrow range is a fundamental homeostatic process to optimize cellular and tissue function, and to improve survival in adverse thermal environments. Body temperature is maintained during a broad range of environmental and physiological challenges by central nervous system circuits that process thermal afferent inputs from the skin and the body core to control the activity of thermoeffectors. These include thermoregulatory behaviors, cutaneous vasomotion (vasoconstriction and, in humans, active vasodilation), thermogenesis (shivering and brown adipose tissue), evaporative heat loss (salivary spreading in rodents, and human sweating). This review provides an overview of the central nervous system circuits for thermoregulatory reflex regulation of thermoeffectors.
... Systemic infection or inflammation or systemic injection of lipopolysaccharide in experimental fever stimulates biosynthesis of the pyrogenic mediator, PGE 2 , in endothelial cells of brain blood vessels (137) and in some peripheral tissues (138). Local and blood-borne PGE 2 acts via EP3Rs in POA neurons distributed in both MnPO and MPA (139,140) (Figure 2b) to trigger thermoeffector responses, including CVC, and BAT and shivering (chills) thermogenesis (7,53,141) that mimic cold-defense responses. ...
Maintenance of a homeostatic body core temperature is a critical brain function accomplished by a central neural network. This orchestrates a complex behavioral and autonomic repertoire in response to environmental temperature challenges or declining energy homeostasis and in support of immune responses and many behavioral states. This review summarizes the anatomical, neurotransmitter, and functional relationships within the central neural network that controls the principal thermoeffectors: cutaneous vasoconstriction regulating heat loss and shivering and brown adipose tissue for heat production. The core thermoregulatory network regulating these thermoeffectors consists of parallel but distinct central efferent pathways that share a common peripheral thermal sensory input. Delineating the neural circuit mechanism underlying central thermoregulation provides a useful platform for exploring its functional organization, elucidating the molecular underpinnings of its neuronal interactions, and discovering novel therapeutic approaches to modulating body temperature and energy homeostasis. Expected final online publication date for the Annual Review of Physiology Volume 81 is February 10, 2019. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... The results might represent a possible mechanism of temperature self-regulation. PGE 2 is believed to exert its effects by activation of G proteins through binding to EP3 receptor [1,3], the main subtype receptor involved in the development of febrile response [24,77]. This process can modulate intracellular levels of cAMP in addition to enhancing calcium levels. ...
... Febrile command signaling from the POA is triggered by an action of prostaglandin E 2 (PGE 2 ) on neurons in the POA. PGE 2 , which is biosynthesized in brain endothelial cells in response to immune signaling stimulated by infection [45,99,100], acts on prostaglandin EP3 receptors expressed in neurons in the POA [55,56] to trigger fever [31]. Because the EP3 receptor has been shown in cultured cells as a metabotropic receptor coupled to the inhibitory GTP-binding protein, G i [72], the action of PGE 2 on POA neurons through EP3 receptors likely inhibits their firing activity. ...
Energy homeostasis of mammals is maintained by balancing energy expenditure within the body and energy intake through feeding. Several lines of evidence indicate that brown adipose tissue (BAT), a sympathetically activated thermogenic organ, turns excess energy into heat to maintain the energy balance in rodents and humans, in addition to its thermoregulatory role for the defense of body core temperature in cold environments. Elucidating the central circuit mechanism controlling BAT thermogenesis dependent on nutritional conditions and food availability in relation to energy homeostasis is essential to understand the etiology of symptoms caused by energy imbalance, such as obesity. The central thermogenic command outflow to BAT descends through an excitatory neural pathway mediated by hypothalamic, medullary and spinal sites. This sympathoexcitatory thermogenic drive is controlled by tonic GABAergic inhibitory signaling from the thermoregulatory center in the preoptic area, whose tone is altered by body core and cutaneous thermosensory inputs. This circuit controlling BAT thermogenesis for cold defense also functions for the development of fever and psychological stress-induced hyperthermia, indicating its important role in the defense from a variety of environmental stressors. When food is unavailable, hunger-driven neural signaling from the hypothalamus activates GABAergic neurons in the medullary reticular formation, which then block the sympathoexcitatory thermogenic outflow to BAT to reduce energy expenditure and simultaneously command the masticatory motor system to promote food intake—effectively commanding responses to survive starvation. This article reviews the central mechanism controlling BAT thermogenesis in relation to the regulation of energy and thermal homeostasis dependent on food availability.
... Integration of temperature information from spinothalamocortical pathways as well as from the lateral parabrachial nucleus, located at the junction of pons and midbrain, occurs in the hypothalamic preoptic area (POA) (Morrison and Nakamura, 2011). POA neurons are uniquely sensitive to the influence of pyrogenic mediators, such as prostaglandin E2, and this area controls body temperature set point (Nakamura et al., 1999). ...
Hyperthermia from a central cause is associated with increased morbidity and mortality. Dysfunction of brainstem thermoregulatory pathways may explain the intractable rise in temperature. Antipyretics, dantrolene, bromocriptine, and surface and intravascular cooling devices have been attempted for temperature control. We report the case of a 54-year-old woman with history of hypertension who presented with pontine hemorrhage with extension into the midbrain and medulla. On days 8–9 of her hospital admission, she developed intractable fever and expired the same day despite aggressive treatment of hypothermia, including antipyretics, ice lavage, cold fluid boluses, surface cooling, dantrolene, and bromocriptine. Hyperthermia from brainstem hemorrhage can be difficult to manage with current treatment options. Early recognition of those patients who may develop hyperthermia could lead to early intervention and possibly better outcomes. More evidence from prospective randomized controlled trials will elucidate the risk–benefit profile of achieving normothermia with aggressive fever control in these patients.
... Thus, distribution of EP3 appears to overlap with those of three GABA A receptor genes, and we selected ␣2 and ␥2 as candidate subunits colocalized with EP3 receptors in the POA. Although an antirat EP3 antibody was used to detect EP3-expressing neurons in the POA, this antibody could not detect mouse EP3 receptor protein effectively (25). Moreover, because all anti-mouse EP3 antibodies that we tested were not suitable for immunohistochemistry, we used EP3 ϩ/Ϫ mice in which the -galactosidase (-gal) gene was "knocked in" at the EP3 gene locus (7). ...
... These studies and those by others demonstrated that EP3 receptors are highly localized to the MnPO appearing at rostral levels as an inverted Y-shaped structure, capping the OVLT. Lower levels of expression of the EP3 receptor were found in the medial part of the OVLT, the medial preoptic nucleus (MPO), and the caudal aspects of the VMPO (Ek et al., 2000;Nakamura et al., 1999;Nakamura et al., 2000;Yoshida et al., 2003). The localization of intense expression in the MnPO provides an important clue to the genesis of fever responses, as the EP3 receptor is probably the most critical for producing fever. ...
... Fever typically occurs when cells of the immune system respond to exogenous or endogenous insults by producing and releasing specific cytokines that ultimately lead to the production of the pyrogenic prostaglandin E2 (PGE 2 ) in either the brain vasculature or peripheral tissues [1,2]. PGE 2 elicits febrile responses largely through stimulating prostaglandin E receptor 3 (EP3) on neurons of the medial and the median preoptic nuclei (MPO and MnO, respectively) of the preoptic area (POA), leading to disinhibition of thermogenic neurons in caudal brain regions and activation of thermoregulatory effectors to increase heat production and reduce heat loss [3][4][5][6][7][8][9][10][11][12][13][14][15][16]. Indeed, PGE 2 -lowering cyclooxygenase (COX) inhibitors, such as aspirin and ibuprofen, have been used for over a century as fever-lowering agents. ...
Cyclooxygenase inhibitors such as ibuprofen have been used for decades to control fever through reducing the levels of the pyrogenic lipid transmitter prostaglandin E2 (PGE2). Historically, phospholipases have been considered to be the primary generator of the arachidonic acid (AA) precursor pool for generating PGE2 and other eicosanoids. However, recent studies have demonstrated that monoacyglycerol lipase (MAGL), through hydrolysis of the endocannabinoid 2-arachidonoylglycerol, provides a major source of AA for PGE2 synthesis in the mammalian brain under basal and neuroinflammatory states. We show here that either genetic or pharmacological ablation of MAGL leads to significantly reduced fever responses in both centrally or peripherally-administered lipopolysaccharide or interleukin-1β-induced fever models in mice. We also show that a cannabinoid CB1 receptor antagonist does not attenuate these anti-pyrogenic effects of MAGL inhibitors. Thus, much like traditional nonsteroidal anti-inflammatory drugs, MAGL inhibitors can control fever, but appear to do so through restricted control over prostaglandin production in the nervous system.
... Da: immunohistochemical localization of the EP3 receptor in the rat MnPO and MPA (left panel). Modified, with permission, from (237). Selective genetic deletion of the EP3 receptor in the MnPO of Ptger 3 CNS /NesCre mice prevents the rise in body temperature observed in wild-type mice following icv PGE 2 (right panel). ...
Thermogenesis, the production of heat energy, in brown adipose tissue is a significant component of the homeostatic repertoire to maintain body temperature during the challenge of low environmental temperature in many species from mouse to man and plays a key role in elevating body temperature during the febrile response to infection. The sympathetic neural outflow determining brown adipose tissue (BAT) thermogenesis is regulated by neural networks in the CNS which increase BAT sympathetic nerve activity in response to cutaneous and deep body thermoreceptor signals. Many behavioral states, including wakefulness, immunologic responses, and stress, are characterized by elevations in core body temperature to which central command-driven BAT activation makes a significant contribution. Since energy consumption during BAT thermogenesis involves oxidation of lipid and glucose fuel molecules, the CNS network driving cold-defensive and behavioral state-related BAT activation is strongly influenced by signals reflecting the short- and long-term availability of the fuel molecules essential for BAT metabolism and, in turn, the regulation of BAT thermogenesis in response to metabolic signals can contribute to energy balance, regulation of body adipose stores and glucose utilization. This review summarizes our understanding of the functional organization and neurochemical influences within the CNS networks that modulate the level of BAT sympathetic nerve activity to produce the thermoregulatory and metabolic alterations in BAT thermogenesis and BAT energy expenditure that contribute to overall energy homeostasis and the autonomic support of behavior. © 2014 American Physiological Society. Compr Physiol 4: 1677-1713, 2014.
... At infection, stimulated immune cells release cytokines, which then induce expression of cyclooxygenase-2 and microsomal prostaglandin (PG) E synthases in brain vasculature to synthesize PGE 2 (Yamagata et al., 2001). The produced PGE 2 acts on the EP3 subtype of PGE receptor expressed in neurons in the median preoptic nucleus and medial preoptic area (Nakamura et al., 1999(Nakamura et al., , 2000Lazarus et al., 2007). These EP3 receptor-expressing neurons are mostly GABAergic neurons that likely provide tonic inhibition on hypothalamic and medullary sympathoexcitatory neurons when PGE 2 is absent under nonfebrile conditions (Nakamura et al., 2002(Nakamura et al., , 2005. ...
The anxiolytic diazepam selectively inhibits psychological stress-induced autonomic and behavioral responses without causing noticeable suppression of other central performances. This pharmacological property of diazepam led us to the idea that neurons that exhibit diazepam-sensitive, psychological stress-induced activation are potentially those recruited for stress responses. To obtain neuroanatomical clues for the central stress circuitries, we examined the effects of diazepam on psychological stress-induced neuronal activation in broad brain regions. Rats were exposed to a social defeat stress, which caused an abrupt increase in body temperature by up to 2°C. Pretreatment with diazepam (4 mg/kg, i.p.) attenuated the stress-induced hyperthermia, confirming an inhibitory physiological effect of diazepam on the autonomic stress response. Subsequently, the distribution of cells expressing Fos, a marker of neuronal activation, was examined in 113 forebrain and midbrain regions of these rats after the stress exposure and diazepam treatment. The stress following vehicle treatment markedly increased Fos-immunoreactive cells in most regions of the cerebral cortex, limbic system, thalamus, hypothalamus and midbrain, which included parts of the autonomic, neuroendocrine, emotional and arousal systems. The diazepam treatment significantly reduced the stress-induced Fos expression in many brain regions including the prefrontal, sensory and motor cortices, septum, medial amygdaloid nucleus, medial and lateral preoptic areas, parvicellular paraventricular hypothalamic nucleus, dorsomedial hypothalamus, perifornical nucleus, tuberomammillary nucleus, association, midline and intralaminar thalami, and median and dorsal raphe nuclei. In contrast, diazepam increased Fos-immunoreactive cells in the central amygdaloid nucleus, medial habenular nucleus, ventromedial hypothalamic nucleus and magnocellular lateral hypothalamus. These results provide important information for elucidating the neural circuitries that mediate the autonomic and behavioral responses to psychosocial stressors.
... (A) Intense immunoreactivity for the EP3-R in the rat POA is particularly distributed in the MnPO, MPA, and parastrial nucleus (PS). Modified from Nakamura et al. (1999). (B) Counting of double-labeled neurons in MnPO and MPA that were EP3-R immunoreactive and retrogradely labeled following injection (D) of Alexa488-conjugated CTb and Alexa594-conjugated CTb in DMH and in rRPa, respectively, thereby suggesting their role in mediating the febrile response. ...
From mouse to man, brown adipose tissue (BAT) is a significant source of thermogenesis contributing to the maintenance of the body temperature homeostasis during the challenge of low environmental temperature. In rodents, BAT thermogenesis also contributes to the febrile increase in core temperature during the immune response. BAT sympathetic nerve activity controlling BAT thermogenesis is regulated by CNS neural networks which respond reflexively to thermal afferent signals from cutaneous and body core thermoreceptors, as well as to alterations in the discharge of central neurons with intrinsic thermosensitivity. Superimposed on the core thermoregulatory circuit for the activation of BAT thermogenesis, is the permissive, modulatory influence of central neural networks controlling metabolic aspects of energy homeostasis. The recent confirmation of the presence of BAT in human and its function as an energy consuming organ have stimulated interest in the potential for the pharmacological activation of BAT to reduce adiposity in the obese. In contrast, the inhibition of BAT thermogenesis could facilitate the induction of therapeutic hypothermia for fever reduction or to improve outcomes in stroke or cardiac ischemia by reducing infarct size through a lowering of metabolic oxygen demand. This review summarizes the central circuits for the autonomic control of BAT thermogenesis and highlights the potential clinical relevance of the pharmacological inhibition or activation of BAT thermogenesis.
... Since LPS is detected in plasma within 15 min after its i.p. injection (Lenczowski et al., 1997), it might act on CD14 receptors present on brain blood vessels (Lacroix et al., 1998) to induce COX-2. The prostaglandin EP-3 receptor is necessary for IL-1b-and LPS-induced fever (Ushikubi et al., 1998), and is found dorsally and laterally from the VMPO (Nakamura et al., 1999). However, LPS-induced Fos expression was not observed in the preoptic area in vagotomized animals. ...
Cytokines act on the brain to induce fever and behavioural depression after infection. Although several mechanisms of cytokine-to-brain communication have been proposed, their physiological significance is unclear. We propose that behavioural depression is mediated by the vagus nerve activating limbic structures, while fever would primarily be due to humoral mechanisms affecting the preoptic area, including interleukin-6 (IL-6) action on the organum vasculosum of the laminae terminalis (OVLT) and induction of prostaglandins. This study assessed the effects of subdiaphragmatic vagotomy in rats on fever, behavioural depression, as measured by the social interaction test, and Fos expression in the brain. These responses were compared with induction of the prostaglandin-producing enzyme cyclooxygenase-2 and the transcription factor Stat3 that translocates after binding of IL-6. Vagotomy blocked behavioural depression after intraperitoneal injection of recombinant rat IL-1β (25 µg/kg) or lipopolysaccharide (250 µg/kg; LPS) and prevented Fos expression in limbic structures and ventromedial preoptic area, but not in the OVLT. Fever was not affected by vagotomy, but associated with translocation of Stat3 in the OVLT and cyclooxygenase-2 induction around blood vessels. These results indicate that the recently proposed vagal link between the immune system and the brain activates limbic structures to induce behavioural depression after abdominal inflammation. Although the vagus might play a role in fever in response to low doses of LPS by activating the ventromedial preoptic area, it is likely to be overridden during more severe infection by action of circulating IL-6 on the OVLT or prostaglandins induced along blood vessels of the preoptic area.
... In a cold environment, the descending inhibition from the POA is attenuated by MnPO neurons that are activated by cutaneous cool signals (Nakamura & Morrison, 2008b). In the case of infection, immune signalling results in the brain vasculature production (Matsumura et al. 1998) of prostaglandin (PG) E 2 , a pyrogenic mediator, which can act through the EP3 subtype of PGE receptors in the POA (Nakamura et al. 1999(Nakamura et al. , 2000(Nakamura et al. , 2002Lazarus et al. 2007) to potentially attenuate the activity of descending GABAergic projection neurons. The coolingor pyrogen-triggered attenuation of the tonic descending inhibition from the POA is hypothesized to disinhibit the BAT sympathoexcitatory neurons in the DMH and sympathetic premotor neurons in the rostral medullary raphe region, including the rostral raphe pallidus nucleus (rRPa), which provide the excitatory drive for the sympathetic outflow determining BAT thermogenesis (Nakamura et al. 2002(Nakamura et al. , 2004(Nakamura et al. , 2005bMorrison, 2003;Madden & Morrison, 2003Zaretskaia et al. 2003;Nakamura & Morrison, 2007). ...
Shivering is a remarkable somatomotor thermogenic response that is controlled by brain mechanisms. We recorded EMGs in anaesthetized rats to elucidate the central neural circuitry for shivering and identified several brain regions whose thermoregulatory neurons comprise the efferent pathway driving shivering responses to skin cooling and pyrogenic stimulation. We simultaneously monitored parameters from sympathetic effectors: brown adipose tissue (BAT) temperature for non-shivering thermogenesis and arterial pressure and heart rate for cardiovascular responses. Acute skin cooling consistently increased EMG, BAT temperature and heart rate and these responses were eliminated by inhibition of neurons in the median preoptic nucleus (MnPO) with nanoinjection of muscimol. Stimulation of the MnPO evoked shivering, BAT thermogenesis and tachycardia, which were all reversed by antagonizing GABA(A) receptors in the medial preoptic area (MPO). Inhibition of neurons in the dorsomedial hypothalamus (DMH) or rostral raphe pallidus nucleus (rRPa) with muscimol or activation of 5-HT1A receptors in the rRPa with 8-OH-DPAT eliminated the shivering, BAT thermogenic, tachycardic and pressor responses evoked by skin cooling or by nanoinjection of prostaglandin (PG) E2, a pyrogenic mediator, into the MPO. These data are summarized with a schematic model in which the shivering as well as the sympathetic responses for cold defence and fever are driven by descending excitatory signalling through the DMH and the rRPa, which is under a tonic inhibitory control from a local circuit in the preoptic area. These results provide the interesting notion that, under the demand for increasing levels of heat production, parallel central efferent pathways control the somatic and sympathetic motor systems to drive thermogenesis.
... In these POA subregions, the EP3 subtype of PGE receptor is localized on many neuronal somata and dendrites ( Figure 8A) (71,72). Although mRNA expression for the EP1 and EP4 subtypes is also detected in the POA (73), analyses of mice lacking each of the known PGE receptor subtypes showed that only EP3 receptor-deficient mice completely failed to show a febrile response to PGE 2 , interleukin-1 beta, or endotoxin ( Figure 8B) (74) and EP1 receptor-deficient mice showed a partial attenuation of endotoxin-induced fever (75). ...
Central neural circuits orchestrate a homeostatic repertoire to maintain body temperature during environmental temperature challenges and to alter body temperature during the inflammatory response. This review summarizes the functional organization of the neural pathways through which cutaneous thermal receptors alter thermoregulatory effectors: the cutaneous circulation for heat loss, the brown adipose tissue, skeletal muscle and heart for thermogenesis and species-dependent mechanisms (sweating, panting and saliva spreading) for evaporative heat loss. These effectors are regulated by parallel but distinct, effector-specific neural pathways that share a common peripheral thermal sensory input. The thermal afferent circuits include cutaneous thermal receptors, spinal dorsal horn neurons and lateral parabrachial nucleus neurons projecting to the preoptic area to influence warm-sensitive, inhibitory output neurons which control thermogenesis-promoting neurons in the dorsomedial hypothalamus that project to premotor neurons in the rostral ventromedial medulla, including the raphe pallidus, that descend to provide the excitation necessary to drive thermogenic thermal effectors. A distinct population of warm-sensitive preoptic neurons controls heat loss through an inhibitory input to raphe pallidus neurons controlling cutaneous vasoconstriction.
... There is evidence that cytokines activate the preoptic area through PGE 2 synthesis and release in the organum vasculosum laminae terminalis (the circumventricular organ system), surrounding the preoptic area but located outside of the blood-brain barrier (Rotondo et al., 1988;Stitt, 1986). In any case, PGE 2 mediated effects through prostanoid EP3 receptors in the hypothalamus play a pivotal role in fever (Nakamura et al., 1999). The vagal nerve (Blatteis et al., 2000;Romanovsky et al., 2000) and the anteroventral third ventricular region are also known to influence lipopolysaccharide-induced fever Hunter, 1997;Scammell et al., 1996;Whyte and Johnson, 2007), although their exact role remains to be elucidated. ...
In anxiety research, the search for models with sufficient clinical predictive validity to support the translation of animal studies on anxiolytic drugs to clinical research is often challenging. This review describes the stress-induced hyperthermia (SIH) paradigm, a model that studies the activation of the autonomic nervous system in response to stress by measuring body temperature. The reproducible and robust SIH response, combined with ease of testing, make the SIH paradigm very suitable for drug screening. We will review the current knowledge on the neurobiology of the SIH response, discuss the role of GABAA and serotonin (5-HT) pharmacology, as well as how the SIH response relates to infectious fever. Furthermore, we will present novel data on the SIH response variance across different mice and their sensitivity to anxiolytic drugs. The SIH response is an autonomic stress response that can be successfully studied at the level of its physiology, pharmacology, neurobiology and genetics and possesses excellent animal-to-human translational properties.
... Interestingly, Scammell and colleagues (68) reported that intrapreoptic injections of PGE 2 evoked increased expression of c-Fos in the PVN that was largely restricted to the parvocellular PVN, while in the present study, microinjection of muscimol into the mPOA elicited marked increases in c-Fos expression in both subdivisions of the PVN. The difference may reflect the fact that muscimol inhibits the vast majority of adult mammalian neurons, while PGE 2 most likely acts on the subpopulation of neurons in the POA possessing EP3 receptors (45,46). Thus, somewhat different populations of neurons in the mPOA may be responsive to microinjection of muscimol and of PGE 2 , and this difference may account for the differential effects on c-Fos expression in the magnocellular PVN, as well as on locomotor activity noted here and previously (94). ...
Previous studies suggest that sympathetic responses evoked from the preoptic area in anesthetized rats require activation of neurons in the dorsomedial hypothalamus. Disinhibition of neurons in the dorsomedial hypothalamus in conscious rats produces physiological and behavioral changes resembling those evoked by microinjection of muscimol, a GABA(A) receptor agonist and neuronal inhibitor, into the medial preoptic area. We tested the hypothesis that all of these effects evoked from the medial preoptic area are mediated through neurons in the dorsomedial hypothalamus by assessing the effect of bilateral microinjection of muscimol into the DMH on these changes. After injection of vehicle into the dorsomedial hypothalamus, injection of muscimol into the medial preoptic area elicited marked increases in heart rate, arterial pressure, body temperature, plasma ACTH, and locomotor activity and also increased c-Fos expression in the hypothalamic paraventricular nucleus, a region known to control the release of ACTH from the adenohypophysis. Prior bilateral microinjection of muscimol into the dorsomedial hypothalamus produced a modest depression of baseline heart rate and body temperature but completely abolished all changes evoked from the medial preoptic area. Microinjection of muscimol just anterior to the dorsomedial hypothalamus had no effect on autonomic and neuroendocrine changes evoked from the medial preoptic area. Thus, activity of neurons in the dorsomedial hypothalamus mediates a diverse array of physiological and behavioral responses elicited from the medial preoptic area, suggesting that the latter region represents an important source of inhibitory tone to key neurons in the dorsomedial hypothalamus.
... Da der Wirkung von PGE 2 im ZNS eine Schlüsselrolle bei der Vermittlung zentralnervös kontrollierter Krankheitssymptome zukommt, waren und sind die Verteilung der einzelnen PGE-Rezeptoren im Gehirn sowie die Analyse ihrer funktionellen Bedeutung Gegenstand zahlreicher Untersuchungen (Scammell et al., 1996a;Ericsson et al., 1997;Oka, 2001). So ist es nicht verwunderlich, dass sich die so genannten "Prostaglandin-sensitiven" Regionen des Gehirns, wie das Organum vasculosum laminae terminalis (OVLT) und die Area praeoptica (POA) durch eine besonders hohe Dichte an PGE-Rezeptoren auszeichnen (Nakamura et al., 1999;Zhang & Rivest, 1999;Oka et al., 2000). ...
Ziel dieser Doktorarbeit war die Untersuchung von Signalwegen zur Fieberentstehung im Rahmen lokaler oder systemischer Entzündungsreaktionen. Die spezifische Rolle von Interleukin-6 (IL-6) und Prostaglandinen in diesen beiden Prozessen wurden analysiert. Im Hinblick auf den humoralen Mechanismus der Fieberentstehung wurde während systemischer oder lokaler Entzündungsreaktion eine mögliche Lipopolysaccharid (LPS)-induzierte Aktivierung des pleiotropen Zytokins IL-6 und des IL-6-induzierten Transkriptionsfaktors STAT3 untersucht. Bei Meerschweinchen verursachten intraarterielle (i.a., 10 µg/kg) oder intraperitoneale (i.p., 30 µg/kg) LPS-Injektionen eine systemische Entzündungsreaktion, die von robustem Fieber beleitet war. Eine Fieberreaktion wurde ebenfalls durch LPS-Applikation in eine subkutan implantierte, künstliche Teflonkammer induziert (s.c., 100 oder 10 µg/kg), die ein Experimentalmodell einer lokalen Entzündungsreaktion darstellt. 60 min. vor LPS- oder Lösungsmittel-Injektion waren basale Spiegel an biologisch aktiven IL-6 von 35-80 internationalen Einheiten pro ml. (I.U./ml.) im Blut messbar. 90 min. nach LPS-Gabe erhöhten sich die IL-6-Plasmakonzentrationen in den i.a. oder i.p. behandelten Tiergruppen etwa 1000-fach, 50-fach in der Gruppe, die mit 100 µg/kg LPS s.c. behandelt wurde und lediglich 5-fach bei Meerschweinchen, denen die niedrige LPS-Dosis (10µ/kg) in die subkutane Kammer injiziert wurde. Zu diesem Zeitpunkt konnte in verschiedenen Gehirnstrukturen eine distinkte Verteilung nukleärer Translokationen des Transkriptionsfaktors STAT3 beobachtet werden. Hierzu gehörten Gehirnstrukturen mit einer unvollständigen Blut-Hirn-Schranke (BBB), die als sensorische zirkumventrikuläre Organe (sCVOs) bezeichnet werden: die Area postrema, das Organum vasculosum laminae terminalis, das Organum subfornicale sowie zusätzlich auch der hypothalamische Nucleus supraopticus, welche in den i.a. oder i.p. mit LPS behandelten Gruppen starke nukleäre STAT3-Aktivierung aufwiesen. Im Gegensatz dazu wurde nach s.c. LPS-Applikation eine moderate Anzahl (hohe LPS-Dosis, 100 µg/kg) bzw. sogar keine (niedrige LPS-Dosis) nukleären STAT3-Signale in den genannten Hirnstrukturen nachgewiesen. Diese Ergebnisse lassen vermuten, dass STAT3-vermittelte Aktivierung der Transkription von Ziel-Genen in Gehirnzellen nur dann stattfindet, wenn bei systemischen oder lokalen Entzündungsreaktionen ausreichend hohe zirkulierende IL-6-Konzentrationen auftreten.
Zell-Phänotypen der Zytokin-responsiven Zielzellen im Gehirn, die durch IL-6 genomisch aktiviert wurden, konnten durch immunhistologische Analyse des Zytokin-induzierten Transkriptionsfaktors in Kombination mit spezifischen zellulären Markerproteinen nachgewiesen werde. Zwei für die Zytokin-abhängige Vermittlung von Immuneffektorfunktionen kritische Gehirnanteile konnten identifiziert werden. Eine Gruppe responsiver Zellen war entlang der BBB lokalisiert, eine andere Gruppe von Zellen befand sich in den bereits erwähnten sCVOs. Die markanteste STAT3-Aktivierung erfolgte in zwei Zielstrukturen des Meerschweinchengehirns und zwar (1) in Endothelzellen des gesamten Gehirns und (2) in Astrozyten der sCVOs. Beide Strukturen stellen entscheidende Gehirnregionen bzw. Zellen der Zytokin-vermittelten Wirkung bei LPS-induzierten Entzündungsreaktionen dar. STAT3-kontrollierte transkriptionelle Aktivierung scheint bei diesem Prozess mitzuwirken. Die funktionelle Konsequenz STAT3-abhängiger genomischer Aktivierung von Gehirnzellen nach LPS-Gabe wird bislang nur unvollständig verstanden. Basierend auf neuern Daten aus der Literatur konnte eine Hypothese formuliert werden, welche STAT3 als einen antiinflammatorischen Mediator speziell im Endothel vorschlägt. Die Ergebnisse dieser Doktorarbeit demonstrieren deutlich, dass ganz besonders das Gehirnendothel in dieses neue Konzept integriert werden muss.
Zur Untersuchung des nervalen Signalwegs zur Fieberentstehung bei lokalen Entzündungsreaktionen und speziell der Rolle von Prostaglandinen in diesem Experimentalmodell wurden Fieberreaktionen durch Injektionen einer hohen (100 µg/kg) oder niedrigen (10 µg/kg) LPS-Dosis in die künstliche subkutan implantierte Teflonkammer induziert. Beide eingesetzten LPS-Dosierungen riefen außerdem eine im subkutanen Entzündungsareal lokalisierte markante Bildung von Prostaglandin E2 (PGE2) hervor. Die Gabe von Diclofenac, einem nicht selektiven Zyklooxygenase-Inhibitor (COX) in unterschiedlichen Dosierungen (5, 50, 500, 5000 µg/kg), verringerte oder unterdrückte LPS-induziertes Fieber und hemmte die LPS-induzierte lokale PGE2-Bildung. Die niedrigste Diclofenac-Dosierung (5 µg/kg) schwächte das Fieber nach LPS-Applikation von 10 µg/kg s.c. nur dann ab, wenn Diclofenac direkt in die subkutane Kammer injiziert wurde, nicht aber bei subkutaner Applikation in den contralateral zur subkutanen Kammer gelegenen Bereich. Diese Beobachtung wies darauf hin, dass eine lokalisierte PGE2-Bildung im Entzündungsgebiet einen Teil der Fieberreaktion vermittelte, die durch Injektion von 10 µg/kg LPS in die subkutane Kammer ausgelöst wurde. Eine Erhärtung dieser Hypothese ergab sich aus Beobachtungen, dass es nicht möglich war, COX-2 mRNA im Gehirn des Meerschweinchens nach Injektion von 10 µg/kg LPS in die subkutane Kammer nachzuweisen. Hingegen induzierte eine subkutane Gabe von 100 µg/kg LPS wie auch systemische LPS-Applikation (i.a., i.p.) eine deutliche Expression des COX-2-Genes im Meerschweinchengehirn, was durch in situ Hybridisierung dokumentiert wurde. Folglich wurde die Fieberreaktion bei subkutaner Injektion von 10 µg/kg LPS zumindest anteilig durch Aktivierung nervaler Signalvermittlung vom lokalen Entzündungsmodell zum Gehirn hervorgerufen.
Als Schlussfolgerung hieraus ergibt sich, dass Fieberreaktionen bei moderaten lokalen Entzündungsreaktionen anteilig sowohl von humoralen als auch nervalen Komponenten der Kommunikation des aktivierten Immunsystems mit dem Gehirn entstehen. Die nervale Beteiligung ist dann nicht mehr detektierbar, wenn die humoralen Signale so stark sind, dass nervale Komponenten zur Fieberinduktion verdeckt werden. Im Bezug auf die humorale Komponente könnte IL-6 eine zentrale Rolle bei der Regulation der Fieberreaktion spielen, obwohl die präzise funktionelle Konsequenz IL-6-induzierter genomischer Aktivierung von Gehirnzellen noch abzuklären bleibt. Purpose of the present study was to investigate signaling pathways for fever induction during localized or systemic inflammation. The specific roles of Interleukin-6 (IL-6) and prostaglandins in both of these processes were analyzed. With regard to the humoral mechanisms of fever generation, a possible lipopolysaccharide (LPS)-induced activation of brain cells mediated by the pleiotropic cytokine IL-6 and its transcription factor STAT3 during systemic or localized inflammation was studied. In guinea pigs, intra-arterial (i.a., 10 µg/kg) or intraperitoneal (i.p., 30 µg/kg) injections of bacterial LPS caused a systemic inflammatory response which was accompanied by a robust fever. A febrile response was also induced by administration of LPS into artificial subcutaneously implanted Teflon chambers (s.c. 100 or 10 µg/kg), which reflects an experimental model that mimics local tissue inflammation. Baseline plasma levels of bioactive IL-6 determined 60 min. prior to injections of LPS or vehicle amounted to 35-80 international units per ml (I.U./ml). Within 90 min. after LPS-injection plasma IL-6 rose about 1000-fold in the groups injected i.a. or i.p., about 50-fold in the group injected s.c. with 100 µg/kg LPS, and only 5-fold in guinea pigs injected with the lower dose of LPS s.c. (10 µg/kg). At this time point, a distinct nuclear translocation pattern of the transcription factor STAT3 became evident in several brain structures. Amongst those the sensory circumventricular organs (sCVOs) known to lack a tight blood-brain barrier, such as the area postrema, the vascular organ of the lamina terminalis and the subfornical organ, as well as the hypothalamic supraoptic nucleus showed intense nuclear STAT3 signals in the i.a. or i.p. injected groups. In contrast, a moderate (s.c. group, 100 µg/kg) or even no (s.c. group, 10 µg/kg) nuclear STAT3 translocation occurred in response to s.c. injections of LPS. These results suggest that STAT3-mediated genomic activation of target gene transcription in brain cells occurred only in those cases in which sufficiently high concentrations of circulating IL-6 were formed during systemic (i.a. and i.p. groups) or localized (s.c. group, 100 µg/kg) inflammation.
Cell phenotypes of cytokine-responsive brain target cells that were genomically activated by IL-6 were mapped by histological analysis of cytokine-induced transcription factors in combination with specific cellular marker proteins. Critical sites mediating cytokine-dependent immuneffector functions could be divided into two groups, one group of responding cells situated along the tight blood-brain barrier (BBB), and a second group of cells in the before mentioned sCVOs. A marked STAT3 activation in two target structures of the guinea pig brain (1) within the entire brain endothelium and (2) within astrocytes of the sCVOs were shown. Both structures represent critical brain sites or cells mediating cytokine action during LPS-induced inflammation. STAT3-controlled transcriptional activation seems to be involved in this process. The functional consequences of STAT3-dependent genomic actions during LPS-challenge are still poorly understood. However, based on a recently published study from another laboratory, a new hypothesis is presented, which involves STAT3 as an anti-inflammatory mediator in the endothelium. The results of this thesis clearly demonstrate that specifically the endothelium of the brain has to be incorporated into this novel concept. To investigate the potentially involved neuronal signaling pathway for fever induction during localized inflammation and the role of prostaglandins in this experimental model, dose-dependent febrile responses were induced by injection of a high (100 µg/kg) or a low (10 µg/kg) dose of bacterial LPS into artificial subcutaneously implanted Teflon chambers. Both of the applied LPS-doses further induced a pronounced formation of prostaglandin E2 (PGE2) at the site of localized subcutaneous inflammation. Administration of diclofenac, a non-selective cyclooxygenase (COX) inhibitor, at different doses (5, 50, 500, 5000 µg/kg) attenuated or abrogated LPS-induced fever and depressed LPS-induced local PGE2 formation. The lowest dose of diclofenac (5 µg/kg) attenuated fever in response to 10 µg/kg LPS only when administered directly into the subcutaneous chamber, but not when injected into the contralateral subcutaneous site of the chamber position. This observation indicated that a localized formation of PGE2 at the site of inflammation mediated a portion of the febrile response which was induced by injection of 10 µg/kg LPS into the subcutaneous chamber. Further support for this hypothesis derived from the observation that I failed to detect induction of COX-2 mRNA in the brain of guinea pigs injected subcutaneously with 10 µg/kg LPS, while subcutaneous injections of 100 µg/kg LPS as well as systemic injections of LPS (i.p. or i.a. routes) readily caused expression of the COX-2 gene in the guinea pig brain as demonstrated by in situ hybridization. Therefore, fever in response to subcutaneous injection of 10 µg/kg LPS may, in part, have been evoked by a neuronal rather than a humoral pathway from the local site of inflammation to the brain.
In conclusion, fever in response to moderate local inflammatory reactions seems to be due to the participation of a humoral as well as a neuronal component of immune-to-brain communication. The neuronal participation is not detectable at all, when humoral signals are so strong that they are overriding the neural component for fever induction. With regard to the humoral component, IL-6 might play an important role in the regulation of the febrile response although the precise functional consequences of IL-6-induced genomic activation of brain cells still has to be clarified.
... Während in den Zellkulturmodellen eine EP3 Rezeptor immunoreaktive Bande mit einem scheinbaren Molekulargewicht von 72 kDa detektiert werden konnte, zeigte sich in der Western Blot Analysen der hippocampalen Homogenisate eine EP3 spezifische immunoreaktive Bande mit einem scheinbaren Molekulargewicht von 60 kDa. Dieser Unterschied im Laufverhalten bei elektrophoretischer Auftrennung in der SDS-PAGE zwischen dem, in Zellkulturmodellen exprimierten EP3 Rezeptorprotein und einem zerebral exprimierten EP3 Rezeptor wurde erstmals von Nakamura und Mitarbeitern beschrieben(Nakamura, et al. 1999). In einer späteren Studie konnte die Differenz dann auf Unterschiede in der Glykosylierung des Rezeptors zurückgeführt werden(Nakamura, et al. 2000). ...
Eine Überexpression des Zytokins Interleukin-1 beta (IL-1b) durch aktivierte Mikrogliazellen und reaktive Astrozyten ist ein charakteristisches Merkmal der Neuroinflammation. Neuroinflammatorische Reaktionen begleiten chronisch progressive neurodegenerative Erkrankungen wie die Alzheimer Demenz oder Prionenerkrankungen, treten aber auch im Rahmen akuter Prozesse wie der zerebralen Ischämie auf. Studien, die protektive Eigenschaften von Cyclooxygenase (COX) Inhibitoren in neuroinflammatorischen Prozessen zeigen, deuten auf eine entscheidende Rolle der Prostaglandine in der Pathogenese dieser Erkrankungen hin. Prostaglandin E2 (PGE2) vermittelt seine Effekte über vier, an verschiedene G-Proteine gekoppelte Rezeptoren (EP1, EP2, EP3, EP4). Eine Änderung des EP Rezeptorexpressionsprofils kann den Einfluss des PGE2 auf eine Zelle grundlegend verändern. Bisher gibt es jedoch kaum Erkenntnisse über die Regulation von EP Rezeptoren im ZNS, so sollte in dieser Arbeit untersucht werden, ob das Zytokin IL-1b die Expression des EP3 Rezeptors, der maßgeblich an der Vermittlung zentraler Effekte des PGE2 beteiligt ist, beeinflusst. Während IL-1b keinen Einfluß auf die Expression des EP3 Rezeptors in neuronalen Zellen und Mikrogliazellen ausübte, konnte in astrozytären Zellen (humane primäre Astrozyten, U373 MG Astrozytomazellen) und gemischtglialen Primärkulturen der Ratte eine deutliche Induktion von EP3 Rezeptor Protein und mRNA nach Stimulation mit IL-1b detektiert werden. Eine Induktion der EP3 Rezeptorsynthese in Astrozyten war abhängig von einer Aktivierung der Protein Kinase C und des Transkriptionsfaktors NF-kB. Eine Beteiligung der p38 MAPK, p42/44 MAPK oder JNK konnte ausgeschlossen werden. Während die Induktion der EP3 Rezeptorexpression in gemischtglialen Kulturen der Ratte von einem Anstieg der COX-2 Expression begleitet wurde, konnte kein Einfluss des Zytokins auf die Expression der COX-2 in U373 MG Astrozytomazellen beobachtet werden. So konnte ein indirekter, PGE2 abhängiger Effekt des IL-1b auf die Expression des Rezeptors ausgeschlossen werden. In einem Rattenmodell konnte ein deutlicher Anstieg der EP3 Rezeptorexpression im Hippocampus nach intrazerebroventrikulaerer IL-1b Injektion beobachtet werden. Dies zeigt, dass IL-1b nicht nur in vitro sondern auch in vivo ein potenter Induktor der EP3 Rezeptorsynthese ist. Eine Modulation der EP3 Rezeptorexpression durch das Zytokin IL-1b könnte von entscheidener Bedeutung für PGE2 vermittelte Effekte in neuroinflammatorischen Prozessen sein und bisher unbekannte Mechanismen aufzeigen, die zur Entwicklung neuer Therapieansätze beitragen könnten. Both interleukin-1 beta (IL-1beta) and prostaglandins (PGs) are important mediators of physiological and pathophysiological processes in the brain. PGE2 exerts its effects by binding to four different types of PGE2 receptors named EP1-EP4. The prostaglandin E receptor subtype EP3 has found to be expressed in neurons, whereas expression of EP3 in glial cells has not been reported in the brain yet. This study describes IL-1beta-induced EP3 receptor expression in human astrocytoma cells, primary astrocytes of rat and human origin and in rat brain. Using western blot, we found a marked up-regulation of EP3 receptor synthesis in human and rat primary glial cells by IL-1 beta. Intracerebroventricular administration of IL-1beta stimulated the EP3 receptor synthesis in rat hippocampus. The analysis of involved signal transduction pathways by pathway-specific inhibitors revealed an essential role of protein kinase C and nuclear factor-kappaB in astrocytic IL-1 beta-induced EP3 synthesis. Our data suggest that PGE2 signaling in the brain may be altered after IL-1beta release due to up-regulation of EP3 receptors. This might play an important role in acute and chronic conditions such as cerebral ischemia, traumatic brain injury, HIV-encephalitis, Alzheimer's disease and prion diseases in which a marked up-regulation of IL-1beta is followed by a prolonged increase of PGE2 levels in the brain.
... Also, differences exist regarding the pharmacological substrates involved in SIH and fever. There is ample evidence for a role of the GABA A receptor in the SIH response [2], while evidence points to a role for prostanoid EP3 receptors in fever [13]. Indeed, fever can be effectively reversed by prostaglandin-blocking drugs such as non-steroidal anti-inflammatory drugs (NSAIDS) which block the synthesis of prostaglandins [14]. ...
Stress exposure activates the autonomic nervous system and leads to a body temperature increase (stress-induced hyperthermia, SIH). On the other hand, an activation of the immune system in response to an infection leads to fever. Both processes increase body temperature, and the relation between SIH and infection-induced fever has been subject to debate. It is not clear whether SIH is a form of fever, or whether both processes are more or less distinct. We therefore examined the relation between SIH and infection-induced fever by looking at the effects of a GABA(A) receptor agonist (diazepam) and a prostaglandin-synthesis blocking drug (acetylsalicylic acid, aspirin) on both the SIH response and fever in rats and mice. The present study shows that the benzodiazepine diazepam but not the prostaglandin-synthesis blocking drug aspirin dose-dependently attenuated the SIH response in both rats and mice. In contrast, aspirin reduced both LPS- and IL-1beta induced fever, whereas diazepam had little effect on these fever states. Altogether, our findings support the hypothesis that stress-induced hyperthermia and infection-induced fever are two distinct processes mediated largely by different neurobiological mechanisms.
Prostanoid receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Prostanoid Receptors [661]) are activated by the endogenous ligands prostaglandins PGD2, PGE1, PGE2 , PGF2α, PGH2, prostacyclin [PGI2] and thromboxane A2. Differences and similarities between human and rodent prostanoid receptor orthologues, and their specific roles in pathophysiologic conditions are reviewed in [423]. Measurement of the potency of PGI2 and thromboxane A2 is hampered by their instability in physiological salt solution; they are often replaced by cicaprost and U46619, respectively, in receptor characterization studies.
The pharmacology of aspirin is unique in both, pharmacokinetic and pharmacodynamics aspects. This chapter describes the pharmaco-kinetics of aspirin and salicylate, focusing on the bioavailability of the active drug(s), their plasma and tissue distribution, metabolism, and clearance from blood and other body fluids. This is followed by a discussion of the multiple modes of action of aspirin and salicylate at the cellular and subcellular levels and includes pharmacodynamic actions of the compounds on mediator systems, cellular signal generation and transmission, and cellular energy metabolism. Only those concentrations of the compound are of interest that can also be obtained at therapeutic doses in vivo, that is, help to explain the multiple clinical actions of aspirin discussed in detail. The chapter talks about the three most interesting pharmacodynamic actions: the antiplatelet/antithrombotic action, the analgesic/antipyretic/anti-inflammatory action, and the action on tumor cells.
Prostanoids comprising prostaglandins (PGs) and thromboxanes exert diverse actions by acting on their specific receptors. Recently, physiological roles of these receptors have been clarified using knockout mice for each receptor as well as receptor-selective agonists and antagonists. In the central nervous system (CNS), prostanoids have been shown to regulate not only fever, but also neuroinflammation, and to play a role in the pathogenesis of many neurodegenerative diseases. In this report, we review the recent research on the roles and molecular mechanisms of prostanoids and their receptors in the CNS and discuss their possibilities as therapeutic targets.
Cytokines and prostaglandins are two groups of molecules whose importance in fever has been well known for a long time. Until 1990, however, there had been little information as to where these molecules are expressed and how they are regulated in the brain during fever. During the last decade of the twentieth century, application of neuroanatomical techniques to fever research provided a large amount of information about the locations of these fever-related molecules in the brain. This chapter gives a short overview of the history of fever research at the molecular level and reviews neuroanatomical studies that were aimed at these fever-related molecules. The results are summarized as follows: (1) proinflammatory cytokines are produced by nonneuronal cells in the brain, especially those associated with the blood-brain interface and microglia, in response to immunological challenge; (2) cytokine receptors are constitutively expressed mainly in nonneuronal cells including brain endothelial cells, and are, in some cases, upregulated by immunological challenge; (3) prostaglandin E2 receptor is constitutively expressed in neurons, and its density is particularly high in the anterior wall of the third ventricle and in some other brain regions involved in autonomic regulation and nociception; and (4) cyclooxygenase 2, an enzyme essential for the biosynthesis of prostaglandin E2, is expressed by immunological challenge in brain endothelial cells. These studies highlighted the brain cells potentially involved in fever and allowed us to further elucidate the molecular and cellular events occurring in the brain during fever.
Many types of psychological stress induce hyperthermia. The stress-induced elevation of body temperature is caused by sympathetic responses including brown adipose tissue thermogenesis, tachycardia, and cutaneous vasoconstriction as well as by neuroendocrine responses including stress hormone release via the hypothalamo-pituitary-adrenal (HPA) axis. Recent studies have revealed that the hypothalamic and medullary neural circuitry for driving these stress responses. In this circuitry, the dorsomedial hypothalamus serves as a hub for the central stress signaling: first, it connects the sympathetic efferents with medullary sympathetic premotor neurons to drive the sympathetic responses; second, it connects the neuroendocrine efferents with the HPA axis to drive the stress hormone release. The findings from the animal experiments would be relevant to understand the etiology of the chronic stress-induced hyperthermia "psychogenic fever", a psychosomatic symptom in humans. In this review, I describe the current understanding of the central circuit mechanism for the development of psychological stress-induced hyperthermia, incorporating recent important discoveries.
The heat shock response (HSR) is an ancient and highly conserved process that is essential for coping with environmental stresses, including extremes of temperature. Fever is a more recently evolved response, during which organisms temporarily subject themselves to thermal stress in the face of infections. We review the phylogenetically conserved mechanisms that regulate fever and discuss the effects that febrile-range temperatures have on multiple biological processes involved in host defense and cell death and survival, including the HSR and its implications for patients with severe sepsis, trauma, and other acute systemic inflammatory states. Heat shock factor-1, a heat-induced transcriptional enhancer is not only the central regulator of the HSR but also regulates expression of pivotal cytokines and early response genes. Febrile-range temperatures exert additional immunomodulatory effects by activating mitogen-activated protein kinase cascades and accelerating apoptosis in some cell types. This results in accelerated pathogen clearance, but increased collateral tissue injury, thus the net effect of exposure to febrile range temperature depends in part on the site and nature of the pathologic process and the specific treatment provided. © 2014 American Physiological Society. Compr Physiol 4:109-148, 2014.
Previous studies in rats have demonstrated that microsomal prostaglandin E synthase-1 (mPGES-1) is induced in brain vascular cells that also express inducible cyclooxygenase-2, suggesting that such cells are the source of the increased PGE2 levels that are seen in the brain following peripheral immune stimulation, and that are associated with sickness responses such as fever, anorexia and stress hormone release. However, while most of what is known about the functional role of mPGES-1 for these centrally evoked symptoms is based on studies on genetically modified mice, the cellular localization of mPGES-1 in the mouse brain has not been thoroughly determined. Here, using a newly developed antibody that specifically recognizes mouse mPGES-1, and dual-labeling for cell-specific markers, we report that mPGES-1 is constitutively expressed in the mouse brain, being present not only in brain endothelial cells, but also in several other cell types and structures, such as capillary-associated pericytes, astroglial cells, leptomeninges and the choroid plexus. Regional differences were seen with particularly prominent labeling in autonomic relay structures such as the area postrema, the subfornical organ, the paraventricular hypothalamic nucleus, the arcuate nucleus and the median preoptic nucleus. Following immune stimulation, mPGES-1 in brain endothelial cells, but not in other mPGES-1 positive cells, was co-expressed with cyclooxygenase-2, whereas there was no co-expression between mPGES-1 and cyclooxygenase-1. These data imply a wide-spread synthesis of PGE2 or other mPGES-1 dependent product in the mouse brain that may be related to inflammation-induced sickness symptom as well as other functions, such as blood flow regulation. J. Comp. Neurol., 2014. © 2014 Wiley Periodicals, Inc.
1. The rostral medullary raphe pallidus contains sympathetic premotor neurons controlling thermogenesis in brown adipose tissue (BAT).2. Disinhibition of neurons in the dorsomedial hypothalamus (DMH) stimulates BAT thermogenesis through activation of neurons in raphe pallidus.3. An increase in BAT sympathetic outflow and BAT thermogenesis following microinjection of prostaglandin E2 into the preoptic area requires activation of both DMH neurons and raphe pallidus neurons.4. DMH contains a population of neurons receiving a tonically- active GABAergic inhibition which mediate increases in BAT thermogenesis through stimulation of BAT sympathetic premotor neurons in raphe pallidus.
1. Fever is the widely known hallmark of disease and induced by the action of the nervous system. 2. It is generally accepted that the action of prostaglandin E2 in the preoptic area (POA) triggers the stimulation of the sympathetic nervous system, and this results in the production of fever. 3. Recent findings have demonstrated that the rostral medullary raphe regions are essential for the pyrogenic signal transmission from the POA to sympathetic output neurons. 4. Here, I review the functional role of these raphe regions in febrile responses and also discuss the pyrogenic transmission from the POA to the raphe regions.
Administration of galanin-like peptide (GALP) leads to a decrease in both total food intake and body weight 24 h after injection, compared to controls. Moreover, GALP induces an increase in core body temperature. To elucidate the mechanism by which GALP exerts its effect on energy homeostasis, urethane-anesthetized rats were intracerebroventricularly injected with GALP or saline, after which oxygen consumption, heart rate, and body temperature were monitored for 4 h. In some cases, animals were also pretreated with the cyclooxygenase (COX) inhibitor, diclofenac, via intracerebroventricular (i.c.v.) or intravenous (i.v.) injection. c-Fos expression in the brain was also examined after injection of GALP, and the levels of COX and prostaglandin E(2) synthetase (PGES) mRNA in primary cultured astrocytes treated with GALP were analyzed by using qPCR. The i.c.v. injection of GALP caused biphasic thermogenesis, an effect which could be blocked by pretreatment with centrally (i.c.v.), but not peripherally (i.v.) administered diclofenac. c-Fos immunoreactivity was observed in astrocytes in the periventricular zone of the third ventricle. GALP treatment also increased COX-2 and cytosolic PGES, but not COX-1, microsomal PGES-1, or microsomal PGES-2 mRNA levels in cultured astrocytes. We, therefore, suggest that GALP elicits thermogenesis via a prostaglandin E(2)-mediated pathway in astrocytes of the central nervous system.
The prostaglandin EP3 receptor (EP3R) subtype is believed to mediate large portions of diverse physiologic actions of prostaglandin E2 in the nervous system. However, the distribution of EP3R protein has not yet been unveiled in the peripheral or central nervous systems. The authors raised a polyclonal antibody against an amino-terminal portion of rat EP3R that recognized specifically the receptor protein. In this study, immunoblotting analysis with this antibody showed several immunoreactive bands with different molecular weights in rat brain extracts and in membrane fractions of recombinant EP3R-expressing culture cells, and treatment with N-glycosidase shifted those immunoreactive bands to an apparently single band with a lower molecular weight, suggesting that EP3R proteins are modified posttranslationally with carbohydrate moieties of various sizes. The authors performed immunohistochemical investigation of EP3R in the rat brain, spinal cord, and peripheral ganglia by using the antibody. EP3R-like immunoreactivity was observed in many and discrete regions of the rostrocaudal axis of the nervous system. The signals were particularly strong in the anterior, intralaminar, and midline thalamic nuclear groups; the median preoptic nucleus; the medial mammillary nucleus; the superior colliculus; the periaqueductal gray; the lateral parabrachial nucleus; the nucleus of the solitary tract; and laminae I and II of the medullary and spinal dorsal horns. Sensory ganglia, such as the trigeminal, dorsal root, and nodose ganglia, contained many immunopositive neurons. Neuronal cells in the locus coeruleus and raphe nuclei exhibited EP3R-like immunoreactivity. This suggests that EP3R plays regulatory roles in the noradrenergic and serotonergic monoamine systems. Autonomic preganglionic nuclei, such as the dorsal motor nucleus of the vagus nerve, the spinal intermediolateral nucleus, and the sacral parasympathetic nucleus, also contained neuronal cell bodies with the immunoreactivity, implying modulatory functions of EP3R in the central autonomic nervous system. The characteristic distribution of EP3R provides valuable information on the mechanisms for various physiologic actions of prostaglandin E2 in the central and peripheral nervous systems. J. Comp. Neurol. 421:543–569, 2000. © 2000 Wiley-Liss, Inc.
The systemic administration of lipopolysaccharide (LPS), an experimental model of systemic bacterial infection is known to modulate nociception. It increases the prostaglandin E2 (PGE2) levels in the preoptic area of the hypothalamus (POA) and the microinjection of PGE2 into the POA and the neighboring basal forebrain induces hyperalgesia. We, therefore, hypothesized that the PGE2 synthesized in these regions mediates intravenous (i.v.) LPS-induced hyperalgesia. To test this hypothesis, we microinjected cyclooxygenase (COX) inhibitors into several sites in the rat hypothalamus and observed their effects on the LPS (0.1–100 μg/kg, i.v.)-induced changes in nociceptive behavior as assessed by a plantar test. LPS (10 and 100 μg/kg, i.v.) reduced the paw-withdrawal latency at 90 min and 45–60 min after injection, respectively, both thus indicating a hyperalgesic effect. This hyperalgesia was observed only in the period before the development of fever which started 120–135 min after the LPS injection. The LPS (100 μg/kg, i.v.)-induced hyperalgesia was completely abolished by pretreatment with the microinjection of diclofenac (an inhibitor of COX-1 and 2) at 1.0 ng into the bilateral POA. Furthermore, it was also blocked by the microinjection of NS-398 (a selective COX-2 inhibitor) at 1.0 ng into the bilateral POA and the horizontal limb of the diagonal band of Broca (DBB), but not the lateral hypothalamic area, the paraventricular hypothalamic nucleus, and the ventromedial hypothalamic nucleus. These findings suggest that LPS (i.v.)-induced hyperalgesia is mediated predominantly through a COX-2 induced prostanoids in the POA and the DBB in rats.
Histamine and prostaglandins (PGs) play a variety of physiological roles as autacoids, which function in the vicinity of their sources and maintain local homeostasis in the body. They stimulate target cells by acting on their specific receptors, which are coupled to trimeric G proteins. For the precise understanding of the physiological roles of histamine and PGs, it is necessary to clarify the molecular mechanisms involved in their synthesis as well as their receptor-mediated responses. We cloned the cDNAs for mouse L-histidine decarboxylase (HDC) and 6 mouse prostanoid receptors (4 PGE2 receptors, PGF receptor, and PGI receptor). We then characterized the expression patterns and functions of these genes. Furthermore, we established gene-targeted mouse strains for HDC and PG receptors to explore the novel pathophysiological roles of histamine and PGs. We have here summarized our research, which should contribute to progress in the molecular biology of HDC and PG receptors.
(Communicated by Osamu HAYAISHI, M.J.A.)
The brain's three sensory circumventricular organs, the subfornical organ, organum vasculosum of the lamina terminalis and the area postrema lack a blood brain barrier and are the only regions in the brain in which neurons are exposed to the chemical environment of the systemic circulation. Therefore they are ideally placed to monitor the changes in osmotic, ionic and hormonal composition of the blood. This book describes their. General structure and relationship to the cerebral ventricles Regional subdivisions Vasculature and barrier properties Neurons, glia and ependymal cells Receptors, neurotransmitters, neuropeptides and enzymes Neuroanatomical connections Functions.
This review presents an overview of the emerging field of prostaglandin signaling in neurological diseases, focusing on PGE(2) signaling through its four E-prostanoid (EP) receptors. A large number of studies have demonstrated a neurotoxic function of the inducible cyclooxygenase COX-2 in a broad spectrum of neurological disease models in the central nervous system (CNS), from models of cerebral ischemia to models of neurodegeneration and inflammation. Since COX-1 and COX-2 catalyze the first committed step in prostaglandin synthesis, an effort is underway to identify the downstream prostaglandin signaling pathways that mediate the toxic effect of COX-2. Recent epidemiologic studies demonstrate that chronic COX-2 inhibition can produce adverse cerebrovascular and cardiovascular effects, indicating that some prostaglandin signaling pathways are beneficial. Consistent with this concept, recent studies demonstrate that in the CNS, specific prostaglandin receptor signaling pathways mediate toxic effects in brain but a larger number appear to mediate paradoxically protective effects. Further complexity is emerging, as exemplified by the PGE(2) EP2 receptor, where cerebroprotective or toxic effects of a particular prostaglandin signaling pathway can differ depending on the context of cerebral injury, for example, in excitotoxicity/hypoxia paradigms versus inflammatory-mediated secondary neurotoxicity. The divergent effects of prostaglandin receptor signaling will likely depend on distinct patterns and dynamics of receptor expression in neurons, endothelial cells, and glia and the specific ways in which these cell types participate in particular models of neurological injury.
In previous studies we demonstrated telomerase activity in frozen tissue from head and neck squamous cell carcinoma (HNSCC) and their tumor-free tumor margins. In the present study frozen sections from the same tissues were examined for in situ presence of hTERT. In preliminary investigations we established that the most suitable method of tissue preparation was fixation in acetone and methanol followed by steaming and visualization by APAAP. Most of the assays involved eleven anti-hTERT antibodies and were supplemented with the inclusion of antibodies Ki-67, anti-nucleolin and CD45. hTERT expression was investigated in the tissues of 61 patients with HNSCC and 37 patients without tumor. Semi-quantitative immunoreactive scores were correlated with telomerase activity. We examined the prognostic significance of hTERT expression with Kaplan-Meier curves and tested the immunological specificity of the antibodies by immunoabsorption with two hTERT peptides and a nucleolin peptide. Nuclear staining of satisfactory distribution and intensity was achieved in seven anti-hTERT antibodies both in the carcinomas and in the squamous epithelia of the tumor resection margins and in the control tissues. Proof of hTERT did not differ from telomerase activity. The telomerase activity demonstrated in tumor-free resection margins and in control tissues did, however, correlate with lymphocytic-monocytic infiltration (CD45 expression). This telomerase activity might be related to nuclear hTERT expression in the squamous epithelium, given that the hTERT score values in the connective tissue tended to be negative. The prognostic significance of hTERT expression demonstrated on paraffin sections from different tumor localizations was not confirmed for the frozen sections of patients with HNSCC. The hTERT specificity of the monoclonal NCL-L-hTERT, whose use as an antibody against hTERT has been questioned, was re-examined with immunohistochemical methods, but the intensity of its immunoabsorption with the nucleolin peptide did not exceed that observed in the other anti-hTERT antibodies.
Prostaglandin D(2) (PGD(2)) is involved in a variety of physiological and pathophysiological processes, but its role in fever is poorly understood. Here we investigated the effects of central PGD(2) administration on body temperature and prostaglandin levels in the cerebrospinal fluid (CSF) of rats. Administration of PGD(2) into the cisterna magna (i.c.m) evoked a delayed fever response that was paralleled by increased levels of prostaglandin E(2) (PGE(2)) in the CSF. The elevated PGE(2) levels were not caused by an increased expression of cyclooxygenase 2 or microsomal prostaglandin E synthase-1 in the hypothalamus. Interestingly, i.c.m. pretreatment of animals with PGD(2) considerably sustained the pyrogenic effects of i.c.m. administered PGE(2). These data indicate that PGD(2) might control the availability of PGE(2) in the CSF and suggest that centrally produced PGD(2) may play a role in the maintenance of fever.
Quantitative autoradiography was performed to investigate the mapping of prostaglandin E2 binding sites in the Macaca fuscata fuscata diencephalon. Autoradiographs were prepared by incubation of 10-micron-thick serial frozen sections with 3H-prostaglandin E2 and were processed by using a rotating drum-scanner and a computer-assisted image-processing system with 3H-microscales as standards. The localization of prostaglandin E2 binding sites was remarkably discrete in the diencephalon. The highest concentrations were found in the median and medial preoptic areas, supramammilary nucleus of the hypothalamus, and centromedian nucleus of the thalamus. High density was observed in the medial and dorsal hypothalamic areas; paraventricular, anterior, dorsomedial, and infundibular nuclei of the hypothalamus; and in the anteroventral, periventricular, paraventricular, laterodorsal, and habenular nuclei of the thalamus. The distribution correlates well with the known effects of prostaglandin E2 and may also give us useful clues in unveiling the novel role of prostaglandin E2 in a variety of brain functions.
Fever is thought to be initiated by pyrogenic cytokines inducing the production of prostaglandin E2 (PGE2) in the preoptic area (POA); PGE2 may act as a paracrine mediator that stimulates the neural pathways that raise body temperature. This essential role for prostaglandins in fever first was proposed 25 years ago, but the specific preoptic cell groups at which PGE2 acts and the pathways through which fever is produced remain poorly understood. To better define the role of preoptic PGE2 in fever, we developed a new method for combining acute brain injections with Fos immunohistochemistry. We microinjected a threshold dose of PGE2 to construct an anatomically detailed map of fever-producing preoptic sites. The most pyrogenic preoptic sites were clustered along the ventromedial aspect of the POA, surrounding and just anterior to the organum vasculosum of the lamina terminalis. We then used Fos immunohistochemistry to identify the pattern of neural activation induced by fever-producing preoptic injections of PGE2 and compared it with the Fos pattern seen after systemic immune stimulation. PGE2 fever was accompanied by Fos induction in the ventromedial POA and the parvicellular subnuclei of the paraventricular nucleus of the hypothalamus (PVH). In contrast to the Fos pattern seen after intravenous lipopolysaccharide administration, PGE2 injection did not induce Fos in the circumventricular organs or the magnocellular subnuclei of the PVH. These observations establish a potential site of PGE2 action during fever and help define candidate pathways through which fever occurs.
The expression and characteristics of the dopamine D3 receptor protein were studied in brain and in stably transfected GH3 cells. Monoclonal antibodies were used for immunoprecipitation
and immunoblot experiments. Immunoprecipitates obtained from primate and rodent brain tissues contain a low molecular weight
D3 protein and one or two larger protein species whose molecular mass are integral multiples of the low molecular weight protein
and thus appear to have resulted from dimerization and tetramerization of a D3 monomer. Whereas D3receptor multimers were found to be abundantly expressed in brain, the major D3 immunoreactivity expressed in stable D3-expressing rat GH3 cells was found to be a monomer. However, multimeric D3 receptor species with electrophoretic mobilities similar to those expressed in brain were also seen in D3-expressing GH3 cells when a truncated D3-like protein (named D3nf) was co-expressed in these cells. Furthermore, results from immunoprecipitation experiments with D3- and D3nf-specific antibodies show that the higher-order D3 proteins extracted from brain and D3/D3nf double transfectants also contain D3nf immunoreactivity, and immunocytochemical studies show that the expression of D3 and D3nfimmunoreactivities overlaps substantially in monkey and rat cortical neurons. Altogether, these data show oligomeric D3 receptor protein expression in vivo and they suggest that at least some of these oligomers are heteroligomeric protein complexes containing D3 and the truncated D3nfprotein.
We previously reported that the activation of prostaglandin E receptor EP3 subtype caused neurite retraction via small GTPase
Rho in the EP3B receptor-expressing PC12 cells (Katoh, H., Negishi, M., and Ichikawa, A. (1996) J. Biol. Chem.271, 29780–29784). However, a potential downstream effector of Rho that induces neurite retraction was not identified. Here
we examined the morphological effect of p160 RhoA-binding kinase ROKα, a target for RhoA recently identified, on the nerve
growth factor-differentiated PC12 cells. Microinjection of the catalytic domain of ROKα rapidly induced neurite retraction
similar to that induced by microinjection of a constitutively active Rho, RhoV14, whereas microinjection of the kinase-deficient catalytic domain of ROKα did not induce neurite retraction. This morphological
change was observed even though C3 exoenzyme, which was known to inactivate Rho, had been preinjected. On the other hand,
microinjection of the Rho-binding domain or the pleckstrin homology domain of ROKα inhibited the EP3 receptor-induced neurite
retraction. These results demonstrate that ROKα induces neurite retraction acting downstream of Rho in neuronal cells.
Fever is induced in response to the entrance of pathogenic microorganisms into the body and is thought to be mediated by cytokines. Because these pathogens most commonly invade the body through its natural barriers and because body temperature is regulated centrally, these mediators are presumed to be produced peripherally and transported by the bloodstream to the brain, to act. It is generally considered that their febrigenic messages are further modulated there by prostaglandin E2 (PGE2). However, the detailed mechanism by which these cytokines signal the brain and activate the febrile response is not yet clear. Indeed, the specific role of each cytokine has been difficult to establish due to complex interactions among them. Furthermore, recent evidence suggests that different pyrogens may induce different cytokines; for example, i.v. LPS (a model of systemic bacterial infection) induces large increases in IL-6, but only small rises in IL-1 and TNF plasma levels. Moreover, their appearance lags the fever onset. We recently found that subdiaphragmatic vagotomy, decomplementation, and blockade of Kupffer cells suppress the febrile response of guinea pigs to i.v. LPS, and that i.v. LPS rapidly stimulates the release of norepinephrine (NE) and, hence, of PGE2 in their preoptic-anterior hypothalamus (POA, the brain region containing the thermoregulatory controller). Based on these and other data in the literature, we hypothesize that LPS fever may be initiated as follows: i.v., LPS → complement → Kupffer cells → cytokines?→ vagal afferents → n. tractus solitarius?→ A1/A2 cell groups?→ ventral noradrenergic bundle?→ POA → NE → PGE2→ fever.
A guinea pig antibody against a C-terminal peptide of rat μ-opioid receptor (MOR) was produced to examine the distribution of MOR in the rat caudate-putamen (CP). The anti-peptide antibody recognized a protein of Mr 69 000 in Triton X-100 extract of rat brain and in the membrane fraction of MOR-expressing culture cells. Intense MOR-like immunoreactivity (LI) was observed in island-like areas of the CP. Some MOR-LI was located on the cell bodies and dendrites of CP neurons. Double immunofluorescence study revealed that the intensely MOR-immunoreactive areas showed weak calbindin-LI, surrounded by intensely calbindin-positive regions. The results indicate that MOR-LI is enriched in the ‘patches’ of the neostriatal mosaic compartmentation.
In the guinea pig brain, LH-RH-containing cell bodies are located not only within the classical hypophysiotrophic area but also in the medial preoptic area, septum and olfactory tubercle. LH-RH fiber tracts project not only to the primary portal plexus in the median eminence but also throughout the limbic forebrain and limbic midbrain regions. Using radiofrequency lesions in different brain regions, the projections of LH-RH cell bodies were determined. Cells in the medial preoptic area project ot the organum vasculosum of the lamina terminalis (OVLT), the suprachiasmatic nucleus, the mammillary body complex and the ventral tegmental area. LH-RH neurons in both the medial septal nucleus and medial preoptic area project via the stria medullaris to the medial habenular nucleus and from there via the fasciculus retroflexus to the interpeduncular nucleus of the midbrain. Other LH-RH neurons in the medial septal nucleus, nucleus of the diagonal band of Broca and olfactory tubercle are congregated in small clusters around large blood vessels which penetrate into this area, and they do not appear to send axons outside their immediate vicinity. The types of LH-RH axonal terminations and the roles of these peptide-containing neurons are discussed.
Basal levels of prostaglandin E2 in the rat brain were determined by radioimmunoassay to be 0.68-0.79 pmol/g brain. About one-third of the prostaglandin E2 (0.23-0.28 pmol/g) was resistant to extraction with ethanol, but could be recovered with a mixture of ethanol and 1 N HCl (9:1, v/v), indicating that a tightly bound form of prostaglandin E2 exists in the brain. The amount of the bound form of prostaglandin E2 was almost unchanged by pentylenetetrazole-induced convulsion or by transcardial perfusion with a formaldehyde solution, although these treatments resulted in 40- to 80-fold increases in prostaglandin E2 content extracted with ethanol at neutral pH. A polyclonal antibody against prostaglandin E2-albumin conjugates recognized the bound form of prostaglandin E2, giving a punctate appearance in many neuronal cell bodies in the brain. Although almost all of the neuronal perikarya were immunoreactive for prostaglandin E2, intense immunoreactivity was observed in the mitral cell layer of the olfactory bulb, layer V of the cerebral neocortex, anterodorsal and reticular nuclei of the thalamus, supraoptic, paraventricular, accessory neurosecretory and lateral mammaillary nuclei of the hypothalamus, mesencephalic trigeminal nucleus, nucleus of the trapezoid body and deep cerebellar nuclei. When the cerebral neocortical regions were observed electron microscopically, immunoreaction products were seen as fine granules which were clustered into small patches in the cytoplasm of neuronal cell bodies and proximal dendrites. No immunoreaction products were seen in glial cells or endothelial cells. These results suggest that prostaglandin E2 is involved in fundamental processes of neurons.
Prostaglandin E2 (PGE2) exerts a potent hyperthermic action when injected into the preoptic-hypothalamic area (POHA) and is considered to be a central mediator of fever. To determine the exact functional sites of PGE2, we used in vitro quantitative autoradiography of [3H]PGE2 binding sites in the rat POHA. The highest density of [3H]PGE2 binding was found in the regions of the anterior wall of the 3rd ventricle (A3V). Within the A3V, binding density was especially high in regions closest to the third ventricle or surrounding the organum vasculosum laminae terminalis (OVLT) but was relatively low within the OVLT itself. It seems likely that the A3V PGE2 binding sites identified in this study are responsible for PGE2 mediation of fever.
Studies were conducted to determine if the stimulatory effect that norepinephrine (NE) exerts on the release of prostaglandin E2 (PGE2) and LHRH from the median eminence is mediated by alpha- or beta-adrenergic receptors. Incubation of median eminence fragments from adult male rats with different concentrations of phentolamine, an alpha-receptor antagonist, resulted in a dose-related inhibition of the release of both PGE2 and LHRH induced by NE, with an IC50 of 3.5 X 10(-7) and 0.9 X 10(-7) M for PGE2 and LHRH, respectively. Complete suppression of the NE effects was observed with a phentolamine concentration of 5 X 10(-6) M. This dose also reduced basal PGE2 and LHRH release. In contrast to phentolamine, the beta-receptor antagonist propranolol, tested at a concentration of 5 X 10(-6) M, was completely ineffective in altering the stimulatory effect of NE on either PGE2 or LHRH release. Moreover (and as previously observed for LHRH), blockade of dopaminergic receptors with Pimozide (10(-6) M) failed to inhibit the release of PGE2 induced by NE, thus indicating that the stimulatory effect of NE is not mediated by a dopaminergic mechanism. It is concluded that NE stimulates the release of PGE2 and LHRH from nerve terminals of the median eminence by first interacting with an alpha-adrenergic receptor.
This review summarizes recent advances in the molecular characterization of prostanoid receptors. Prostanoids exert versatile actions in diverse tissues and cells through specific cell surface receptors. Molecular biological studies revealed the primary structure of eight types and subtypes of prostanoid receptor from various species. These include the thromboxane A2 receptor, prostacyclin receptor, prostaglandin (PG) F receptor, PGD receptor and four subtypes of PGE receptors. They are coupled to different signal transduction systems. In addition, multiple isoforms of PGE receptor EP3 subtype have been identified in various species. They are produced through alternative RNA splicing from a single gene and differ only in their carboxy-terminal tails. These isoforms differ in the efficiency of G protein activation, in the specificity of coupling to G proteins or in sensitivity to desensitization. This molecular characterization is useful for understanding the diverse physiological roles of prostanoids.
Distribution of the messenger RNA for the prostaglandin E receptor subtype EP3 was investigated by in situ hybridization in the nervous system of the mouse. The hybridization signals for EP3 were widely distributed in the brain and sensory ganglia and specifically localized to neurons. In the dorsal root and trigeminal ganglia, about half of the neurons were labeled intensely. In the brain, intensely labeled neurons were found in Ammon's horn, the preoptic nuclei, lateral hypothalamic area, dorsomedial hypothalamic nucleus, lateral mammillary nucleus, entopeduncular nucleus, substantia nigra pars compacta, locus coeruleus and raphe nuclei. Moderately labeled neurons were seen in the mitral cell layer of the main olfactory bulb, layer V of the entorhinal and parasubicular cortices, layers V and VI of the cerebral neocortex, nuclei of the diagonal band, magnocellular preoptic nucleus, globus pallidus and lateral parabrachial nucleus. In the thalamus, moderately labeled neurons were distributed in the anterior, ventromedial, laterodorsal, paraventricular and central medial nuclei. Based on these distributions, we suggest that EP3 not only mediates prostaglandin E2 signals evoked by blood-borne cytokines in the areas poor in the blood-brain barrier, but also responds to those formed intrinsically within the brain to modulate various neuronal activities. Possible EP3 actions are discussed in relation to the reported neuronal activities of prostaglandin E2 in the brain.
Three isoforms of the mouse prostaglandin-E-receptor EP3 subtype (EP3), EP3 alpha, EP3 beta and EP3 gamma, with different C-termini, which are produced through alternative splicing, showed different efficiencies with respect to heterotrimeric GTP-binding protein activation and adenylate cyclase inhibition [Sugimoto, Y., Negishi, M., Hayashi, Y., Namba, T., Honda, A., Watabe, A., Hirata, M., Narumiya, S. & Ichikawa, A. (1993) J. Biol. Chem. 268, 2712-2718; Irie, A., Sugimoto, Y., Namba, T., Harazono, A., Honda, A., Watabe, A., Negishi, M., Narumiya, S. & Ichikawa, A. (1993) Eur. J. Biochem. 217, 313-318]. To assess the role of the C-terminus in GTP-binding protein coupling, we truncated the C-terminus of EP3 at an alternative splicing site and expressed the mutant receptor. The truncated receptor retained the ability to physically associate with Gi2, forming an agonist/receptor/Gi2 ternary complex, and to undergo the characteristic conversion of its agonist-binding affinity, mediated by a guanine nucleotide from a low-affinity state to a high-affinity state. However, sulprostone, an EP3 agonist, failed not only to inhibit the forskolin-induced cAMP accumulation in the mutant receptor-expressing cells but also to stimulate the GTPase activity in the mutant receptor-expressing cell membrane. These results indicated that the C-terminus of EP3 is essential for the activation of GTP-binding protein.
Two different clones, named rEP3A (approximately 2.2 kb) and rEP3B (approximately 5.2 kb), were isolated from a rat kidney cDNA library by a homology screening approach. rEP3A was shown to encode the rat kidney prostaglandin E receptor EP3 subtype (rEP3A receptor) (Takeuchi, K. et al. B.B.R.C. (1993) 194: 885). rEP3B receptor differs only in its carboxyl-terminal tail (Ile-336 to Pro-364) from rEP3A receptor. Southern blot analysis of genomic DNA has suggested that the EP3 receptor gene is a single copy gene. RT-PCR using microdissected nephron segments showed co-expression of both receptor mRNAs specifically in distal nephron segments such as mTAL, cTAL, CCD and IMCD, whereas no significant expression of both receptor mRNAs was detected from GL, PCT, and PST. In conclusion, we have cloned an isoform of the rat kidney EP3 receptor, rEP3B receptor. rEP3A and rEP3B receptors are suggested to be derived by alternative RNA splicing, and both receptors are co-localized to distal tubules exerting an effect on water and electrolyte metabolism.
A rat kidney cDNA library was screened with a mouse prostaglandin (PG) E2 receptor EP3 alpha subtype cDNA as a probe, and a 2.2-kilobase pair cDNA was isolated. The cDNA encodes 365 amino acids with 97.0% sequence identity to mouse EP3 alpha receptor. Specific binding of [3H]PGE2 was found in COS-7 cells transfected with the cDNA (Kd = 3.2nM) and was displaced with unlabeled prostaglandins in the order of PGE2 = PGE1 > PGF2 alpha > PGD2. Thus, a cDNA for rat EP3 (rEP3) receptor was cloned. In situ hybridization revealed expression of rEP3 receptor principally in tubules of renal medulla. Consistent with this observation, the reverse transcription and polymerase chain reaction (RT-PCR) technique using dissected nephron segments showed the receptor expression specifically in medullary thick ascending limbs of Henle's loop (mTAL), cortical TAL (cTAL), cortical collecting ducts (CCD) and inner medullary collecting ducts (IMCD), indicating a possible diuretic and natriuretic role of rEP3 receptor at the distal nephron segments.
Polyclonal antibodies were raised against 15-residue sequences in the carboxyl terminal region of mouse EP1, EP2, and EP3 subtypes. The selected sequences are well conserved in different species. Using the antibodies, the localization of the receptor subtypes in porcine uveal tissues was investigated by immunoperoxidase reaction (by light microscopy) and immunogold labeling (by electron microscopy). EP1 immunoreactivity was found in ciliary nonpigmented epithelium and iris muscles (both sphincter and dilator). EP2 was localized to ciliary nonpigmented epithelium and muscle, iris sphincter muscle, and trabecular meshwork. EP3 immunoreactivity was detected in all uveal tissues examined.
Expression level of messenger RNAs (mRNAs) for prostanoid EP3, FP, and TP receptors was investigated in cultured rat astrocytes, oligodendrocytes, and microglia, as well as in meningeal fibroblasts, rat glioma C6 cells, rat pheochromocytoma PC12 cells, whole brain, and several peripheral tissues by reverse transcriptase-polymerase chain reaction. Cultured astrocytes and oligodendrocytes expressed mRNAs for 3 prostanoid receptors examined. In contrast, cultured microglia and pheochromocytoma PC12 cells expressed EP3 and TP receptor mRNAs, but not FP receptor mRNA. Glioma C6 cells expressed only TP receptor mRNA among 3 prostanoid receptors with the same expression level as that in astrocytes. Cultured meningeal fibroblasts expressed 3 receptor transcripts, and their expression levels were lower than those in astrocytes. Expression level of mRNA for each prostanoid receptor in cultured glial cells was higher than that in whole brain. These observations suggest that each prostanoid has its specific roles in each glial cell type of the brain.
Fever is induced in response to the entrance of pathogenic microorganisms into the body and is thought to be mediated by cytokines. Because these pathogens most commonly invade the body through its natural barriers and because body temperature is regulated centrally, these mediators are presumed to be produced peripherally and transported by the bloodstream to the brain, to act. It is generally considered that their febrigenic messages are further modulated there by prostaglandin E2 (PGE2). However, the detailed mechanism by which these cytokines signal the brain and activate the febrile response is not yet clear. Indeed, the specific role of each cytokine has been difficult to establish due to complex interactions among them. Furthermore, recent evidence suggests that different pyrogens may induce different cytokines; for example, i.v. LPS (a model of systemic bacterial infection) induces large increases in IL-6, but only small rises in IL-1 and TNF alpha plasma levels. Moreover, their appearance lags the fever onset. We recently found that subdiaphragmatic vagotomy, decomplementation, and blockade of Kupffer cells suppress the febrile response of guinea pigs to i.v. LPS, and that i.v. LPS rapidly stimulates the release of norepinephrine (NE) and, hence, of PGE2 in their preoptic-anterior hypothalamus (POA, the brain region containing the thermoregulatory controller). Based on these and other data in the literature, we hypothesize that LPS fever may be initiated as follows: i.v., LPS-->complement-->Kupffer cells-->cytokines?-->vagal afferents -->n. tractus solitarius?-->A1/A2 cell groups?-->ventral noradrenergic bundle? -->POA-->NE-->PGE2-->fever.
Cyclooxygenase-2 (COX-2) is now considered to be the major constitutively expressed COX isozyme in the central nervous system. The present immunocytochemical study details localization of COX-2 immunoreactivity in rat spinal cord along with the expression of prostaglandin E2 receptor subtype EP3. Prominent COX-2 staining was observed in the nuclear envelope of neurons throughout the spinal cord, especially in the superficial dorsal horn laminae and motoneurons of lamina IX, as well as in glial cells of the white matter. Expression of EP3 receptor was strictly confined to afferent terminal areas in the superficial dorsal horns.
Fever, a hallmark of disease, is elicited by exogenous pyrogens, that is, cellular components, such as lipopolysaccharide (LPS), of infectious organisms, as well as by non-infectious inflammatory insults. Both stimulate the production of cytokines, such as interleukin (IL)-1beta, that act on the brain as endogenous pyrogens. Fever can be suppressed by aspirin-like anti-inflammatory drugs. As these drugs share the ability to inhibit prostaglandin biosynthesis, it is thought that a prostaglandin is important in fever generation. Prostaglandin E2 (PGE2) may be a neural mediator of fever, but this has been much debated. PGE2 acts by interacting with four subtypes of PGE receptor, the EP1, EP2, EP3 and EP4 receptors. Here we generate mice lacking each of these receptors by homologous recombination. Only mice lacking the EP3 receptor fail to show a febrile response to PGE2 and to either IL-1beta or LPS. Our results establish that PGE2 mediates fever generation in response to both exogenous and endogenous pyrogens by acting at the EP3 receptor.
Molecular mechanisms of diverse actions of prostanoid receptors
- M Negishi
- Y Sugimoto
- A Ichikawa
Negishi, M., Sugimoto, Y. and Ichikawa, A., Molecular mechanisms of diverse actions of prostanoid receptors, Biochim. Biophys. Acta, 1259 (1995) 109–120.
Molecular mechanisms of diverse actions of prostanoid receptors
- Negishi