MyD88 signaling in brain endothelial cells is essential for the neuronal activity and glucocorticoid release during systemic inflammation.

Activation of neuronal circuits involved in the control of autonomic responses is critical for the host survival to immune threats. The brain vascular system plays a key role in such immune-CNS communication, but the signaling pathway and exact type of cells within the blood-brain barrier (BBB) mediating these functions have yet to be uncovered. To elucidate this issue we used myeloid differentiation factor 88 (MyD88)-deficient mice, because these animals do not show any responses to the cytokine interleukin-1beta (IL-1beta). We created chimeric mice with competent MyD88 signaling in either the BBB endothelium or perivascular microglia of bone marrow origin and challenged them with IL-1beta. Systemic treatment with the cytokine caused a robust transcriptional activation of genes involved in the prostaglandin E(2) (PGE(2)) production by vascular cells of the brain. Upregulation of these genes is dependent on a functional MyD88 signaling in the endothelium, because MyD88-deficient mice that received bone marrow stem cells from wild-type animals (for example, functional perivascular microglia) exhibited no response to systemic IL-1beta administration. MyD88 competent endothelial cells also mediate neuronal activation and plasma release of glucocorticoids, whereas chimeric mice with MyD88-competent perivascular microglia did not show a significant increase of these functions. Moreover, competent endothelial cells for the gene encoding Toll-like receptor 4 (TLR4) are essential for the release of plasma corticosterone in response to low and high doses of lipopolysaccharide. Therefore, BBB endothelial cells and not perivascular microglia are the main target of circulating inflammatory mediators to activate the brain circuits and key autonomic functions during systemic immune challenges.

Neuroprotective role of the innate immune system by microglia.

Innate immunity is a rapid series of reactions to pathogens, cell injuries and toxic proteins. A key component of this natural response is the production of inflammatory mediators by resident microglia and infiltrating macrophages. There is accumulating evidence that inflammation contributes to acute injuries and more chronic CNS diseases, though other studies have shown that inhibition of microglia is, in contrast, associated with more damages or less repair. The controversies regarding the neuroprotective and neurodegenerative properties of microglia may depend on the experimental approaches. Neurotoxic substances are frequently used to produce animal models of acute injuries or diseases and they may activate microglia either directly or indirectly by their ability to cause neuronal death and demyelination. Whether microglia and the immune response play a direct role in such processes still remains an open question. On the other hand, there are data supporting the role of resident microglia and those derived from the bone marrow in the stimulation of myelin repair, removal of toxic proteins from the CNS and the prevention of neurodegeneration in chronic brain diseases. The ability of glucocorticoids to provide a negative feedback on nuclear factor kappa B pathways in microglia may be a determinant mechanism underlying the ultimate fate of the inflammatory response in the CNS. This review presents new concepts regarding the neuroprotective role of the innate immune response in the brain and how microglia can be directed to improve recovery after injuries and prevent/delay neurodegeneration.

Innate immune receptor expression in normal brain aging.

Brain aging often results in cognitive impairment and is considered to be a major risk factor for neurodegenerative diseases. Earlier studies reported inflammatory responses in aged brain that could contribute to age-related neurodegeneration. Recently, innate immune receptors such as toll-like receptors (TLRs), so far implicated in defense against microorganisms, have been linked to pathogenesis of Alzheimer's disease. Therefore, we asked whether the transcription of TLRs (1-9) and CD14, could also be altered in physiological brain aging. Using real-time polymerase chain reaction (PCR), we indeed observed that TLR1, TLR2, TLR4, TLR5, TLR7 and CD14 expression was up-regulated in mouse brain in correlation with age. In contrast, transcriptions of TLR3, TLR6 and TLR8 were unchanged and the one of TLR9 was down-regulated. In situ hybridization further confirmed these results and identified the cellular source of TLR2 and TLR7 as mononuclear phagocytes. Together, this first systematic analysis demonstrates altered regulation of those innate immune receptors even in normal brain aging, which might be of relevance for understanding susceptibility to neurodegenerative processes associated with aging.

Role of microglial-derived tumor necrosis factor in mediating CD14 transcription and nuclear factor kappa B activity in the brain during endotoxemia.

Systemic injection of the endotoxin lipopolysaccharide (LPS) upregulates the gene encoding CD14 early in the circumventricular organs (CVOs) and later in the brain parenchyma. The present study tested the hypothesis that the parenchymal production of the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) by microglial cells is responsible for triggering CD14 transcription in an autocrine/paracrine loop-like manner. In a first set of experiments, Sprague Dawley rats were killed 1, 3, 6, and 12 hr after an intracerebroventricular administration of recombinant rat TNF-alpha or vehicle solution. Second, anti-rat TNF-alpha-neutralizing antibody or vehicle solution was administrated into the lateral ventricle 10 hr before an intraperitoneal injection of LPS. Central administration of the cytokine caused a strong induction of IkappaBalpha, TNF-alpha, and CD14 mRNA in parenchymal microglial cells. The hybridization signal for these transcripts was localized to the edge of the ventricles and the site of infusion. The time-related expression of each mRNA suggested that TNF-alpha has the ability to trigger its own production followed by the transcription of the LPS receptor; the signal for IkappaBalpha, TNF-alpha, and CD14 peaked at 1, 3, and 6 hr, respectively. The genes encoding TNF-alpha and mCD14 were also induced in the CVOs and within microglial cells across the brain parenchyma in response to intraperitoneal LPS administration. This induction in parenchymal cells of the brain was prevented in animals that received the anti-TNF-antisera intracerebroventricularly 10 hr before the systemic treatment with the endotoxin. The present data provide the evidence that microglial-derived TNF-alpha is responsible for the production of the LPS receptor CD14 during endotoxemia. This autocrine/paracrine stimulatory loop may be of great importance in controlling the inflammatory events that take place in the CNS during innate immune response as well as under pathological conditions.

An essential role of interleukin-1beta in mediating NF-kappaB activity and COX-2 transcription in cells of the blood-brain barrier in response to a systemic and localized inflammation but not during endotoxemia.

When released into the bloodstream, proinflammatory cytokines have the ability to trigger the transcription of different genes in cells of the blood-brain barrier (BBB), including members of the nuclear factor kappa B (NF-kappaB) family and cyclooxygenase-2 (COX-2), the limiting enzyme for the formation of prostaglandins (PGs). The present study investigated the possibility that interleukin-1beta (IL-1beta) plays an essential role in these events during a systemic inflammatory response. Both wild-type and IL-1beta-deficient mice were killed at different times after two different immunogenic stimuli, i.e., intraperitoneal lipopolysaccharide (LPS) injection and intramuscular turpentine injection, used here as a model of systemic localized inflammatory insult. The inhibitory factor kappaBalpha (IkappaBalpha, index of NF-kappaB activity) and COX-2 transcripts were detected throughout the brain by means of in situ hybridization. Systemic LPS injection caused a strong and rapid expression of IkappaBalpha in endothelial cells lining the BBB of large and small blood vessels and thereafter within parenchymal microglia across the brain. This treatment also provoked a transient expression of COX-2 along cells of the vascular system, and the expression pattern and intensity of the signal for both transcripts were essentially the same in wild-type and IL-1beta-deficient animals. In contrast, the induction of these genes that was quite selective to the cells of the BBB in response to intramuscularly turpentine insult was completely abolished in IL-1beta-deficient mice. Indeed, a late and prolonged expression of IkappaBalpha and COX-2 mRNAs was found along the cerebral blood vessels in response to the sterile and localized inflammation in wild-type mice, whereas such induction was absent in the brain of IL-1beta-deficient animals. These results indicate that IL-1beta has an obligatory role in the activation of NF-kappaB molecules and PGs within endothelial cells of the BBB in an experimental model of intramuscularly turpentine-induced inflammation but not during endotoxemia.

The LPS receptor (CD14) links innate immunity with Alzheimer's disease.

To rapidly respond to invading microorganisms, humans call on their innate immune system. This occurs by microbe-detecting receptors, such as CD14, that activate immune cells to eliminate the pathogens. Here, we link the lipopolysaccharide receptor CD14 with Alzheimer's disease, a severe neurodegenerative disease resulting in dementia. We demonstrate that this key innate immunity receptor interacts with fibrils of Alzheimer amyloid peptide. Neutralization with antibodies against CD14 and genetic deficiency for this receptor significantly reduced amyloid peptide induced microglial activation and microglial toxicity. The observation of strongly enhanced microglial expression of the LPS receptor in brains of animal models of Alzheimer's disease indicates a clinical relevance of these findings. These data suggest that CD14 may significantly contribute to the overall neuroinflammatory response to amyloid peptide, highlighting the possibility that the enormous progress currently being made in the field of innate immunity could be extended to research on Alzheimer's disease.

Corticotropin-releasing factor activates c-fos, NGFI-B, and corticotropin-releasing factor gene expression within the paraventricular nucleus of the rat hypothalamus.

Studies examining the regulation of hypothalamic CRF biosynthesis have provided substantial information regarding the relevance of this peptide in neuroendocrine homeostasis. However, the consequences of elevated CRF levels within the mammalian central nervous system on regulation of CRF production within the paraventricular nucleus (PVN) of the hypothalamus remain unclear. The expression of the immediate early gene c-fos has been used and validated as a marker for neural systems activated by a variety of extracellular stimuli and has been especially useful when examining activation of central neuroendocrine systems such as those involved in the response to stressful stimuli. The present study investigates the effects of injecting CRF into the lateral ventricle of conscious rats, firstly on the expression of two separate immediate early genes, c-fos and NGFI-B within the hypothalamic PVN, and secondly on the expression of CRF mRNA itself, as determined by quantitative in situ hybridization. Expression of Fos protein was also examined by immunohistochemical techniques. Intracerebroventricular (icv) injection of CRF increased the gene expression of both c-fos and NGFI-B in the parvocellular division of the PVN 30 min after injection. Fos immunoreactivity increased in this same region between 30-60 min, whereas expression of the CRF gene itself increased 2-fold 60 min after injection and remained elevated 2 h after treatment. A positive hybridization signal for CRF was observed over Fos-immunoreactive neurons within the parvocellular division of the PVN. Finally, we observed that all CRF-induced changes in gene expression were abolished by pretreatment with the competitive CRF antagonist alpha-helical CRF-(9-41). The time-related changes in expression of the genes measured imply that the expression of both c-fos and NGFI-B occurs before a significant increase in the expression of CRF. The results also suggest that CRF may act in a positive manner to regulate its own biosynthesis.

Inhibitory action of nitric oxide on circulating tumor necrosis factor-induced NF-kappaB activity and COX-2 transcription in the endothelium of the brain capillaries.

Circulating tumor necrosis factor alpha (TNF-alpha) has a profound stimulatory influence on mitogen-activated protein kinases that lead to nuclear factor kappa B (NF-kappaB) activity and transcription of the cyclooxygenase 2 (COX-2) gene in cells associated with the blood-brain barrier (BBB). This study investigated the hypothesis that nitric oxide (NO) acts as an endogenous modulator of TNF-induced NF-kappaB signaling and COX-2 transcription in the endothelium of the cerebral capillaries. To this end, rats were pretreated with the nonselective inhibitor of NO synthase (NOS) N(G)-nitro-L-arginine methyl ester (L-NAME) and killed 15, 45, and 90 minutes (min) after an i.v. injection of recombinant rat TNF-alpha. De novo expression of the inhibitory factor kappa B alpha (IkappaB alpha) was used as an index of NF-kappaB activity, whereas COX-2 mRNA induction was evaluated throughout the brain by in situ hybridization combined with immunohistochemistry. A single i.v. bolus of TNF caused a rapid expression of IkappaB alpha transcript first along large arterioles and small capillaries and thereafter within microglia across the brain parenchyma. The proinflammatory cytokine also provoked a strong transcriptional activation of the COX-2 gene that was quite specific to the cerebral endothelium as revealed by dual labeling using an antisera directed against the von Willebrand factor. Inhibition of NO synthesis did not by itself activate these proinflammatory molecules, but it enhanced the effects of circulating TNF-alpha in the BBB; the IkappaB alpha and COX-2 signal was significantly higher in microvascular-associated cells of animals that received both L-NAME and TNF-alpha treatments than those challenged with the proinflammatory cytokine alone. Rats treated with specific NOS inhibitors provided the evidence that these effects were mediated via the constitutive endothelial NOS (eNOS) and not the inducible form. These results indicate that eNOS-derived NO acts as an endogenous inhibitor of TNF-alpha-induced NF-kappaB activity and COX-2 transcription in the endothelium of the cerebral capillaries. This autoregulatory feedback of NO on these proinflammatory signal transduction events may be an essential element to prevent an exaggerated response that takes place in cells of the BBB during systemic immune challenges.

Regulation of the gene encoding tumor necrosis factor alpha (TNF-alpha) in the rat brain and pituitary in response in different models of systemic immune challenge.

Tumor necrosis factor (TNF)-alpha is usually referred to as a proinflammatory cytokine that plays a central role in initiating the cascade of other cytokines and factors for an appropriate immune response to infection. Like systemic phagocytes, recent studies have reported that specific cellular populations of the CNS have the ability to express and release the proinflammatory cytokine in response to peripheral administration of the bacterial endotoxin lipopolysaccharide (LPS). Whether such phenomenon represents a general mechanism of systemic immunogenic stimuli and how the severity of the challenge may influence TNF-alpha transcription in the brain has yet to be defined. Adult male rats were sacrificed 1, 3, 6, 12, 24 and 48 hours (h) after intraperitoneal (IP) injection of LPS (25-250 microg/100 g) or intramuscular (IM) injection of turpentine. Brains and pituitary glands were removed, cut, and TNF-alpha mRNA assayed by in situ hybridization using a full-length rat cRNA probe. The results show no positive signal under basal conditions or following sterile inflammation into the left hind limb. Systemic LPS caused a profound increase in the expression of the gene encoding TNF-alpha in the leptomeninges, choroid plexus (chp) and all sensorial circumventricular organs (CVOs). Interestingly, a migratory-like pattern of TNF-alpha-positive cells became apparent around the sensorial CVOs at 3 h, while a ubiquitous-like positive signal was found throughout the brain 6 h after the injection with the highest dose of LPS. The IP LPS injection also stimulated TNF-alpha transcription in the anterior pituitary lobe; the signal was maximal 1 h after the injection and returned gradually to basal levels at 12 h, whereas the mRNA encoding the cytokine was detected later in the neurohypophysis, i.e. 3 and 6 h post challenge. Dual-labeling procedure provided the evidence of an LPS-dependent induction of TNF-alpha in different phagocytic cellular populations of the brain, including parenchymal microglial cells during severe endotoxemia. The fact that these myeloid-derived cells have the ability to express the LPS receptor CD14 in the brain may well explain the transcriptional activation of the cytokine in response to the bacterial endotoxin, but not to systemic localized inflammation.

Influence of the paraventricular nucleus of the hypothalamus in the alteration of neuroendocrine functions induced by intermittent footshock or interleukin.

The documented ability of physical stress and cytokines to increase the secretion of corticotropin-releasing factor CRF by the paraventricular nucleus of the hypothalamus (PVN), coupled with our earlier demonstration that CRF acts within the brain to interfere with reproductive functions, led us to investigate the effect of lesions of the PVN on LH, testosterone, ACTH, and corticosterone (CORT) secretion. Bilateral lesions of the PVN were done electrolytically, and 2 weeks later a series of acute and chronic experiments were performed in intact or castrated male rats bearing indwelling jugular and/or intracerebroventricular cannulas. The first study involved a single 2-h exposure of intact male rats to footshocks (2 mA, 2-sec duration, 4 per min). Although PVN lesions did not measurably alter the ability of intermittent footshock to lower plasma testosterone levels, this treatment attenuated the rise in plasma ACTH and CORT. In a second study, which was done in castrated rats, shocks were delivered 2 h daily for 7 days, and circulating hormone levels were measured at the end of the last shock period. Plasma LH levels of stressed rats showed statistically comparable decreases in both sham- and PVN-lesioned animals. Chronic exposure to footshocks induced smaller increases of ACTH and CORT secretion in PVN-lesioned compared to sham-lesioned rats, but the lesions did not completely abolish stress-induced activation of the hypothalamic-pituitary adrenal (HPA) axis. The third experiment involved the central injection of the vehicle or 40 ng interleukin-1 beta (IL-1 beta) to castrated rats. As expected, IL-1 beta dramatically decreased plasma LH values and increased circulating ACTH and CORT levels measured 2 h later. Bilateral PVN lesion did not influence LH secretion after injection of the vehicle or IL-1 beta. In contrast, destruction of the PVN completely blocked the increase of HPA axis activity observed in sham-operated rats 2 h after the intracerebroventricular infusion of IL-1 beta. These results confirm our previous observation that exposure to either footshocks or interleukins alters the activity of the hypothalamic-pituitary gonadal and HPA axis. Additionally, the present studies suggest that the PVN, which represents the predominant hypothalamic nucleus controlling the response of the HPA axis to a number of stimuli, does not appear to be necessary for the antireproductive effects of intermittent footshock or immune activation.

Interleukin-6 is a needed proinflammatory cytokine in the prolonged neural activity and transcriptional activation of corticotropin-releasing factor during endotoxemia.

Interleukin-6 (IL-6) is a proinflammatory cytokine that plays multiple roles in the central nervous system during infections and injuries. Although this molecule is capable of stimulating the release of ACTH and glucocorticoids, it has been demonstrated that a single injection of IL-6 fails to activate the paraventricular nucleus (PVN) neurons that control the hypothalamic-pituitary-adrenal axis. The observation that IL-6 receptor (IL-6R) is up-regulated in the brain during endotoxemia led us to hypothesize that prior induction of IL-6R synthesis could amplify the effect of circulating IL-6 on the neuroendocrine response. Rats received a first iv injection of either bacterial lipopolysaccharide (LPS; 5 microg) or vehicle solution. After a 6-h waiting period, they received a second iv injection of either recombinant rat IL-6 or vehicle solution and were killed 1 h thereafter. Using in situ hybridization, we observed that IL-6R was barely expressed in the PVN under basal conditions, but was rapidly produced in response to LPS. IL-6 itself was also able to induce the synthesis of its own receptor along cerebral blood vessels, and this effect extended to several parenchymal structures, including the PVN, when the cytokine was administrated after LPS. In agreement with our hypothesis, we found that IL-6 injected in LPS-pretreated rats stimulated PVN neurons, as revealed by the expression of CRF primary transcript and c-fos messenger RNA, an immediate early gene used as a marker of cellular activation. A significant increase in plasma corticosterone levels was also found in animals that received iv IL-6 injection after being pretreated 6 h before with the very low dose of LPS. The fact that IL-6 alone or injected after LPS treatment was unable to induce cyclooxygenase-2 synthesis is an argument in favor of a PG-independent mechanism. The relative contribution of IL-6 in stimulating CRF expression in the PVN and neural activity throughout the brain during endotoxemia was also investigated in IL-6-deficient mice after an ip injection of LPS. The endotoxin induced similar c-fos and CRF expression patterns in knockout and wild-type mice, but the expression levels were generally higher and/or lasted longer in wild-type animals. Taken together, physiological changes that may include the induction of IL-6R synthesis seem to be necessary for IL-6 to activate PVN neurons. Moreover, although IL-6 does not appear essential during the early phases of endotoxemia, this cytokine is required during the later phases to prolong the activation of neural cells throughout the brain and to maintain CRF expression in the PVN neurons that control the hypothalamic-pituitary-adrenal axis.

Selective involvement of interleukin-6 in the transcriptional activation of the suppressor of cytokine signaling-3 in the brain during systemic immune challenges.

Cytokine-inducible proteins named as suppressors of cytokine signaling (SOCS) are rapidly induced by interleukin-6 (IL-6) and other members sharing the gp130 receptor subunit after activation of the Janus kinases (JAK) and the signal transducers and activators of transcription (STAT). These inhibitory proteins generally prevent tyrosine phosphorylation of IL-6 receptor signaling subunit gp130, specific JAK and STAT or in acting at steps distal to JAK activation. Expression of these inhibitory proteins is therefore a useful tool to investigate the signaling events occurring in the brain during immunogenic stimuli that involve cytokines of the IL-6 family. This study investigated the effect of ip lipopolysaccharide (LPS) administration on the expression of one key member of the SOCS family, SOCS-3, in both rats and mice. In rats, the endotoxin caused a profound transcriptional activation of the inhibitory factor in the circumventricular organs subfornical organ, organum vasculosum of the lamina terminalis, arcuate nucleus/median eminence, area postrema, choroid plexus, leptomeninges, ependymal lining cells, and along the endothelium of the brain blood vessels. The hybridization signal for SOCS-3 messenger RNA was low at 1 h, but robust at 3 and 6 h and declined to return to basal levels 12 h after the single ip LPS injection. The pattern of SOCS-3 expression was similar in the brain of wild-type mice, although induction of the inhibitory factor was no longer observed in the ependymal lining cells of the cerebral ventricles and the blood microvessels of IL-6-deficient animals at all the times evaluated, i.e. from 1-8 h post-LPS injection. The endothelium of the brain capillaries also exhibited up-regulation of both IL-6 receptor and gp130 subunits during systemic inflammation, which allowed SOCS-3 expression in response to circulating IL-6. The present data indicate that the JAK/STAT transduction pathways that lead to SOCS-3 transcription are activated within cells accessible from the blood circulation, but not within deep parenchymal elements of the brain during endotoxemia. Induction of SOCS-3 followed the cascade of events that take place during the acute phase response and the contribution of IL-6 in activating the inhibitory factor is site specific and not generalized throughout the central nervous system.

Effect of immune and metabolic challenges on the luteinizing hormone-releasing hormone neuronal system in cycling female rats: an evaluation at the transcriptional level.

The purpose of this study was to investigate the influence of immune (systemic endotoxin administration) and metabolic (fasting) challenges on LHRH neuronal activity and transcription in the organum vasculosum of the lamina terminalis/medial preoptic area as well as on the expression of the LHRH receptor (LHRH-R) in the anterior pituitary of cycling female rats. The reproductive stages of adult female rats (200-250 g; 14 h of light; lights on at 0600 h) were verified by daily vaginal smears taken every morning for a minimum of three or four cycles before the experiment. The acute-phase response was induced via an i.p. injection of lipopolysaccharide (LPS; 200 microg/100 g BW), whereas the metabolic challenge consisted of food deprivation for at least 48 h. Control and challenged rats were killed at specific times in the ovulatory cycle (1200, 1500, and 1800 h on proestrus and diestrous day 2). Frozen brains and pituitaries were mounted on a microtome, cut into 30-microm slices, and then processed for the detection of transcripts encoding either LHRH or LHRH-R by means of in situ hybridization histochemistry using intronic (heteronuclear RNA) and exonic [messenger RNA (mRNA)] riboprobes. Dual immunocytochemistry to detect Fos-immunoreactive (ir) nuclei in LHRH-ir perikarya and colocalization of LHRH mRNA with Fos protein during the day of proestrus were performed by using both in situ hybridization and immunocytochemistry techniques on the same brain sections. The percentage of LHRH-ir and LHRH-expressing neurons displaying positive Fos-ir nuclei during the afternoon of proestrus was significantly inhibited 3 h after endotoxin administration. Rats exhibited an increase in the levels of LHRH primary transcript in the organum vasculosum of the lamina terminalis/medial preoptic area structure at 1500 h on proestrus, a phenomenon significantly attenuated by LPS injection only at this phase of the estrous cycle. On the other hand, fasting did not affect LHRH neuronal activity or gene expression in intact cycling rats, but affected these cells in animals exhibiting a disruption of the ovulatory cycle. Interestingly, LPS caused a profound down-regulation of LHRH-R gene expression in the anterior pituitary throughout the entire estrous cycle. Although food deprivation provoked a more variable pattern of LHRH-R mRNA in cycling rats, the signal for this transcript in the adenohypophysis was deeply altered in those showing a perturbed cycle. These results provide evidence that immune challenge interferes with the LHRH system at both hypothalamic and pituitary levels, whereas alteration of that neuroendocrine system in food-deprived rats seems highly associated with the impairment of reproductive cyclicity.

Ovulatory cycle influences the stimulatory effect of stress on the expression of corticotropin-releasing factor receptor messenger ribonucleic acid in the paraventricular nucleus of the female rat hypothalamus.

The purpose of this study was to investigate whether the ovulatory cycle interferes with the effect of immobilization exposure on the expression of the type I CRF receptor (CRF-R) gene in the brains of female rats. The reproductive stages of adult female rats (200-250 g; 14 h of light; lights on at 0600 h) were verified by daily vaginal smears taken every morning for a minimum of three or four cycles before the experiment. Three hours after beginning the 90-min immobilization session, rats were deeply anesthetized and transcardially perfused with a solution of 4% paraformaldehyde in the morning (1100 h) and the afternoon (1700 h) of proestrus and diestrous day 2. Frozen brains were mounted on a microtome, cut in 30-microns slices, and processed for the detection of CRF-R messenger RNA (mRNA) by in situ hybridization histochemistry. Strand-specific antisense riboprobe was generated by in vitro transcription using T7 RNA polymerase and [35S]UTP. Basal levels of CRF-R transcripts were observed in several defined regions of the brain, such as the medial septal nucleus, nucleus of the diagonal band, basolateral and medial nuclei of the amygdala, red nucleus, pontine gray, and various layers of the cerebral cortex. Although control animals displayed almost undetectable levels of CRF-R mRNA in the paraventricular nucleus (PVN), immobilization stress induced a marked expression of the gene encoding CRF-R in the dorsomedial parvocellular division of this hypothalamic structure in immobilized female rats. The large majority of CRF-R-positive cells were colocalized in CRF-immunoreactive neurons. Interestingly, stress-induced CRF-R transcription was significantly higher in the morning of proestrus than in the afternoon and on diestrous day 2. Apart from the PVN, none of the other sites exhibiting basal signals of CRF-R mRNA in the brain was affected by acute exposure to immobilization. These results provide evidence that neurogenic stress can stimulate the transcription of the type I CRF-R selectively in the parvocellular PVN, a phenomenon influenced by the neuroendocrine events regulating the reproductive cyclicity in female rats.

The chronic intracerebroventricular infusion of interleukin-1 beta alters the activity of the hypothalamic-pituitary-gonadal axis of cycling rats. I. Effect on LHRH and gonadotropin biosynthesis and secretion.

We have previously reported that the acute injection of interleukin-1 beta (IL-1 beta) into the brain ventricles of intact female rats promptly decreases LHRH release and inhibits gene expression of this peptide in the medial preoptic area (MPOA). The present studies were therefore designed to determine whether continuous exposure to the cytokine would disrupt the estrous cycle. IL-1 beta was injected intracerebroventricularly for 4-6 days at a rate of 4 ng/h. Daily vaginal smears were obtained to follow the cycle; pituitary LH and FSH secretion were measured at regular intervals. Steady state levels of LH and FSH messenger RNA (mRNA) in the pituitary, and LHRH gene expression in the MPOA, were measured at the end of the treatment. Infusion of IL-1 beta caused a total disruption of the estrous cycle, characterized by persistent smears indicative of the diestrus stage. When compared to animals treated with the vehicle, rats infused with IL-1 beta showed a significant decrease in circulating LH concentrations, which was accompanied by lowered mRNA levels in the pituitary. This statistical difference (P < 0.01) persisted even when treated rats were compared to control in a similar stage of the cycle (i.e. diestrus). Plasma FSH levels remained low at all times after IL-1 beta infusion but showed the expected cyclic changes in control animals. At the end of treatment, LHRH gene expression was also markedly suppressed in LHRH neurons distributed between the rostral preoptic area/organum vasculosum of the lamina terminalis and the MPOA of these animals. These results indicate that prolonged infusion of IL-1 beta into brain ventricles disrupts the estrous cycle, an event accompanied by decreased biosynthesis/release of LHRH and gonadotropins. We report in a related study that IL-1-treated rats also show increased plasma progesterone levels. However, it is improbable that this change was responsible for the interruption of the cycle described here; indeed we have previously observed that the central administration of IL-1 beta to intact rats resulted in an immediate blockade of the spontaneous activity of LHRH perikarya during the afternoon of proestrus and significantly decreased LHRH mRNA levels in gonadectomized animals. Taken together, these data suggest that the primary effect of IL-1 beta is at the level of LHRH perikarya, and that the resulting interruption of the cycle is caused by altered LHRH neuronal activity and blunted gonadotropin secretion.

Regulation of corticotropin-releasing factor type 1 (CRF1) receptor messenger ribonucleic acid in the paraventricular nucleus of rat hypothalamus by exogenous CRF.

The present study sought to examine the effects of intracerebroventricular (icv) administration of corticotropin-releasing factor (CRF) on the expression of CRF1 receptor messenger RNA (mRNA) within the hypothalamus as determined by quantitative in situ hybridization histochemistry. Adult male Sprague-Dawley rats were stereotaxically implanted with guide cannulae directed towards the right lateral ventricle. After 8-10 days of recovery, either 10 microliters CRF (5 micrograms) or vehicle solution was injected into the lateral ventricle over a 2-min period. The rats were then deeply anesthetized at 15, 60, and 180 min after icv injection, transcardially perfused, and their brains cut into 30-micron coronal sections. Brain sections were then processed using standard radioactive in situ hybridization histochemistry revealing the expression of the CRF1 receptor mRNA. Low to moderate basal levels of CRF1 receptor transcript were observed in several regions of the forebrain. However, the hybridization signal for the mRNA encoding the CRF1 receptor was barely detectable in the paraventricular nucleus of the hypothalamus (PVN) of vehicle-injected rats. In contrast, 180 min after icv administration of CRF, a significant increase in CRF1 receptor transcript was measured specifically in the PVN, despite having virtually any hybridization signal before 180 min. This increase in the level of receptor transcription by CRF was restricted to the type 1 receptor subtype because the hybridization signal for the CRF2 alpha receptor mRNA was unaffected in the brain regions in which it was located. Moreover, we confirmed previous findings of a CRF-induced neuronal activation of parvocellular neurosecretory cells of the PVN, as assessed by c-fos mRNA expression. This neuronal activation induced by exogenous CRF was also associated with a rapid and strong induction of CRF heteronuclear RNA selectively in the rat PVN, a phenomenon abolished by a pretreatment with a CRF receptor antagonist. These results provide evidence that elevated levels of central CRF may trigger CRF1 receptor transcription selectively in the PVN. This positive feedback of CRF on its own receptor may represent a functional adaptation of the hypothalamic-pituitary-adrenal axis in response to stress.

Expression of corticotropin-releasing factor and its receptors in the brain of lean and obese Zucker rats.

Expression of CRF messenger RNA (mRNA) and heteronuclear RNA (hnRNA) as well as the mRNAs encoding the CRF receptors of type 1 (CRF1R) and type 2 alpha (CFR2R) in the brain has been investigated in lean (Fa/?) and obese (fa/fa) Zucker rats. Exonic and intronic in situ hybridization histochemistry was employed to measure the mRNA and hnRNA levels in rats killed before (resting state), during, and 120 min after a treadmill running session. The resting expression of CRF hnRNA in the hypothalamic paraventricular nucleus (PVN) of obese rats was minimal and comparable to that of lean rats. However, during treadmill running, this expression was higher in obese than in lean rats. In obese rats, the transcription of the CRF1R mRNA in the PVN was high under resting conditions, dropped considerably during running, and rose again to elevated levels 120 min after the treadmill session. In lean rats, CRF1R mRNA in the PVN was minimal before and during running, but rose to a value similar to that in obese rats 120 min after running. In the PVN of obese rats, expression of the CRF1R gene measured during resting conditions was comparable to the level seen after running and proved to be dependent upon the feeding state of the rats. Expression of the CRF2R transcript was reduced in the ventromedial nucleus of the hypothalamus (VMH) of the obese rat. Plasma ACTH concentrations during treadmill running were lower in obese than in lean animals. Basal and postrunning levels of circulating corticosterone were higher in fa/fa than in Fa/? rats. However, there was no difference in corticosterone levels between lean and obese animals during running. The present results provide evidence for differences between lean and obese rats in the expression of CRF and its receptor within selective hypothalamic nuclei. Given the anorectic and thermogenic properties of CRF and the roles of PVN and VMH in the regulation of energy balance, it can be argued that the observed alterations in the biosynthesis of CRF and its receptors within the PVN and VMH might be related to the development of obesity.

Luteinizing hormone secretion and corticotropin-releasing factor gene expression in the paraventricular nucleus of rhesus monkeys following cortisol synthesis inhibition.

Corticotropin-releasing Factor (CRF) is an important inhibitory neuromodulator of GnRH/LH secretion, and mediates in part the inhibitory effects of stress on the hypothalamic-pituitary-gonadal axis. The purpose of the present study was to further investigate CRF's role in regulating LH secretion in primates. This was accomplished by examining LH secretion in ovariectomized rhesus monkeys (n = 7) following cortisol synthesis inhibition with metyrapone. Infusion of metyrapone (5 mg/kg per h) for 4 h decreased cortisol levels to less than 20% of controls while increasing ACTH approximately 10-fold. LH concentrations were not affected by this acute activation of the hypothalamic-corticotroph axis. In a second experiment, metyrapone was infused for 10 h before collecting serial blood samples every 15 min for 6 h. Although this protocol produced a sustained increase in ACTH, no apparent effect on pulsatile LH secretion compared with saline controls was observed. Mean LH (+/- SEM) levels calculated for consecutive 2-h increments were 87.6 +/- 9.2 (0-2 h) 82.1 +/- 5.5 (2-4 h), and 80.7 +/- 4.8 (4-6 h) ng/ml in saline pretreated animals compared with 83.6 +/- 4.9, 79.8 +/- 5.8, and 72.5 +/- 6.2 ng/ml, respectively, in metyrapone pretreated monkeys. The same regimen of metyrapone infusion increased CRF messenger RNA levels in the paraventricular nucleus by approximately 33% (P < 0.0002). In a final experiment designed to examine the potential synergy between CRF and cortisol, the LH response to insulin-induced hypoglycemia was contrasted in saline and metyrapone pretreated monkeys. LH concentrations were reduced to approximately 40% of basal levels following insulin in both metyrapone and saline pretreated monkeys. Therefore, even though inhibition of cortisol synthesis leads to an increase in CRF messenger RNA in the paraventricular nucleus and a robust increase in ACTH secretion in rhesus monkeys, presumably due in part to increased neuroendocrine CRF secretion, LH secretion was not inhibited during either the acute or more chronic phase of corticotroph activation. Absence of LH inhibition was not due to low cortisol concentrations resulting from metyrapone because metyrapone did not prevent hypoglycemia-induced suppression of LH secretion. We conclude that increased neuroendocrine CRF secretion following metyrapone does not inhibit LH secretion under these conditions. Several explanations for this result are discussed.

The bacterial endotoxin lipopolysaccharide has the ability to target the brain in upregulating its membrane CD14 receptor within specific cellular populations.

Systemic injection of the bacterial endotoxin lipopolysaccharide (LPS) provides a very good mean for increasing the release of proinflammatory cytokines by circulating monocytes and tissue macrophages. There is now considerable evidence that LPS exerts its action on mononuclear phagocytes via the cell surface receptor CD14. The aim of the present study was to verify the hypothesis that the brain has also the ability to express the gene encoding the LPS receptor, which may allow a direct action of the endotoxin onto specific cellular populations during blood sepsis. Adult male Sprague-Dawley rats were sacrificed 1, 3, 6 and 24 h after systemic (i.v. or i.p.) injection of LPS or the vehicle solution. Brains were cut from the olfactory bulb to the medulla in 30-microm coronal sections and mRNA encoding rat CD14 was assayed by in situ hybridization histochemistry using a specific 35S-labeled riboprobe. The results show low levels of CD14 mRNA in the leptomeninges, choroid plexus and along blood vessels of the brain microvasculature under basal conditions. Systemic injection of the bacterial endotoxin caused a profound increase in the expression of the gene encoding CD14 within these same structures as well as in the circumventricular organs (CVOs) the organum vasculosum of the lamina terminalis, subfornical organ, median eminence and area postrema. In most of these structures, the signal for CD14 mRNA was first detected at 1 h, reached a peak at 3 h post-injection, declined at 6 h, and return to basal levels 24 h after LPS treatment. Quite interestingly, a migratory-like pattern of CD14 positive cells was observed from all sensorial CVOs to deeper parenchymal brain 3 and 6 h after LPS injection. At 6 h post-challenge, small positive cells were found throughout the entire parenchymal brain and dual-labeling procedure indicated that different cells of myeloid origin have the ability to express CD14 in response to systemic LPS. These included CVO microglia, choroid plexus and leptomeninge macrophages, parenchymal and perivascular-associated microglial cells, although specific nonmyeloid cells were also positive for the LPS receptor. These results provide the very first evidence of a direct role of LPS on specific cell populations of the central nervous system, which is likely to be responsible for the transcription of proinflammatory cytokines; first within accessible structures from the blood and thereafter through scattered parenchymal cells during severe sepsis.

Functional circuitry in the brain of immune-challenged rats: partial involvement of prostaglandins.

This study investigated the role of prostaglandins (PGs) on the neuronal activity and the transcription of corticotropin-releasing factor (CRF) in the brain of conscious immune-challenged rats. Intravenous (i.v.) administration of indomethacin, an inhibitor of PG synthesis, was performed prior to and after the intraperitoneal (i.p.) injection of different doses [250 microg, 25 microg, and 2.5 microg/100 g body weight (b.w.)] of the immune activator lipopolysaccharide (LPS). Systemic administration of the high and middle doses of LPS caused a robust and widespread induction of both immediate-early genes (IEGs), c-fos and nerve growth factor-inducible gene B (NGFI-B) mRNAs, whereas injection of the low dose selectively triggered c-fos expression within the sensorial circumventricular organs. Pretreatment with indomethacin did not prevent c-fos transcription in the rat brains challenged with the high dose of LPS at 3 hours postinjection. Inhibition of PG formation was more effective for interruption of the neuronal activation in animals injected with 25 microg LPS/100 g b.w., although the influence depended on the structures and the groups of activated cells. Indeed, PG inhibition significantly altered LPS-induced c-fos mRNA expression in the medial preoptic area/organum vasculosum of the lamina terminalis, the periventricular nucleus, the paraventricular nucleus of the hypothalamus (PVN), and the ventrolateral medulla (VLM) but not in many other regions, including the subfornical organ, the central nucleus of the amygdala, the arcuate nucleus/median eminence, the parabrachial nucleus, the choroid plexus, and the nucleus of the solitary tract (NTS). In the hypothalamic PVN, inhibition of both c-fos and NGFI-B transcripts by indomethacin was also associated to an abolished influence of the endotoxin on the transcription of neuroendocrine CRF; induction of CRF primary transcript by the middle dose of LPS was selective to the PVN and was completely blocked by pretreatment with indomethacin. Moreover, a large number of tyrosine hydroxylase (TH)-immunoreactive neurons of the VLM (A1/C1) and the NTS (A2/C2) were positive for c-fos mRNA in immune-challenged rats, an effect that was largely prevented by indomethacin in the VLM but not in the NTS. These results indicate that the role of PGs in mediating the stimulatory influence of the acute-phase response depends on the severity of the systemic stressful situation, the brain regions, and the cell groups as well as the activated target genes.