Nathalie Laflamme

Expression and neuropeptidergic characterization of estrogen receptors (ER? and ER?) throughout the rat brain: Anatomical evidence of distinct roles of each subtype

The recent cloning of a second estrogen receptor (ER) provided a new tool to investigate and clarify how estrogens are capable of communicating with the brain and influence gene expression and neural function. The purpose of the present study was to define the neuroanatomical organization of each receptor subtype using a side-by-side approach and to characterize the cellular population (s) expressing the ERbeta transcript in the endocrine hypothalamus using immunohistochemistry combined with in situ hybridization. Axonal transport inhibition was accomplished to cause neuropeptide accumulation into the cytoplasm and thus facilitate the detection of all positive luteinizing hormone-releasing hormone (LHRH), corticotropin-releasing factor (CRF), vasopressin (AVP), oxytocin (OT), gastrin-related peptide (GRP), and enkephalin (ENK) neurons. The genes encoding either ERalpha or -beta were expressed in numerous limbic-associated structures, and fine differences were found in terms of intensity and positive signal. Such phenomenon is best represented by the bed nucleus of the stria terminalis (BnST) and preoptic area/anterior hypothalamus, where the expression pattern of both transcripts differed across subnuclei. The novel ER was also found to be expressed quite exclusively in other hypothalamic nuclei, including the supraoptic (SON) and selective compartments (magnocellular and autonomic divisions) of the paraventricular nucleus (PVN). A high percentage of the ERbeta-expressing neurons located in the ventro- and dorsomedial PVN are of OT type; 40% of the OT-ir cells forming the medial magnocellular and ventromedial parvocellular PVN showed a clear hybridization signal for ERbeta mRNA, whereas a lower percentage (15-20%) of OT neurons were positive in the caudal parvocellular PVN and no double-labeled cells were found in the rostral PVN and other regions of the brain with the exception of the SON. Very few AVP-ir neurons expressing ERbeta transcript were found throughout the rat brain, although the medial PVN displayed some scattered double-labeled cells (<5%). Quite interestingly, the large majority of the ERbeta-positive cells in the caudal PVN were colocalized within CRF-ir perikarya. Indeed, more than 60-80% of the CRF-containing cells located in the caudolateral division of the parvocellular PVN exhibited a positive hybridization signal for ERbeta mRNA, whereas very few (<5%) neuroendocrine CRF-ir parvocellular neurons of the medial PVN expressed the gene encoding ERbeta. A small percentage of ERbeta-expressing cells in the dorsocaudal and ventromedial zones of the parvocellular PVN were also ENK positive. The ventral zone of the medial parvocellular PVN also displayed GRP-ir neurons, but no convincing hybridization signal for ERbeta was detected in this neuronal population. Finally, as previously described for the gene encoding the classic ER, LHRH neurons of both intact and colchicine-pretreated animals did not express the novel estrogen receptor. This study shows a differential pattern of expression of both receptors in the brain of intact rats and that ERbeta is expressed at various levels in distinct neuropeptidergic populations, including OT, CRF, and ENK. The influence of estrogen in mediating genomic and neuronal responses may therefore take place within these specific cellular groups in the brains of cycling as well as intact male mammals.

Effects of Systemic Immunogenic Insults and Circulating Proinflammatory Cytokines on the Transcription of the Inhibitory Factor κBα Within Specific Cellular Populations of the Rat Brain

Abstract: Expression of the inhibitory factor κBα (IκBα) reflects the activity of nuclear factor κB(NF‐κB) and is a powerful tool to investigate the regulation of the transcription factor within the CNS. IκBα mRNA was evaluated in the rat brain by means of in situ hybridization following different immunogenic stimuli; i.e., intraperitoneal (i.p.) and intravenous (i.v.) lipopolysaccharide (LPS), i.v. recombinant rat interleukin (IL) 1β, IL‐6, or tumor necrosis factor‐α (TNF‐α), and intramuscular (i.m.) turpentine injection, used here as a model of systemic localized inflammatory insult. Systemic LPS, IL‐1β, and TNF‐α caused a rapid and transient transcriptional activation of IκBα along the blood vessels of the entire brain; the signal was very intense 30‐60 min after the i.v. injections and returned to undetectable levels from 2 to 12 h depending on the challenge. Double‐labeling procedure provided the anatomical evidence that IκBα‐expressing cells within the microvasculature were essentially of the endothelial type, as they were immunoreactive to the von Willebrand factor. Scattered small cells were also found across the brain of LPS‐, IL‐1β‐, and TNF‐α‐injected rats at time 1‐3 h, and microglial (OX‐42)‐immunoreactive cells were positive for the transcript. Such expression within parenchymal microglia was nevertheless not observed in the brain following a localized and sterile inflammatory insult. Indeed, i.m. turpentine administration stimulated IκBα transcription quite uniquely within the endothelium of the brain capillaries, an effect that paralleled the swelling of the injection site and lasted up to 24 h after the aggression. In contrast to these immunogenic challenges, i.v. IL‐6 injection failed to activate the gene encoding IκBα in the rat brain. These results indicate that NF‐κB may play a crucial role in specific cellular populations of the CNS to trigger transcription of immune‐related genes and that IκBα resynthesis may act as a dynamic intracellular inhibitory feedback to avoid exaggeration of the response. It is possible that IκBα expression in cells of the blood‐brain barrier is a general mechanism that takes place during systemic inflammation, whereas the participation of NF‐κB‐related molecules within parenchymal cells of the CNS is solicited during more severe conditions such as blood sepsis and endotoxemia.

Circulating cell wall components derived from gram‐negative, not gram‐positive, bacteria cause a profound induction of the gene‐encoding Toll‐like receptor 2 in the CNS

The recent characterization of human homologs of Toll may be the missing link for the transduction events leading to nuclear factor‐κB (NF‐κB) activity and proinflammatory gene transcription during innate immune response. Mammalian cells may express as many as 10 distinct Toll‐like receptors (TLRs), although TLR2 is a key receptor for recognizing cell wall components of Gram‐positive bacteria. The present study investigated the effects of circulating bacterial cell wall components on the expression of the gene‐encoding TLR2 across the mouse brain. Surprisingly, while Gram‐negative components caused a robust increase in TLR2 transcription within the cerebral tissue, peptidoglycan (PGN) and lipoteichoic acid (LTA), either alone or combined, failed to modulate the receptor transcript. Indeed, the mRNA levels for TLR2 in the choroid plexus and few other regions of the brain remained similar between vehicle‐, LTA‐, PGN‐, and LTA/PGN‐administered mice at all the times evaluated (i.e. 30 min to 24 h post‐intraperitoneal injection). This contrasts with the profound de novo expression of TLR2 following a single systemic injection of the lipopolysaccharide (LPS). The signal was first detected in regions devoid of blood–brain barrier and few blood vessels and microcapillaries. A second wave of TLR2 expression was also detected from these structures to their surrounding parenchymal cells that stained for a microglial marker iba1. The rapid induction of IκBα (index of NF‐κB activity) and up‐regulation of the adaptor protein MyD88 suggest that LPS‐induced TLR2 transcription may be dependent on the NF‐κB pathway. These data provide the evidence that TLR2 is not only present in the brain, but its encoding gene is regulated by cell wall components derived from Gram‐negative, not Gram‐positive, bacteria. The robust wave of TLR2‐expressing microglial cells may have a determinant impact on the innate immune response that occurs in the brain during systemic infection by Gram‐negative, not Gram‐positive, bacteria.

Cooperation between toll-like receptor 2 and 4 in the brain of mice challenged with cell wall components derived from gram-negative and gram-positive bacteria.

In this study we investigated whether induction of toll‐like receptor 2 (TLR2) amplifies the effect of a cell wall component derived from gram‐positive bacteria, namely peptidoglycan (PGN). Mice received a first systemic lipopolysaccharide (LPS) injection to pre‐induce TLR2 in various regions of the brain, and 6 h later, a second administration of either LPS or PGN. The data show a robust transcriptional activation of TLR2, TNF‐α and monocyte chemotactic protein‐1 (MCP‐1) in microglial cells of mice challenged twice with LPS, whereas PGN essentially abolished this response. TLR4 plays a critical role in this process, because C3H/HeJ mice no longer responded to LPS but exhibited a normal reaction to PGN. Conversely, a robust signal for genes encoding innate immune proteinswas found in the brain of TLR2‐deficient mice challenged with LPS. However, the second LPS bolus failed to trigger TNF‐α and IL‐12 in TLR2‐deficient mice, while the same treatment caused a strong induction of these genes in the cerebral tissue of wild‐type littermates. The present data provide evidence that cooperation exists between TLR4 and TLR2. While TLR4 is absolutely necessary to engage the innate immune response in the brain, TLR2 participates in the regulation of genes encoding TNF‐α and IL‐12 during severe endotoxemia. Such collaboration between TLR4 and TLR2 may be determinant for the transfer from the innate to the adaptive immunity within the CNS of infected animals.

Regulation of the gene encoding the monocyte chemoattractant protein 1 (MCP-1) in the mouse and rat brain in response to circulating LPS and proinflammatory cytokines

Accumulating evidence supports the existence of an innate immune response in the brain during systemic inflammation that is associated with a robust induction of proinflammatory cytokines and chemokines by specific cells of the central nervous system. The present study investigated the genetic regulation and fine cellular distribution of the monocyte chemoattractant protein‐1 (MCP‐1) in the brain of mice and rats in response to systemic immune insults. MCP‐1 belongs to a superfamily of chemokines that have a leading role in the early chemotaxic events during inflammation. In situ hybridization histochemistry failed to detect constitutive expression of the chemokine transcript in the cerebral tissue except for the area postrema (AP) that exhibited a low signal under basal conditions. This contrasts with the strong and transient induction of the mRNA encoding MCP‐1 following a single systemic bolus of lipopolysaccharide (LPS), recombinant interleukin‐1β (IL‐1β) and tumor necrosis factor alpha (TNF‐α). These stimuli rapidly triggered (30 to 90 minutes) MCP‐1 transcription in all the circumventricular organs (CVOs), the choroid plexus (chp), the leptomeninges, and along the cerebral blood vessels. The time‐related induction and intensity of the signal differed among the challenges, route of administration and species, but MCP‐1‐expressing cells were always found in vascular‐associated structures and those devoid of blood‐brain barrier. At later times, few isolated microglia across the brain parenchyma depicted positive signal for MCP‐1 mRNA. A dual‐labeling procedure also provided convincing anatomical evidence that endothelial cells of the microvasculature and a few myeloid cells of the CVOs and chp were positive for the transcript during endotoxemia. This gene is under a sophisticated transcriptional regulation, as the hybridization signal returned to undetectable levels 12 to 24 hours after all the treatments in both species. Of interest are the data that only ligands that triggered nuclear factor kappa B (NF‐κB) signaling had the ability to increase MCP‐1 gene expression, because high doses of IL‐6 remained without effects. These data provide the anatomical evidence that MCP‐1 is expressed within specific populations of cells in response to systemic inflammatory molecules that use NF‐κB as intracellular signaling system. This chemokine may therefore play a critical role in the cerebral innate immune response and contribute to the early chemotaxic events during chronic cerebral inflammation. J. Comp. Neurol. 434:461–477, 2001.

Inefficient clearance of myelin debris by microglia impairs remyelinating processes

Lampron et al. use a cuprizone mouse model of demyelination/remyelination to show that in CX3CR1-deficient mice, the clearance of myelin debris by microglia is impaired, affecting the integrity of axon and myelin sheaths.

REDUCED CORTICOTROPIN-RELEASING FACTOR AND ENHANCED VASOPRESSIN GENE EXPRESSION IN BRAINS OF MICE WITH AUTOIMMUNITY-INDUCED BEHAVIORAL DYSFUNCTION

The spontaneous development of autoimmune disease in MRL-lpr mice induces behavioral and endocrine changes that resemble effects of chronic stressors. To further examine the correspondence between autoimmune disease and chronic stress, we asked whether the brains of autoimmune mice show a shift in the corticotropin-releasing factor (CRF) to vasopressin (AVP) ratio. Using in situ hybridization histochemistry with 35S-labelled mouse riboprobes, the levels of mRNA transcripts encoding CRF and AVP were compared between autoimmune MRL-lpr and control MRL +/+ brains. CRF transcript levels were lower in the hypothalamic paraventricular nucleus and in the central nucleus of the amygdala in MRL-lpr mice. AVP transcript levels were higher in the paraventricular and the supraoptic nuclei in MRL-lpr mice compared to controls. CRF mRNA levels were inversely related to performance in stress-sensitive tasks and to measures of autoimmunity. As found previously for behavioral performance, immunosuppressive treatment with cyclophosphamide abolished the group difference in neuropeptide gene expression. These results indicate that an autoimmune disease process is necessary for the shift in the brain CRF:AVP ratio. Furthermore, they support the parallel between chronic stress and chronic autoimmunity/inflammation, and suggest common central mechanisms relevant to endocrine function and behavior.

Effect of dexfenfluramine on the transcriptional activation of CRF and its type 1 receptor within the paraventricular nucleus of the rat hypothalamus

1 The present study investigated the effect of intraperitoneal (i.p.) administration of the indirect 5‐hydroxytryptamine (5‐HT) receptor agonist, dexfenfluramine, on the transcriptional activity of corticotropin‐releasing factor (CRF) and its type 1 receptor in the brains of conscious male Sprague‐Dawley rats via in situ hybridization histochemistry (ISHH) using both intronic and exonic probe technology. 2 The immediate early gene (IEG) c‐fos mRNA was also used as index of cellular activity, whereas localization between CRF‐immunoreactive (ir) perikarya and the IEG was accomplished to determine the site of CRF neuronal activation in the brain of dexfenfluramine‐treated rats. 3 Thirty minutes, 1, 3, and 6 h after a single injection of either dexfenfluramine (10 mg kg−1) or the vehicle solution, adult male rats (230–260 g) were deeply anaesthetized and rapidly perfused with a 4% paraformaldehyde‐borax solution (PF). The brains were removed from the skull, postfixed, and placed in a solution of 4% PF‐10% sucrose overnight at 4°C. Frozen brains were mounted on a microtome and cut from the olfactory bulb to the medulla in 30‐μm coronal sections. 4 Dexfenfluramine induced a general neuronal activation as indicated by the strong signal of c‐fos mRNA in several structures of the brain, including the parietal cortex, caudate putamen, circumventricular organs, medial preoptic area, bed nucleus of the stria terminalis, choroid plexus, choroidal fissure, supraoptic nucleus, paraventricular nucleus of the hypothalamus (PVN), paraventricular nucleus of the thalamus, central nucleus of the amygdala, dorsomedial nucleus of the hypothalamus, laterodorsal tegmental nucleus, locus coeruleus, and several subdivisions of the dorsal vagal complex. In most of these structures, the signal was maximal at 30 min, still strong and positive at 60 min, largely decreased at 3 h, and had completely disappeared 6 h after injection. 5 In the parvocellular division of the PVN, the large majority of CRF‐ir perikarya displayed a positive signal for the mRNA encoding c‐fos, indicating a profound CRFergic activation within this neuroendocrine nucleus after dexfenfluramine administration. 6 Colocalization between CRF‐ir neurones and c‐fos positive cells was not detected in any other regions. This selective activation of PVN CRF neurones was also confirmed by the presence of CRF primary transcript; 30 min after i.p. injection of the indirect 5‐HT agonist, a positive signal for CRF hnRNA was observed, specifically in the parvocellular PVN. 7 Transcription of the gene encoding the type 1 receptor for CRF was highly stimulated in the PVN following 5‐HT activation. Although this hypothalamic nucleus exhibited a barely detectable signal under basal conditions, dexfenfluramine induced a strong signal of CRF1 receptor mRNA in the parvocellular PVN. Interestingly, CRF‐ir neurones displayed a positive signal for the mRNA encoding the CRF1 receptor, 3 and 6 h after systemic treatment with dexfenfluramine. 8 These results indicate that although dexfenfluramine can generate a wide neuronal activation throughout the brain, this 5‐HT agonist triggers the activity of CRF neurones selectively in the parvocellular division of the PVN, a mechanism possibly related to the activity of hypothalamic‐pituitary‐adrenal axis. Induction of CRF1 receptor mRNA in CRF cells of the PVN indicates that neuroendocrine CRF neurones can be targeted by CNS CRF under 5‐HT stimulation.

Involvement of serotonergic pathways in mediating the neuronal activity and genetic transcription of neuroendocrine corticotropin-releasing factor in the brain of systemically endotoxin-challenged rats.

The present study investigated the effect of serotonin depletion on the neuronal activity and transcription of corticotropin-releasing factor in the rat brain during the acute-phase response. Conscious male rats received an intraperitoneal (i.p.) injection with the immune activator lipopolysaccaride (25 microg/100 g body wt) after being treated for three consecutive days with para-chlorophenylalanine (30mg/100 g/day). This irreversible inhibitor of tryptophane-5-hydroxylase decreased hypothalamic serotonin levels by 96%. One, 3 and 6 h after a single i.p. injection of lipopolysaccharide or vehicle solution, rats were killed and their brains cut in 30-microm coronal sections. Messenger RNAs encoding c-fos, nerve-growth factor inducible-B gene, corticotropin-releasing factor and the heteronuclear RNA encoding corticotropin-releasing factor primary transcript were assayed by in situ hybridization using 35S-labeled riboprobes, whereas Fos-immunoreactive nuclei were labeled by immunocytochemistry. Lipopolysaccharide induced a wide neuronal activation indicated by the expression of both immediate-early gene transcripts and Fos protein in numerous structures of the brain. The signal for both immediate-early gene transcripts was low to moderate 1 h after lipopolysaccharide administration, maximal at 3 h and decline at 6 h post-injection, whereas at that time, Fos-immunoreactive nuclei were still detected in most of the c-fos messenger RNA-positive structures. Interestingly, the strong and widespread induction of both immediate-early gene transcripts was almost totally inhibited by para-chlorophenylalanine treatment; in the hypothalamic paraventricular nucleus for example, c-fos messenger RNA signal and the number of Fos-immunoreactive positive cells were reduced by 80 and 48%, respectively, in serotonin-depleted rats treated with the bacterial endotoxin. This blunted neuronal response was also associated with an attenuated stimulation of neuroendocrine corticotropin-releasing factor transcription and plasma corticosterone release. Indeed, lipopolysaccharide caused a selective expression of corticotropin-releasing factor primary transcript in the paraventricular nucleus of the hypothalamus and this effect was significantly reduced by treatment with the serotonin inhibitor. However, basal expression of corticotropin-releasing factor messenger RNA across the brain (bed nucleus of the stria terminalis, medial preoptic area, paraventricular nucleus of the hypothalamus, central nucleus of the amygdala, etc.) was not affected by the para-chlorophenylalanine treatment. These results suggest that the integrity of serotonin pathways plays a role in the neuronal activity triggered by the systemic endotoxin insult. The fact that serotonin depletion largely prevented activation of neurosecretory parvocellular neurons of the paraventricular nucleus of the hypothalamus and neuroendocrine corticotropin-releasing factor gene transcription in response to immunogenic challenge provides the evidence that serotonergic system is part of the brain circuitry involved in the corticotroph axis-immune interface.

A Frequent Regulatory Variant of the Estrogen-Related Receptor α Gene Associated With BMD in French-Canadian Premenopausal Women†

Genes are important BMD determinants. We studied the association of an ESRRA gene functional variant with BMD in 1335 premenopausal women. The ESRRA genotype was an independent predictor of L2‐L4 BMD, with an effect similar to smoking and equivalent to a 10‐kg difference in weight.