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The activation of nitric oxide (NO) signaling pathways in hypothalamic neurons plays a key role in the control of GnRH secretion that is central to reproductive function. It is unknown whether NO directly modulates the firing behavior of GnRH neurons in the preoptic region of the mature brain. Using patch-clamp recordings from GnRH neurons expressing green fluorescent protein in adult mice brain slices, we demonstrate that the NO precursor, l-arginine (Arg), or the NO donor, diethylamine/NO, induced a robust and reversible reduction in the spontaneous firing activity of GnRH neurons, including bursting activity. The effects of l-Arg were prevented by the NO synthase inhibitor Nω-nitro-l-Arg methyl ester hydrochloride. Histochemical studies revealing a close anatomical relationship between neurons producing NO and GnRH perikarya, together with the loss of the l-Arg-mediated inhibition of GnRH neuronal activity via the selective blockade of neuronal NO synthase, suggested that the primary source of local NO production in the mouse preoptic region was neuronal. Synaptic transmission uncoupling did not alter the effect of NO, suggesting that NO affects the firing pattern of GnRH neurons by acting at a postsynaptic site. We also show that the NO-mediated changes in membrane properties in the GnRH neurons require soluble guanylyl cyclase activity and may involve potassium conductance. By revealing that NO is a direct modulator of GnRH neuronal activity, our results introduce the intriguing possibility that this gaseous neurotransmitter may be used by the sexual brain to modulate burst firing patterns. It may set into phase the bursting activity of GnRH neurons at key stages of reproductive physiology.

Most adenoviral vectors (HAdvs) elaborated for gene therapy are derived from serotype 5 viruses that use clathrin-coated vesicle endocytosis for cell entry. However, it appears that adenoviral vectors are able to take advantage of lipid raft/caveolae endocytosis to infect cells. In vivo targeting of a therapeutic gene to specific cells by vector engineering has become a major focus of gene therapy research. Yet, modification of adenoviral tropism, especially fiber gene engineering, can induce deficient intracellular trafficking of the viral particle, with a shift in subcellular localization resulting in extensive exocytosis. In this study we demonstrate that uptake of a fiber-modified adenovirus using lipid raft/caveolae endocytosis leads to non-altered intracellular trafficking without endosomal retention. Moreover, activation of lipid raft structures by this vector leads to the formation of "mega-caveosomes". These results demonstrate that, by forcing adenoviruses to take advantage of a non-clathrin, non-classical endocytic pathway, it is possible to compensate for the deficiency in endosomolysis that is associated with the use of some of the fiber-modified adenoviral constructs. Moreover, it renders such vectors ideal candidates for infecting human coxsackie and adenoviruses receptor (hCAR) negative cells.

It is becoming increasingly apparent that non-neuronal cells play a critical role in generating and regulating the flow of information within the brain. Among these non-neuronal cells, astroglial cells have been shown to play important roles in the control of both synaptic transmission and neurosecretion. In addition to modulating neuronal activity, astroglial cells interact with endothelial cells throughout the central nervous system to define specific functional domains. In the hypothalamus, neurons that release gonadotropin-releasing hormone (GnRH), the neurohormone that controls both sexual development and adult reproductive function, offer an attractive model system in which to study glial-neuronal-endothelial interactions. Within the median eminence of the hypothalamus, alterations of the anatomical relationship that exists between GnRH axon terminals and ependymoglial cell processes belonging to tanycytes regulate the direct access of GnRH neurosecretory axons to the vascular wall. This cell plasticity presumably modulates the release of GnRH into the portal vasculature during the reproductive cycle. Both structural changes and GnRH secretory activity appear to be modulated, at least in part, by specific cell-cell signalling molecules secreted by astrocytes, tanycytes and endothelial cells. It is becoming increasingly clear that among the different factors that may be involved, glial cells use growth factor members of the epidermal growth factor (EGF) family, acting via receptors endowed with tyrosine kinase activity, to produce morphological changes and release neuroactive substances that directly excite nearby neurons, whereas endothelial cells of the median eminence employ nitric oxide to induce neuroglial plasticity and facilitate GnRH release.

Amyloid precursor protein (APP) metabolism is central to the pathogenesis of Alzheimer disease. We showed recently that the amyloid intracellular domain (AICD), which is released by gamma-secretase cleavage of APP C-terminal fragments (CTFs), is strongly increased in cells treated with alkalizing drugs (Vingtdeux, V., Hamdane, M., Bégard, S., Loyens, A., Delacourte, A., Beauvillain, J.-C., Buée, L., Marambaud, P., and Sergeant, N. (2007) Neurobiol. Dis. 25, 686-696). Herein, we aimed to determine the cell compartment in which AICD accumulates. We show that APP-CTFs and AICD are present in multivesicular structures. Multivesicular bodies contain intraluminal vesicles (known as exosomes) when released in the extracellular space. We demonstrate that APP, APP-CTFs, and AICD are integrated and secreted within exosomes in differentiated neuroblastoma and primary neuronal culture cells. Together with recent data showing that amyloid-beta is also found in exosomes, our data show that multivesicular bodies are essential organelles for APP metabolism and that all APP metabolites can be secreted in the extracellular space.

Terminally differentiated keratinocytes are dead enucleated squams. We showed previously that the mitochondria-dependent cell death pathway might be gradually activated as differentiation progresses. In this study, we demonstrated that protoporphyrin IX, staurosporine, and rotenone induced apoptotic-like changes in the mitochondria, and early differentiation of keratinocytes without inducing apoptosis. Kinetics studies established that differentiation-related changes, including growth arrest, flattened morphology, stratification, and keratin 10 (K10) expression, were downstream of mitochondrial depolarization and proliferation, reactive oxygen species (ROS) production, and release of cytochrome c and apoptosis-inducing factor. When these changes were prevented by overexpressing Bcl-2 or pharmacologically decreasing the ROS level, K10 upregulation was inhibited, implying that the differentiated phenotype and K10 expression require apoptotic mitochondria, ROS being the most likely differentiation-mediating factor. Our data also suggest that the same mitochondria-affecting stimuli can induce either differentiation or apoptosis, depending on the keratinocyte's competency to undergo differentiation, a competency that may be controlled by Bcl-2.

Doxorubicin is one of the most largely prescribed antitumor drug for the treatment of breast, liver and colon cancers as well as leukemia, but the cardiotoxicity of this anthracycline derivative limits its clinical use. Although doxorubicin is toxic to both cancer and cardiac cells, there are evidences suggesting that the mechanism of cell death is different for the two cell types. To investigate further this issue, we have compared the proapoptotic effects of doxorubicin and the functionally related anthracenedione compound mitoxantrone, which is also used in the clinic for the treatment of cancer. After evaluating the toxicity of the two drugs to mammary adenocarcinoma MTLn3 cells and H9C2 cardiomyocytes, we dissected the drug-induced apoptotic machinery by measuring the effects on the cell cycle progression, DNA condensation and fragmentation, production of endogenous peroxides and caspase activation. Both doxorubicin and mitoxantrone are potent inducers of apoptosis in H9C2 cardiomyocytes and MTLn3 breast cancer cells, but there are significant differences between the two cell types in terms of kinetics and order of the events. In particular, flow cytometry measurements of drug-induced changes in mitochondrial transmembrane potential and mitochondrial mass with different fluorescent probes suggested that the two drugs induced a progressive increase in mitochondrial mass in the cancer cells but not in the cardiac cells. The hypothesis was validated by means of electron microscopy, which revealed a significant increase in the number of mitochondria in drug-treated MTLn3 but not in H9C2 cells. The mitochondrial proliferation precedes the nuclear apoptosis in doxorubicin-treated MTLn3 cells. The changes in the architecture and number of mitochondria are linked to the drug-induced perturbation of the cell cycle progression and apoptosis. The proliferation of mitochondria could explain the higher toxicity of doxorubicin to cancer cells compared to cardiac cells and this suggests novel therapeutic opportunities to better control the cardiotoxicity of anthracyclines.

In vitro studies using immortalized GT1 cells suggest that hypothalamic astrocytes employ TGFbeta(1) to directly regulate the secretion of GnRH, the neurohormone that controls sexual maturation and adult reproductive function. However, whether such astrocyte-GnRH neuron signaling occurs in vivo is not clear. In the present study, we used in situ hybridization and immunohistochemistry to determine whether astrocytes and GnRH neurons express the molecular components necessary to set in motion communication processes involving TGFbeta(1) signaling. Double-labeling experiments showed that astrocytes in the male rat preoptic region (POA) expressed TGFbeta(1) mRNA and that GnRH perikarya were often found in close association with TGFbeta(1) mRNA-expressing cells. In addition, GnRH neuronal cell bodies in the POA expressed both type II TGFbeta receptors (TGFbeta-RII), which selectively bind TGFbeta, and Smad2/3, one of the primary transducers of TGFbeta signaling, suggesting that they are fully capable of responding directly to TGFbeta(1) stimulation. Consistent with this hypothesis, incubation of POA explants with TGFbeta(1) caused a significant, dose-dependent decrease in GnRH mRNA expression in individual neurons. This effect was observed within 1 h after TGFbeta(1)-treatment and was inhibited by addition of the soluble form of TGFbeta-RII to the incubation medium. In contrast, whereas both TGFbeta(1) and TGFbeta-RII mRNAs were abundantly expressed in both glial cells and capillaries in the median eminence, the projection field of GnRH neurons, TGFbeta-RII immunoreactivity was mainly restricted to the processes of tanycytes and did not colocalize with GnRH-immunoreactive fibers. This observation supports previous in vivo studies showing that TGFbeta(1) is unable to directly modulate decapeptide release from GnRH nerve terminals. Thus, astrocyte-derived TGFbeta(1) may directly influence GnRH expression and/or secretion in vivo by acting on the perikarya, but not the terminals, of GnRH neurons.

De nombreux travaux publies ces dix dernieres annees demontrent que la secretion de GnRH est susceptible d’etre modulee par des facteurs provenant de cellules gliales mais aussi de l’endothelium des capillaires du plexus porte. Parmi les facteurs gliaux susceptibles d’intervenir, on peut distinguer le TGFβ qui peut agir directement sur les cellules a GnRH et les EGFs qui exercent leurs actions clairement stimulantes via la secretion de prostaglandine par les cellules astrocytaires voisines. Pour l’endothelium, il a ete montre qu’il pouvait favoriser la liberation de GnRH via la secretion de NO. En parallele a ces actions, la glie et l’endothelium sont capables de moduler la secretion de GnRH en favorisant l’acces des terminaisons nerveuses a l’espace pericapillaire afin de faciliter le deversement de la neurohormone dans le sang porte. De fait, les terminaisons ne sont en situation de pouvoir deverser leur contenu que l’apres midi du prooestrus, periode ou un pic de GnRH est necessaire afin de provoquer le pic preovulatoire de LH. Cette observation suggere qu’il existe une plasticite morphologique de l’eminence mediane mettant en cause des mouvements des tanycytes (ependymocytes de l’eminence mediane). Cette plasticite peut etre induite par des facteurs de croissances gliaux (TGFβ et TGFα) et par le NO d’origine endotheliale. Au site de liberation de la GnRH, l’interaction neuro-glio-endotheliale apparait donc un element cle de la regulation de secretion de GnRH.

We have recently shown that the mRNA encoding for a type I receptor for transforming growth factor beta and activin – named B1 – is expressed in hypothalamic areas implicated in gonadotropin-releasing hormone regulation, particularly in estrogen-receptive regions. In the present study, we examined whether ovarian steroids may regulate expression of B1 mRNA in the hypothalamus. Comparing relative levels of B1 mRNA expression in ovariectomized (OVX), OVX + estradiol-treated, and OVX + estradiol + progesterone-treated female rats, we observed that estrogen significantly (p < 0.05) downregulated B1 mRNA levels in the anteroventral periventricular nucleus (by 12.5%), medial preoptic nucleus (by 27.5%), and arcuate nucleus (by 29.5%). In contrast, no effects of gonadal steroids were observed in the median preoptic nucleus. We next examined whether cells expressing B1 mRNA may be direct targets for the action of estrogen. Using an in situ hybridization coupled to immunohistochemical labeling, we found that many B1-mRNA-expressing cells also exhibited estrogen receptor alpha immunoreactvity in anteroventral periventricular nucleus, medial preoptic nucleus, and arcuate nucleus. Taken together, these results reveal that estrogen may directly modulate expression of B1 mRNA in the hypothalamus and support the idea that transforming growth factors beta play an important role in the hypothalamic control of gonadotropin-releasing hormone function.

The role of nitric oxide (NO) from vascular endothelium in the control of GnRH release at the median eminence (ME) level is well established. Interactions between NPY receptor/endothelium/nitric oxide are clearly demonstrated. While several studies implicate NPY Y1 receptor in the control of GnRH/LH at the time of the preovulatory LH surge, our results also demonstrate the importance of NPY Y2 receptor in the control of GnRH release via endothelial NO. We conclude that NPY may be one of the elements implicated in the generation of the spontaneous NO/GnRH via Y2 receptor located on endothelium.

The involvement of nitric oxide (NO) as a gaseous neurotransmitter in the hypothalamic control of pituitary LH secretion has been demonstrated. NO, as a diffusible signaling gas, has the ability to control and synchronize the activity of the neighboring cells. NO is secreted at the median eminence (ME), the common termination field for the antehypophysiotropic neurons, under the stimulation of other signaling substances. At the ME, NO stimulates GnRH release from neuroendocrine terminals. The present studies were undertaken to determine whether NO is secreted spontaneously from ME fragments ex vivo and whether its secretion is correlated to GnRH release. To accomplish this, female rats were killed at different time points of the day and/or of the estrous cycle. The spontaneous NO release was monitored in real time, with an amperometric probe, during 4 periods of 30 min, from individual ME fragments (for each time point, n = 4). GnRH levels were measured in parallel for each incubation-period by RIA. The results revealed that NO was released in a pulsatile manner from female ME fragments and, unambiguously, that the amplitude of NO secretion varied markedly across the estrous cycle. Indeed, though the NO pulse period (32 +/- 1 min, n = 36) and duration (21 +/- 2 min, n = 36) did not vary significantly across the estrous cycle, the amplitude of this secretion pulse was significantly higher on proestrus (Pro; 39 +/- 3 nM, n = 20), compared with diestrus (16 +/- 1 nM, n = 8) or estrus (23 +/- 3 nM, n = 8, P0.05). The GnRH levels in the incubation medium were positively correlated to NO secretion across the estrous cycle (r = 0.86, P0.003, n = 9), confirming that NO and GnRH release are coupled. Furthermore, 5 x 10(-7) M L-N(5)-(1-iminoethyl)ornithine (L-NIO), a NO synthase inhibitor, succeeded in inhibiting the strong NO-GnRH secretory coupling and GnRH release on PRO: Because at this concentration, L-NIO selectively inhibits endothelial NO synthase, the results further demonstrate that the major source of NO involved in GnRH release at the ME is endothelial in origin. Additionally, the induction of a massive NO/GnRH release in 15-day ovariectomized rat treated with estradiol benzoate strongly suggested that estradiol is participating in the stimulation of NO release activity between diestrus II and PRO: The present study is the first demonstrating that ME can spontaneously release NO and that NO's rhythm of secretion varies markedly across the estrous cycle. This pulsatile/cyclic ME NO release may constitute the synchronizing link to anatomically scattered GnRH neurons.

The effects of the polypeptide hormone prolactin (PRL) in the development and regulation of benign prostate hyperplasia (BPH) and also in prostate cancer are not very well characterized. This study examines the action of PRL, either alone or in association with androgens [testosterone (T) or dihydrotestosterone (DHT)], in the rat prostate gland. The effects of PRL and androgens were investigated after 30 and 60 days in control, castrated, castrated with a substitutive implant of T or DHT, and sham-operated Wistar rats. To enhance PRL release, we induced hyperprolactinemia by administering chronic injections of sulpiride (40 mg · kg−1 · day−1). Chronic hyperprolactinemia induces enlargement and inflammation of the lateral rat prostate without any histological changes on ventral and dorsal lobes. We also demonstrate that hyperprolactinemia induces Bcl-2 overexpression in the lateral rat prostate and that this could inhibit the level of apoptosis. The in vivo model established here is a useful in vivo approach for studying the hormonal regulation of normal and pathological prostate development.

The forebrain and pituitary sites of synthesis of growth hormone secretagogue-receptor mRNA were identified in four adult lemurs (Microcebus murinus) by in situ hybridisation performed with a radiolabeled cRNA probe transcribed from human Growth Hormone Secretagogue-Receptor cDNA. The cRNA sense and antisense probes were hybridised to cryostat sections containing structures extending from the rostral hypothalamus to its caudal limit as defined by the mammillary bodies. The pituitary gland and areas adjacent to the hypothalamus were also analyzed. For comparative purposes, sections from five adult rats containing these structures were hybridised with the same probes. The results point to a widespread distribution of Growth Hormone Secretagogue-Receptor mRNA in the hypothalamus, hippocampal formation, and cerebellar cortex of both lemurs and rats. As in the rat, specific hybridisation was particularly dense in the arcuate nucleus. Significant species differences were observed in the periventricular nucleus, the ventromedial nucleus, the lateral hypothalamic area, and the pituitary gland. In contrast to the rat, the lemur exhibited marked labelling in the infundibular nucleus, the periventricular nucleus and the pars tuberalis of the pituitary gland, whereas no labeling was detectable in the ventromedial nucleus and the lateral hypothalamic area. These results are discussed in terms of difference between the control of growth hormone secretion, feeding behaviour and seasonal rhythmicity among murine species and primates.

Nitric oxide (NO) as well as beta-endorphin are involved in the neuroendocrine control of gonadotropin-releasing hormone (GnRH) secretion. Recently, morphological and microdialysis experiments have suggested that beta-endorphin may exert an inhibitory influence on NO release in the preoptic area of rat hypothalamus. The present study determines if the mu opioid receptor mRNA is expressed in neuronal NO synthase (nNOS)-immunopositive neurons and if this expression varies among the regions of the basal forebrain being examined. We found, through the use of immunohistochemical and in situ hybridization techniques, that the mu opioid receptor mRNA is expressed in a representative subpopulation of nNOS-immunoreactive neurons in the rat preoptic area. Interestingly, the mu opioid receptor mRNA/nNOS-immunoreactive coexpression is predominant in the rostral and median preoptic area, containing most of GnRH cell bodies. These results strongly suggest that beta-endorphin, via an action through mu opioid receptors, may directly participate in the regulation of NO production in the preoptic area. Our results strengthen the hypothesis that beta-endorphin may participate in GnRH neuronal modulation at the cell body level by regulating NO release from the interneurons of the preoptic area that express nNOS.

Previous studies have shown that galanin-containing fibers make synaptic contacts with POMC neurons in the arcuate nucleus. However, the ability of POMC neurons to express galanin receptors has never been assessed. The present study was designed to investigate whether POMC neurons express galanin receptor messenger RNA (mRNA) and whether testosterone could modulate galanin receptor gene expression. A dual-labeling in situ hybridization histochemistry, using 35S-labeled (galanin receptors GalR1 or GalR2) and digoxigenin-labeled (POMC) riboprobes, was performed on brain sections from intact, castrated, and testosterone-replaced adult male rats. For analysis, the arcuate nucleus was divided into four rostro-caudal areas. The results revealed that both GalR1 and GalR2 mRNAs were expressed in POMC neurons. Most POMC neurons expressing galanin receptor mRNAs were found in the rostral parts of the nucleus. Castration reduced the labeling density of galanin receptor mRNAs in POMC neurons, and testosterone prevented the effects of castration in all rostro-caudal subdivisions of the arcuate nucleus. Taken together, these data indicate that galanin can directly modulate the activity of POMC neurons, via an action on GalR1 or GalR2 receptors, particularly in the rostral-arcuate nucleus. In addition, testosterone can modulate the expression of GalR1 and GalR2. Because POMC neurons located in the rostral part of the nucleus are known to project preferentially to the preoptic area, POMC neurons expressing the galanin receptor genes may play an important role in the regulation of the GnRH neuroendocrine axis.

The distribution of galanin receptor subtype 2 (Gal-R2) mRNA-expressing cells was examined by in situ hybridization in the rat hypothalamus using a full-length rat 35S-riboprobe. Gal-R2 receptor mRNA-expressing cells were found at moderate to high levels of expression in most nuclei and regions of hypothalamus. The labeling was observed within well-defined anatomical nuclei: preoptic, suprachiasmatic, periventricular, paraventricular, arcuate, dorsomedial, mammillary nuclei. The supraoptic and ventromedial nuclei were almost devoid of labeling. Some scattered labeled cells were also observed in the pituitary. This distribution of Gal-R2 mRNA-expressing cells corresponds well with that of galanin binding sites studies. As compared to the distribution of the galanin receptor subtype 1 (Gal-R1), our results indicate that the Gal-R2 type is differentially distributed, although a significant overlap exists in some regions such the preoptic area, arcuate and dorsomedial nuclei. The functional implications of these results are discussed in light of the role of galanin receptors plays in neuroendocrine regulation and feeding behavior.

By in situ hybridization, we show the ability of human neuroblastoma SY 5Y cells to synthesize apolipoprotein E (apoE) mRNA. This synthesis varied during cell NGF-differentiation: the mRNA level decreased during the first 4 days of NGF treatment (NGF 4 days) and then increased during the 3 following days (NGF 7 days). Furthermore, a treatment of 4-day NGF differentiated cells with exogenous apoE during 3 additional days induced a clear decrease in apoE mRNA synthesis when compared with control cells. This effect was more or less pronounced according to the apoE tested variants: apoE4 was more efficient to decrease the apoE mRNA synthesis as compared with the control cells than apoE3 which was itself more efficient than apoE2. These results suggest that apoE mRNA synthesis in human neuronal-type cells could be regulated by different mechanisms such as those induced by NGF- and apoE-treatments.

The present investigation concerns 80–90 day-old male rats born from morphine-exposed mothers (2×10 mg/kg per day from days 11 to 18 of gestation which showed at birth reduced size and activity of the adrenals). This prenatal treatment did not significantly disturb under resting conditions: (1) the postnatal body growth up to week 10 after birth, (2) the activity of the pituitary gonadal axis (circulating luteinizing hormone (LH) and testosterone (T), weight of the testicles and seminal vesicles), (3) the activity of the hypothalamo-pituitary-adrenal axis (HPA) (hypothalamic corticoliberin (CRF) content, plasma adrenocorticotrophic hormone (ACTH) level, adrenal weight and corticosterone (B) content, plasma B level) as well as B max and K d of mineralocorticoid (type I) and glucocorticoid (type II) receptors to B in both the hippocampus and the hypothalamus. In contrast these rats showed reduced content of adrenals in noradrenaline (NA) and adrenaline (A) but increased circulating levels of A.

Following treatment with interleukin-10 (IL-10), basal corticotrophin releasing factor (CRF) levels from rat hypothalamic median eminence (ME) were found to be increased. Our data show: (1) the specificity of stimulation of CRF through the use of recombinant IL-10 and its blockage by monoclonal anti-IL-10 antibody; (2) the requirement of NO in this process through the use of N ω -nitro- l -arginine methyl ester ( l -NAME), a nitric oxide synthase inhibitor; (3) the blockage of IL-10 stimulated NO production by anti-IL-10; and, (4) the presence of IL-10 transcripts in hypothalamic poly A + mRNA. These results provide the first evidence of IL-10 acting in the ME to influence CRF levels and further support our earlier findings of a potential for IL-10 in the hypothalamic-pituitary axis.

Variations in the number of galanin receptor (Gal-R1)-expressing cells and levels of Gal-R1 messenger RNA (mRNA) were determined in the preoptic area in intact female rats throughout the phases of the estrous cycle and compared with those in the male. Female and male Wistar rats were fixed by perfusion with 4% paraformaldehyde. Cryostat sections were hybridized with a 35S-labeled antisense Gal-R1 riboprobe. The number of Gal-R1 mRNA-expressing cells was lower in the rostral preoptic area than in the medial preoptic area. During the estrous cycle, the highest number of Gal-R1 mRNA-expressing cells in the rostral preoptic region was detected at 0800 h on proestrus, whereas in the medial preoptic area, the maximum number was observed at 1800 h on estrus. Gal-R1 mRNA levels in individual cells were low during diestrus and increased at estrus in both areas. In the male, the number of mRNA-expressing cells and the hybridization signal were significantly lower than those in females during estrus. The results dem...

Nitric oxide (NO) is involved in neurohormonal secretion from median eminence neuroendocrine nerve terminals. We report that stimulation of NO release from median eminence fragments including vascular tissues occurs by μ3 receptor activation by morphine, or by cannabinoid type 1 receptor activation by anandamide. The released levels of NO are lower after anandamide than after morphine stimulation. These processes can be blocked by L-NAME, a specific nitric oxide synthase inhibitor, by naloxone for the morphine-stimulated NO release, or SR 141716A, a specific CB1 receptor inhibitor, for the anandamide-stimulated NO release. Furthermore, morphine and anandamide, by this NO dependent process, influences neurohormonal release from median eminence nerve terminals within 10 min. Via this NO dependent process, morphine stimulates both GnRH and CRF release, whereas anandamide selectively stimulates GnRH release. These observations together with previous data suggest that morphine and the anandamide-stimulated NO originates from the vascular endothelium of the portal plexus. These results indicate that endothelial cells of the median eminence may be involved in the release of neurohormones.

The osmoregulator peptide (leech osmoregulatory factor, LORF; IPEPYVWD) was first found in the leech central nervous system (CNS). Given the fact that certain peptides can be found in mammals and invertebrates, e.g., opioid, we examined rat brains to determine if LORF was present. This peptide was found and isolated by successive reversed-phase HPLC purification steps and characterized by electrospray mass spectrometry measurement. It was sequenced by Edman degradation and quantified in different tissues by ELISA. Our results demonstrate the presence of LORF in the hypothalamus, thalamus, and striatum (6 pmol/mg of protein extract) and in other brain areas at lower levels. This octapeptide is also present in the rat duodenum and liver (10 to 14 pmol/mg) and at lower levels in heart, lung, pancreas and caudal spinal cord (5 pmol/mg). The testes, adrenals and kidneys have the lowest levels of all the tissues examined (ca. 0.5 pmol/mg of protein). Furthermore, we also demonstrate that LORF is coupled to nitric oxide (NO) release in leech CNS, rat hypothalamus and human saphenous vein in a manner which is inhibited by a nitric oxide synthase inhibitor as well as an antibody directed toward LORF. The study demonstrates that LORF, and its function in relation to NO release, has been conserved over more than 400 million years of evolution.

The rat TRH precursor contains five copies of TRH separated by connecting peptides. Previous studies have shown that the decapeptide prepro-TRH (160-169; Ps4) potentiates the effect of TRH on TSH secretion. In the present study, we have characterized Ps4 receptors in the rat pituitary by in vitro autoradiography using [125I-Tyr0]Ps4 as a radioligand, and we have investigated the evolution of receptor density during ontogenesis. Incubation of rat pituitary slices with [125I-Tyr0]Ps4 revealed intense binding in the anterior lobe and virtually no binding in the neurointermediate lobe. Biochemical characterization of the Ps4-binding sites suggested the existence of a single class of sites exhibiting high affinity for [Tyr0]Ps4 (IC50 = 8.3 +/- 1.2 nM) and a much lower affinity for Ps4 (IC50 = 9.3 +/- 1.2 microM). Emulsion-coated cytoautoradiography performed on cultured anterior pituitary cells showed that only 26% of the cells possessed [125I-Tyr0]Ps4-binding sites. Immunocytochemical analysis using antibodies against the different anterior pituitary hormones indicated that the cells possessing [125I-Tyr0]Ps4-binding sites did not correspond to TSH-, PRL-, GH-, ACTH-, or LH-secreting cells. In contrast, cells expressing Ps4 receptors were immunoreactive for the S-100 protein, a marker of folliculo-stellate cells. During postnatal development, a 4-fold increase in the concentration of [125I-Tyr0]Ps4-binding sites occurred from birth to weaning in the pituitary, with a marked and transient increase at the time of weaning. Thereafter, the density of sites declined gradually until day 60. In conclusion, the present study shows that folliculo-stellate cells express [125I-Tyr0]Ps4-binding sites in the anterior pituitary, and that these sites are developmentally regulated. The present data suggest that the potentiating effect of Ps4 on TRH-induced TSH secretion is mediated by folliculo-stellate cells.

Using a combination of radioactive and non-radioactive in situ hybridizations, the expression of mu and kappa opioid receptor mRNA was investigated in neurons in the female rat preoptic nucleus expressing galanin and gonadotropin-releasing hormone (GnRH) mRNA. Numerous cells expressing both mu or kappa and galanin were found in the intermediate and rostral regions of the preoptic area whereas little co-localization was observed at the rostral level. The number of kappa/galanin expressing cells was greater than that of mu/galanin cells. mu/galanin co-localization was observed essentially in the anteroventral preoptic nucleus while neurons expressing kappa/galanin were present in both the anteroventral preoptic nucleus and in the periventricular hypothalamic nucleus. Co-localization of GnRH with mu or kappa could not be detected in the preoptic area. These observations showed that galaninergic neurons but not GnRH neurons of the preoptic area might be directly regulated by endogenous opioid peptides.

The aim of this work was to compare the localization of galanin and galanin receptors in the guinea pig median eminence at the light and electron microscopic level. Concerning galanin the highest labeling was shown in the external part of the median eminence. At the ultrastructural level, galanin immunoreactivity was observed only in nerve terminals containing granular vesicles of approximately 120 nm in diameter. Light microscopic autoradiographs of semithin sections exhibited a moderate labeling in the external part of the median eminence. Galanin receptors were labeled in vitro on semithin sections (2 microm) using the highly specific radioligand [125I]galanin. Ultrastructural data showed that most of galanin binding sites overlaid membrane appositions between nerve terminals and also between nerve terminal and tanycyte. By considering the percentages in the distribution of the binding it appeared that galanin receptors were located on some nerve ending membranes. Our observations were not really in favor of a presence of receptors in tanycytes. The presence of galanin nerve endings in the external part suggests that like in the rat the peptide may have a direct hypophysiotrophic role. In contrast, the occurrence of numerous binding sites gives additional arguments in favor of a local action (paracrine and/or autocrine) of galanin occurring via galanin receptors located essentially on the pericapillary nerve terminals in the guinea pig median eminence.

Galanin is a 29-amino acid peptide widely distributed in the mammalian central nervous system. Galanin receptors in the guinea pig brain were visualized using [125I]galanin by in vitro receptor quantitative autoradiography. Scatchard analysis of [125I]galanin binding to slide-mounted sections revealed saturable binding to a single class of high affinity receptors with a KD of approximately 1 nM. Specific [125I]galanin binding sites were detected in a large number of brain areas (concentration range: from non detectable to 99.32 fmol/mg of tissular proteins). The anatomical mapping revealed high densities essentially in the telencephalon (e.g. lateral septal nuclei, amygdala, hippocampal dentate gyrus) and the diencephalon (e.g. the anterodorsal and medial habenular thalamic nuclei, the paraventricular, dorsomedian and median mammillary hypothalamic nuclei, the posterior lobe of the pituitary). Addition of Mg2+ and GTP increased binding in some areas such as the zona incerta, the median eminence and the arcuate nucleus, and decreased it in other areas such as the amygdala, the hippocampus and the mammillary nuclei. This regional heterogeneity in the effect of Mg2+ and GTP can be interpreted as: (1) different rates of galanin receptor occupancy by endogenous peptide; (2) a differential coupling of GTP binding proteins to galanin receptors in the brain structures; and (3) a different nature of receptors. At any rate, this study provides evidence for a specific GTP-sensitive galanin receptor in guinea pig brain with an extensive distribution suggesting various physiological implications. Comparison with studies performed in several mammals shows that the overall distribution of galanin receptors is well preserved among species. These data suggest that galanin may posses similar functional properties in the different species tested so far. Nevertheless, very distinct differences were found in some areas like the cortex, the hippocampus and the pituitary.

Cells immunoreactive to an antiserum specifically directed against vertebrate alpha-Neo-endorphin (alpha-NE) were detected in the internal wall of anterior and posterior suckers of the rhynchobdellid leech Theromyzon tessulatum. These cells have morphological and ultrastructural characteristics close to the "releasing gland cells" of adhesive organs. The epitope recognized by anti-alpha-NE was contained in granules having a diameter of 0.2-0.3 microm. Previous works involving the brain of this leech demonstrate the existence of approximately 14 neurons immunoreactive to the anti-alpha-NE. Following an extensive purification including high pressure gel permeation and reversed-phase high performance liquid chromatography, epitopes contained in both suckers and central nervous system were isolated. Purity of the isolated peptides was controlled by capillary electrophoresis. Their sequences were determined by a combination of automated Edman degradation, electrospray mass spectrometry measurement, and coelution experiments in reversed-phase high performance liquid chromatography with synthetic alpha-NE. The results demonstrate that epitopes recognized by the anti-alpha-NE in the suckers and the central nervous system are identical to vertebrate alpha-NE (YGGFLRKYPK). This finding constitutes the first biochemical characterization of a prodynorphin-derived peptide in invertebrates. Moreover the isolation of this peptide in the annelida establishes the very ancient phylogenetic origin of alpha-NE as well as its conservation in evolution.

Neutral endopeptidase (E.G. 3.4.24.11) was visualized at the ultrastructural level in the external zone of the rat median eminence by using 125|-labelled IgG of a monoclonal serum. A precise analysis of the localization of the immunolabelling, which appears in the form of individual stray silver grains, was undertaken. Among the 1,045 grains counted, 82% were localized over membrane appositions involving nerve endings only and nerve endings plus tanycytes. The difference between the real and a randomly generated population of grains was statistically significant. Our results provide morphological arguments in support of the view of a paracrine action of neuropeptides present in the median eminence especially enkephalins but possibly, substance P, angiotensin, cholecystokinin and neurotensin. These neuropeptides are known to be inactivated by neutral endopeptidase. The action of these peptides may be exerted on nerve endings (autocrine or paracrine) but an intervention on tanycytes cannot be excluded.

The role of Tau phosphorylation in neurofibrillary degeneration linked to Alzheimer's disease remains to be established. While transgenic mice based on FTDP-17 Tau mutations recapitulate hallmarks of neurofibrillary degeneration, cell models could be helpful for exploratory studies on molecular mechanisms underlying Tau pathology. Here, "human neuronal cell lines" overexpressing Wild Type or mutated Tau were established. Two-dimensional electrophoresis highlights that mutated Tau displayed a specific phosphorylation pattern, which occurs in parallel to the formation of Tau clusters as visualized by electron microscopy. In fact, this pattern is also displayed before Tau pathology onset in a well established mouse model relevant to Tau aggregation in Alzheimer's disease. This study suggests first that pathological Tau mutations may change the distribution of phosphate groups. Secondly, it is possible that this molecular event could be one of the first Tau modifications in the neurofibrillary degenerative process, as this phenomenon appears prior to Tau pathology in an in vivo model and is linked to early steps of Tau nucleation in Tau mutants cell lines. Such cell lines consist in suitable and evolving models to investigate additional factors involved in molecular pathways leading to whole Tau aggregation., info:eu-repo/semantics/published

International audience; 26RFa is a novel RFamide peptide originally isolated in the amphibian brain. The 26RFa precursor has been subsequently characterized in various mammalian species but, until now, the anatomical distribution and the molecular forms of 26RFa produced in the CNS of mammals, in particular in human, are unknown. In the present study, we have investigated the localization and the biochemical characteristics of 26RFa-like immunoreactivity (LI) in two regions of the human CNS--the hypothalamus and the spinal cord. Immunohistochemical labeling using specific antibodies against human 26RFa and in situ hybridization histochemistry revealed that in the human hypothalamus 26RFa-expressing neurons are located in the paraventricular and ventromedial nuclei. In the spinal cord, 26RFa-expressing neurons were observed in the dorsal and lateral horns. Characterization of 26RFa-related peptides showed that two distinct molecular forms of 26RFa are present in the human hypothalamus and spinal cord, i.e. 26RFa and an N-terminally elongated form of 43 amino acids designated 43RFa. These data provide the first evidence that 26RFa and 43RFa are actually produced in the human CNS. The distribution of 26RF-LI suggests that 26RFa and/or 43RFa may modulate feeding, sexual behavior and transmission of nociceptive stimuli.

Lamellarin D is a marine alkaloid with a pronounced cytotoxicity against a large panel of cancer cell lines and is a potent inhibitor of topoisomerase I. However, lamellarin D maintains a marked cytotoxicity toward cell lines resistant to the reference topoisomerase I poison camptothecin. We therefore hypothesized that topoisomerase I is not the only cellular target for the drug. Using complementary cell-based assays, we provide evidence that lamellarin D acts on cancer cell mitochondria to induce apoptosis. Lamellarin D, unlike camptothecin, induces early disruption of the inner mitochondrial transmembrane potential (Δψm) in the P388 leukemia cell line. The functional alterations are largely prevented by cyclosporin A, an inhibitor of the mitochondrial permeability transition (MPT), but not by the inhibitor of caspases, benzyloxycarbonyl-Val-Ala-Asp(Ome)-fluoromethylketone. Δψm disruption is associated with mitochondrial swelling and cytochrome c leakage. Using a reliable real-time flow cytometric monitoring of Δψm and swelling of mitochondria isolated from leukemia cells, we show that lamellarin D has a direct MPT-inducing effect. Furthermore, mitochondria are required in a cell-free system to mediate lamellarin D–induced nuclear apoptosis. The direct mitochondrial effect of lamellarin D accounts for the sensitivity of topoisomerase I–mutated P388CPT5 cells resistant to camptothecin. Interestingly, a tumor-active analogue of lamellarin D, designated PM031379, also exerts a direct proapoptotic action on mitochondria, with a more pronounced activity toward mitochondria of tumor cell lines compared with nontumor cell lines. Altogether, this work reinforces the pharmacologic interest of the lamellarins and defines lamellarin D as a lead in the search for treatments against chemoresistant cancer cells. (Cancer Res 2006; 66(6): 3177-87)

The amyloid precursor protein (APP) metabolism is central to pathogenesis of Alzheimer's disease (AD). Parenchymal amyloid deposits, a neuropathological hallmark of AD, are composed of amyloid-beta peptides (Abeta). Abeta derives from the amyloid precursor protein (APP) by sequential cleavages by beta- and gamma-secretases. Gamma-secretase cleavage releases the APP intracellular domain (AICD), suggested to mediate a nuclear signaling. Physiologically, AICD is seldom detected and thus supposed to be rapidly degraded. The mechanisms responsible of its degradation remain unknown. We used a pharmacological approach and showed that several alkalizing drugs induce the accumulation of AICD in neuroblastoma SY5Y cell lines stably expressing APP constructs. Moreover, alkalizing drugs induce AICD accumulation in naive SY5Y, HEK and COS cells. This accumulation is not mediated by the proteasome or metallopeptidases and is not the result of an increased gamma-secretase activity since the gamma-secretase cleavage of Notch1 and N-Cadherin is not affected by alkalizing drug treatments. Altogether, our data demonstrate for the first time that alkalizing drugs induce the accumulation of AICD, a mechanism likely mediated by the endosome/lysosome pathway.

We have studied the mature Alzheimer-like fibers of tau by fluorescence and NMR spectroscopy. Assembly of the protein into paired helical filaments after incubation with heparin at 37 degrees C was verified by electron microscopy and size-exclusion chromatography. NMR spectroscopy on these mature fibers revealed different regions of residual mobility for tau: the N-terminal domain was found to maintain solution-like dynamics and was followed by a large domain of decreasing mobility; finally the core region was distinguished by a solid-like character. Heteronuclear-NOE data indicate that the decreasing mobility is due to both a slowing down of the rapid nanosecond movements and the introduction of slower movements that lead to exchange broadening. Fluorescence spectroscopy confirmed the presence of this rigid core, and some degree of protection from hydrogen exchange for those residues was observed. Hence, our data give a more precise picture of the dynamics of tau when it is integrated into mature filaments and should provide further understanding of the molecular processes that govern aggregation.

During the last decade, compelling evidence has been provided that, in addition of being regulated by transsynaptic inputs, GnRH neuroendocrine secretion is modulated by factors released both by glial cells and the endothelium of pituitary portal blood vessels. Glial cells exert their regulatory influence on GnRH release through the secretion of growth factors, such as TGFbetas and peptides member of the EGF family, that act either directly on GnRH neurons or require prostaglandin release from astrocytes, respectively. On the other hand vascular endothelial cells stimulate GnRH release via NO secretion. In addition, recent studies suggest that both glial cells and endothelial cells of the median eminence can modulate the direct access of GnRH neuroendocrine terminals to the vascular wall and thus control GnRH release efficiency. During the reproductive cycle, direct neurovascular contacts of GnRH nerve endings, that are engulfed in tanycytic endfeet, only occur at periods when massive GnRH release is required, i.e., at the onset of the preovulatory GnRH/LH surge on the day of proestrus. Recent in vitro and in vivo data demonstrate that both glial (TGFalpha and TGFbeta) and endothelial (NO) factors can induce such morphological plasticity. Neuro-glio-endothelial interactions at the median eminence of the hypothalamus thus appear to be key regulatory mechanisms for GnRH neuroendocrine secretion.

The purpose of the present study was to determine whether the septo-preoptico-tuberoinfundibular gonadotropin-releasing hormone (GnRH) pathway comes in close juxtaposition with tyrosine hydroxylase immunoreactive (TH-IR) neurons in the arcuate nucleus of female mice. Immunohistochemical staining with a TH monoclonal antibody coupled with confocal microscopy was employed on vibratome-cut brain sections of female GnRH-green fluorescent protein (GFP) transgenic mice to evaluate possible appositions between GnRH and tuberoinfundibular dopaminergic (TIDA) neurons. TH-IR neurons of the arcuate nucleus received GnRH neuronal appositions in adult female mice at proestrus and estrus stages. In contrast, no GnRH appositions were observed in adult females at diestrus. Subsequently, double immunohistochemical staining for TH and estrogen receptor-α (ERα) was performed to examine the role of estradiol on this relationship. We found that most TH-IR neurons contacted by GnRH fibers were immunoreactive for ERα. Our observations suggest that GnRH neurons communicate directly with TIDA neurons in the adult female. Furthermore, ERα activation in TIDA neurons may be involved in the formation of connections between GnRH neurons and TIDA neurons.

Recent studies from our laboratory suggested that the vascular endothelium of the median eminence was involved via nitric oxide secretion in the modulation of GnRH release during the estrous cycle. To further investigate that issue, we studied the variations of endothelial nitric oxide synthase protein and mRNA in the median eminence of female rats killed at different time points of the day and/or of the estrous cycle. Endothelial nitric oxide synthase protein levels were measured by Western blot, and endothelial nitric oxide synthase mRNA analysis was performed with semiquantitative RT-PCR (for each time point, n = 4). The results revealed that endothelial nitric oxide synthase synthesis varied markedly across the estrous cycle. Indeed, endothelial nitric oxide synthase protein (n = 20) and mRNA (n = 16) levels increase significantly on 0800 h and 1600 h proestrus compared with 1400 h diestrus II. In a second step, quantification analysis were made in median eminence obtained from ovariectomized and ovariectomized, E2 benzoate primed rat. The results show a significant increase in expression of endothelial nitric oxide synthase protein as well as endothelial nitric oxide synthase mRNA in ovx-E2 primed rat median eminence. Concurrently, the levels of the cav-1 protein, a specific endogenous inhibitor of endothelial nitric oxide synthase, were measured in median eminence during estrous cycle and in ME from ovx and ovx-E2 primed rats. A significant decrease of median eminence cav-1 was noted on 1600 h proestrus and in ovx-E2 primed rats when compared with 1400 h diestrus II and ovx, respectively. Altogether, these results strongly suggest that high NO release from median eminence observed on proestrus may be due to an increase of endothelial nitric oxide synthase expression and a decrease of the cav-1 protein levels. These findings demonstrate that E2 is able to modulate endothelial nitric oxide synthase and cav-1 expression both during the estrous cycle and in experimental conditions and consequently reinforce the idea that nitric oxide acting on GnRH release, is essentially endothelial in origin. These results may also imply that variations of endothelial nitric oxide synthase expression are essential for the pulsatile/cyclic nitric oxide median eminence release observed in a previous study.

The purpose of the present study was to determine whether TGF beta, a cytokine secreted by hypothalamic astrocytes, was able to regulate POMC neurons in the arcuate nucleus. In a first set of experiments, mediobasal hypothalamic fragments were exposed to TGF beta(1), and the relative POMC mRNA expression was assessed by in situ hybridization using a radiolabeled POMC riboprobe. The results showed that 4 x 10(-10) M TGF beta(1) was efficient in decreasing significantly the amounts of POMC mRNA (P < 0.01). Interestingly, the decrease of relative POMC mRNA levels was higher in the rostral than in the caudal parts of the arcuate nucleus. In a second set of experiments, we examined the occurrence of TGF beta receptors expression in arcuate POMC neurons. Dual labeling in situ hybridization and in situ hybridization, coupled to immunohistochemical labeling, were performed to examine mRNA expression of the type I serine-threonine kinase receptor for TGF beta and the presence of type II receptor for TGF beta, respectively, in POMC neurons. The results indicated that TGF beta receptor I mRNA and TGF beta receptor II protein were expressed in numerous POMC neurons. Regional analysis revealed that the highest proportion of POMC neurons expressing TGF beta receptors was located in the rostral part of the arcuate nucleus. Using dual labeling immunohistochemistry, we also found that Smad2/3 immunoreactivity, a TGF beta(1) downstream signaling molecule, was present in the cytoplasm and nucleus of some POMC (beta-endorphin) neurons. We next examined whether the number of POMC neurons expressing TGF beta-RI mRNA was affected by sex steroids. Quantification of the number of POMC neurons expressing TGF beta receptor I mRNA in ovariectomized, ovariectomized E2-treated, and ovariectomized E2 plus progesterone-treated animals revealed that estrogen treatment decreased the expression of TGF beta receptor I mRNA in POMC neurons located in the rostral half of the arcuate nucleus, an effect reversed by progesterone in a subset of the most rostral cells. Taken together, these data reveal that TGF beta(1) may directly modulate the activity of POMC neurons through the activation of TGF beta receptors. Therefore, the present study provides additional evidence for the involvement of TGF beta(1) in the regulation of neuroendocrine functions and supports the existence of a glial-to-neurons communication within the arcuate nucleus.

The present investigation concerns 80–90-day-old female rats born from morphine-exposed mothers (2×10 mg/kg per day from day 11–18 of gestation) or saline-treated ones (controls). The former showed reduced size and activity of the adrenals at birth. At adult stage, they present: (1) higher increase of plasma adrenocorticotrophic hormone level on proestrus; (2) significant rise of plasma corticosterone level on diestrus morning and estrus evening; (3) adrenal atrophy which was significant only on diestrus and estrus morning; (4) more corticosterone binding sites of type I (mineralocorticoid receptors) on proestrus morning in the hippocampus; (5) more corticosterone binding sites of type II (glucocorticoid receptors) in the hippocampus on proestrus morning and in the hypothalamus on estrus morning. In both experimental groups, B max for hypothalamic mineralocorticoid receptors were drastically higher on estrus morning than on the other stages of the estrous cycle. The activity of the pituitary–gonadal axis is poorly affected by prenatal morphine-exposition. In both experimental groups drastic and comparable surges of both plasma progesterone and luteinizing hormone were observed during proestrus. Nevertheless morphine-exposed females showed higher levels of plasma estradiol on diestrus morning but lower levels on metestrus morning. In conclusion, prenatal exposition to morphine has long-term effects mainly on pituitary–adrenal axis as well as on binding sites for corticosterone in the hypothalamus and the hippocampus which are dependent on the estrous cycle stages in adult females.

Growth hormone secretagogues (GHSs) act at distinct levels to control growth hormone (GH) secretion. At the pituitary level they reinforce or extend a tonic GH-releasing-hormone (GHRH)-induced activated state by mobilizing intracellular Ca2+ store. At the hypothalamic level GHS actions are more complex than originally anticipated. Chronic treatments with GHS result in loss of responsiveness to the secretagogues, an effect probably accounted for by indirect negative feedback of GHS stimulated plasma GH levels over GHRH release. Moreover, intracerebroven-tricular treatments with GHS can have paradoxical, inhibitory effects on GH secretion. Several mechanisms can account for such dual effects. GHS receptors were found to extend far beyond the arcuate nucleus and are mainly coexpressed, by GHRH, somatostatin, and neuropeptide Y (NPY) neurons. Activation of GHRH neurons by GHS can be direct or indirect. Indeed using antisense strategy we found that sst1 are physiological activators of arcuate GHRH neurons and we propose that activation of SRIH arcuate interneurons by GHS can increase GHRH neuron activity. Moreover, GHS can stimulate distinct populations of NPY neurons having opposite effects on GH secretion: arcuate NPY interneurons, act as indirect facilitators of GHRH release, whereas, on the contrary, a different subset of NPY neurons projecting to the periventricular hypothalamus (those also involved in mediating leptin effects on GH) seems able to activate SRIH release.

It is becoming increasingly clear that nitric oxide (NO), an active free radical formed during the conversion of arginine to citrulline by the enzyme NO synthase (NOS), is a critical neurotransmitter and biological mediator of the neuroendocrine axis. Current evidence suggests that NO modulates the activity of both the hypothalamic-pituitary-gonadal axis and the hypothalamic-pituitary-adrenal axis. Supporting this hypothesis is the finding that the highest expression of neuronal NOS in the brain is found within the hypothalamus in areas where the cell bodies of the neurons from the different neuroendocrine systems are located. In this regard, the influence of neuronal NO on the regulation of the neuroendocrine neural cell body activity has been well-documented whereas little is known about NO signaling that directly modulates neurohormonal release into the pituitary portal vessels from the neuroendocrine terminals within the median eminence, the common termination field of the adenohypophysiotropic systems. Studies in rat suggest that NO is an important factor controlling both gonadotropin-releasing hormone (GnRH) and corticotropin-releasing hormone (CRH) release at the median eminence. The recent use of amperometric NO detection from median eminence fragments coupled to the use of selective NOS inhibitors demonstrated that a major source of NO at the median eminence might be endothelial in origin rather than neuronal. The present article reviews the recent progress in identifying the origin and the role of the NO produced at the median eminence in the control of neurohormonal release. We also discuss the potential implications of the putative involvement of the median eminence endothelial cells in a neurovascular regulatory process for hypothalamic neurohormonal signaling.

The present study was designed to determine whether whole body injection of lipopolysaccharide increases endogenous rat brain morphine levels. High performance liquid chromatography coupled to electrochemical detection and gas-chromatography mass spectrometry reveal that the resting brain morphine level is 7.0+/-3.2ng/g wet weight. In a time dependent manner, intraperitoneal injection of lipopolysaccharide (10 microg), leads to an increase of brain morphine level with a peak reaching after 36h post injection. After 72h following the lipopolysaccharide-injection, morphine levels increased to 66.0+/-5.4ng/g brain wet weight (P0.001). Furthermore, 96h of fasting also increased the brain morphine level to 44.0+/-3. 6ng/g of brain wet weight. These results suggest that rat brain morphinergic processes respond to organismic challenges after a significant latent period.

It has been previously demonstrated that the activity of proopiomelanocortin (POMC)-containing neurons in the rat arcuate nucleus is regulated by opiates, but the expression of opioid receptors in POMC neurons has never been reported. In the present study, we have applied a double-labeling in situ hybridization technique to investigate the occurrence of μ-opioid receptor mRNA on POMC neurons. We have found that 20±3% of arcuate POMC neurons express μ-opioid receptor mRNA and that the proportion of POMC neurons expressing μ-opioid receptor is higher in the caudal than in the rostral portion of the arcuate nucleus. Our data suggest that POMC neurons might be both auto-regulated by β-endorphin, and regulated by enkephalins.

Urotensin II (UII) is a cyclic peptide initially isolated from the caudal neurosecretory system of teleost fish. Subsequently, UII has been characterized from a frog brain extract, indicating that a gene encoding a UII precursor is also present in the genome of a tetrapod. Here, we report the characterization of the cDNAs encoding frog and human UII precursors and the localization of the corresponding mRNAs. In both frog and human, the UII sequence is located at the C-terminal position of the precursor. Human UII is composed of only 11 amino acid residues, while fish and frog UII possess 12 and 13 amino acid residues, respectively. The cyclic region of UII, which is responsible for the biological activity of the peptide, has been fully conserved from fish to human. Northern blot and dot blot analysis revealed that UII precursor mRNAs are found predominantly in the frog and human spinal cord. In situ hybridization studies showed that the UII precursor gene is actively expressed in motoneurons. The present study demonstrates that UII, which has long been regarded as a peptide exclusively produced by the urophysis of teleost fish, is actually present in the brain of amphibians and mammals. The fact that evolutionary pressure has acted to conserve fully the biologically active sequence of UII suggests that the peptide may exert important physiological functions in humans.

The presence of iron in brain tissue in increased concentrations in Parkinson's disease cases, where it might be responsible for oxidative stress, and the parallel observation that the iron transporter lactoferrin (Lf) was present in increased amounts in surviving neurons, led us to study the synthesis of Lf in a mouse model of Parkinson's disease. In this context, the origin and expression of brain Lf in normal, aged and MPTP (1-methyl-4-phenyl-1, 2,3,6-tetrahydropyridine)-treated mice were investigated. Lf immunostaining was observed mainly on microvessels in the cerebral cortex of the adult mice and to a greater extent in older mice. Lf immunoreactivity was also present in the hippocampus only in the aged mouse brains, associated with structures which seemed to be pyramidal neurons and fibers. After RT-PCR (polymerase chain reaction), Lf transcripts were found in mouse brain tissue whatever the age of the animals studied but the level of their expression was very low. No up-regulation of Lf was detectable during aging. Lf distribution and expression in the MPTP-induced Parkinsonian mouse model were also investigated. A marked depletion of dopamine (DA) occurred in the high dose MPTP-treated mice. The level of Lf expression was found to be markedly increased in the same animals and this up-regulation occurred on the first day after MPTP administration. When the brain was stressed by the neurotoxin MPTP, Lf expression increased in line with antioxidant enzymes such as catalase and gamma-glutamylcysteine synthetase, which may permit the protection of brain tissue from oxidative damage induced by the drug.

The semiquantitative distribution of mRNA encoding for rat galanin receptor (GAL-R1) was examined by in situ hybridization in the rat hypothalamus using a 35S-riboprobe. Most hypothalamic nuclei expressed GAL-R1 mRNA. In the anterior hypothalamus, high levels of expression were found in the medial preoptic area, paraventricular and supraoptic nuclei. Numerous cells also expressed the GAL-R1 mRNA with a moderate level of expression in the periventricular region. Very few GAL-R1-expressing cells were present in the suprachiasmatic nucleus. In the medial hypothalamus, numerous expressing cells were detected in the dorsomedial and ventromedial nuclei. The arcuate nucleus was moderately labeled throughout its rostrocaudal extent; labeled cell bodies were visible in the ventromedial and ventrolateral subdivisions as well. These results indicate that the GAL-R1 mRNA is not only expressed in anterior hypothalamic nuclei but also in the mediobasal hypothalamus and periventricular region. This hypothalamic distribution correlates well with that of 125I-GAL-binding sites and GAL-immunoreactive fibers. This distribution represents the morphological substrate for GAL roles in the hypothalamic regulation of neuroendocrine, behavioral and autonomic functions.

Comparison of the localization of immunoreactivities which were obtained with antibodies to ?-endorphin, ?-neoendorphin, Leu- and met-enk and dynorphin with those obtained earlier using antisera to ?-endorphin, somatostatin, CRF, GRF, ?-MSH, CCK/gastrin, VIP and substance P (Dhainaut-Courtois et al., 1985a,b) show the particular status of opiate-like substances in Nereis. Indeed, immunoreactivities for vertebrate opiates were observed quite exclusively in the brain, on paraffin embedded as well as on frozen tissue sections. Furthermore, immunoreactive neurones for dynorphin occur only in light green cells (C II cells) located in nuclei 20; axons may be followed to the infracerebral region (ICR), a neurohemal area where immunoreactive nerve fibres for Leu-enk and met-enk are also present. The question to be answered now is whether the compounds immunologically related to vertebrate opiates are authentic Nereis neurohormones released in the blood vessel or the coelom or if they act only as neuromodulators at the ICR level by controlling the activity of classical neurosecretory cells or infracerebral cells. At present, a coexistence in a single neurone of an uncharacterized Nereis neurohórmone and an opiate-like substance may also be postulated. In this study, colocalization of materials immunologically related to CCK/gastrin and GRF or CCK/gastrin and 5-HT were also demonstrated. In addition to this, the use of antibody to 5-HT allowed us to confirm previous results which were obtained with radioautography and histofluorescence methods (Dhainaut-Courtois et al. 1979a,b) and to specify the distribution of this biogenic amine in the whole CNS and in the peripheral nervous system.