Hypothalamic ghrelin treatment modulates NPY- but not CRH-ergic activity in adrenalectomized rats subjected to food restriction: Evidence of a novel hypothalamic ghrelin effect

It has been proposed that ghrelin induces food intake by a mechanism due to the stimulation of hypothalamic NPY-ergic activity. It is recognized that bilateral adrenalectomy (ADX) enhances hypothalamic CRH-ergic function and reduces appetite. Thus, the aim of the present study was to test whether, icv-administered, ghrelin modulates NPY- and CRH-ergic functions after food restriction (FR) and glucocorticoid deprivation. For this purpose; 1 μg ghrelin was administered icv to ad libitum (AL) eating and to corticosterone (B)-depleted (ADX) and- replete (sham and ADX+B) male animals habituated, for 15 d, to FR. Food intake, hypothalamic function, and peripheral ghrelin, ACTH, and B concentrations were evaluated 2 h after ghrelin administration. Results indicate that while icv ghrelin treatment stimulated 2-h food intake in AL rats, it failed to do so in sham- and ADX+B-FR animals; moreover, 2-h food intake was inhibited by icv ghrelin treatment in ADX-FR rats. Regarding peripheral hormone levels: (a) basal circulating ghrelin levels, already enhanced (vs AL rats) by FR, significantly increased 2 h after icv ghrelin treatment in AL and sham-FR rats; (b) central ghrelin treatment stimulated ACTH secretion in circulation of AL and glucocorticoid-replete-FR rats; and (c) B circulating levels remained unchanged after ghrelin treatment, although they were in relation to the food intake condition of rats. Finally, hypothalamic NPY mRNA expression was enhanced by FR and, in response to icv ghrelin treatment, it decreased in ADX-FR rats only. ADX-enhanced hypothalamic CRH mRNA levels were reduced by ghrelin icv administration only when antimals received B replacement therapy. Our data indicate an inhibitory effect of hypothalamic ghrelin on NPY-ergic activity in FR rats lacking endogenous glucocorticoi

The Fetal Hypothalamus Has the Potential to Generate Cells with a Gonadotropin Releasing Hormone (GnRH) Phenotype

Neurospheres (NS) are colonies of neural stem and precursor cells capable of differentiating into the central nervous system (CNS) cell lineages upon appropriate culture conditions: neurons, and glial cells. NS were originally derived from the embryonic and adult mouse striatum subventricular zone. More recently, experimental evidence substantiated the isolation of NS from almost any region of the CNS, including the hypothalamus. Here we report a protocol that enables to generate large quantities of NS from both fetal and adult rat hypothalami. We found that either FGF-2 or EGF were capable of inducing NS formation from fetal hypothalamic cultures, but that only FGF-2 is effective in the adult cultures. The hypothalamic-derived NS are capable of differentiating into neurons and glial cells and most notably, as demonstrated by immunocytochemical detection with a specific anti-GnRH antibody, the fetal cultures contain cells that exhibit a GnRH phenotype upon differentiation. This in vitro model should be useful to study the molecular mechanisms involved in GnRH neuronal differentiation

A phospholipase A2-related snake venom (from Crotalus durissus terrificus) stimulates neuroendocrine and immune functions: determination of different sites of action

Immune neuroendocrine interactions are vital for the individual's survival in certain physiopathological conditions, such as sepsis and tissular injury. It is known that several animal venoms, such as those from different snakes, are potent neurotoxic compounds and that their main component is a specific phospholipase A type 2 (PLA2). It has been described recently that the venom from Crotalus durissus terrificus [snake venom (SV), in the present study] possesses some cytotoxic effect in different in vitro and in vivo animal models. In the present study, we investigated whether SV and its main component, PLA2 (obtained from the same source), are able to stimulate both immune and neuroendocrine functions in mice, thus characterizing this type of neurotoxic shock. For this purpose, several in vivo and in vitro designs were used to further determine the sites of action of SV-PLA2 on the hypothalamo-pituitary-adrenal (HPA) axis function and on the release of the pathognomonic cytokine, tumor necrosis factor alpha (TNF alpha), of different types of inflammatory stress. Our results indicate that SV (25 microg/animal) and PLA2 (5 microg/animal), from the same origin, stimulate the HPA and immune axes when administered (i.p.) to adult mice; both preparations were able to enhance plasma glucose, ACTH, corticosterone (B), and TNF alpha plasma levels in a time-related fashion. SV was found to activate CRH- and arginine vasopressin-ergic functions in vivo and, in vitro, SV and PLA2 induced a concentration-related (0.05-10 microg/ml) effect on the release of both neuropeptides. SV also was effective in changing anterior pituitary ACTH and adrenal B contents, also in a time-dependent fashion. Direct effects of SV and PLA2 on anterior pituitary ACTH secretion also were found to function in a concentration-related fashion (0.001-1 microg/ml), and the direct corticotropin-releasing activity of PLA2 was additive to those of CRH and arginine vasopressin; the corticotropin-releasing activity of both SV and PLA2 were partially reversed by the specific PLA2 inhibitor, manoalide. On the other hand, neither preparation was able to directly modify spontaneous and ACTH-stimulated adrenal B output. The stimulatory effect of SV and PLA2 on in vivo TNF alpha release was confirmed by in vitro experiments on peripheral mononuclear cells; in fact, both PLA2 (0.001-1 microg/ml) and SV (0.1-10 microg/ml), as well as concavalin A (1-100 microg/ml), were able to stimulate TNF alpha output in the incubation medium. Our results clearly indicate that PLA2-dependent mechanisms are responsible for several symptoms of inflammatory stress induced during neurotoxemia. In fact, we found that this particular PLA2-related SV is able to stimulate both HPA axis and immune functions during the acute phase response of the inflammatory processes.Instituto Multidisciplinario de Biología Celula

Sexual dimorphism in the hypothalamo-pituitary-adrenal (HPA) axis and TNFα responses to phospholipase A2-related neurotoxin (from crotalus durissus terrificus) challenge

Neuroendocrine-immune interactions are vital for the individual’s survival in certain physiopathological conditions such as sepsis and tissular injury. It is known that several snake venoms (SV) are potent neurotoxic compounds and that their main component is a specific phospholipase type 2 (PLA2). It has been recently described that the venom from crotalus durissus terrificus (SV) possesses a cytotoxic effect in different in vitro and in vivo animal models. In the present study we investigated whether SV is able to stimulate both TNFα and neuroendocrine functions in a sexual dimorphic fashion. For this purpose the modulatory role of endogenous sex steroids during neurotoxemia was evaluated. Our results indicate that SV (25 μg/animal) stimulates the hypothalamo-pituitary-adrenal axis and TNFα secretion when administered (ip) to adult male mice, such responses were characterized by a time-related enhance in plasma glucose, ACTH, corticosterone and TNFα levels. SV-stimulated glycemia, corti-costeronemia and adrenal glucocorticoid were sexually dimorphic. Twenty-day gonadectomized mice showed a similar sexual dimorphism to that found in intact animals, however, they additionally showed a sexual dimorphic pattern in cytokine release in plasma 30 min post-SV. Estradiol (E2) treatment, in gonadectomized mice, abolished some characteristics of the sexual dimorphism, such as hyperglycemia, hypercorticosteronemia and hypercytokinemia. Finally, in vitro experiments indicate that: a) gonadectomy increased spontaneous and SV-stimulated cytokine output by incubated peripheral mononuclear cells (PMNC), regardless of the sex; and b) despite E2 treatment, in gonadectomized, did not modify the pattern of basal and SV-elicited TNFα secretion induced by orchidectomy, fully reversed the enhance in basal and SV-stimulated cytokine release found after ovariectomy alone. Our results further indicate that neurotoxemia, due to SV challenge, induces several symptoms common to those of inflammatory stress; they also strongly support that both gender and endogenous sex steroids are responsible for neuroendocrine-immunological sexual dimorphism.Instituto Multidisciplinario de Biología Celula

Representative images of brains sectioned after lateral ventricle administration with either methylene blue or DiI dyes.

<p>A, Coronal section of adult rat brain injected icv with methylene blue, showing the dissected hypothalamus (HT), (LV: lateral ventricle), (3rdV: third ventricle). B, Enlargement of HT showing the region (dotted lines) that was removed prior to starting cell culture. C, Coronal section of adult rat brain after icv DiI injection, laterals and third ventricles are red stained by the dye.</p

Primers used for the PCR analysis.

<p>Primers used for the PCR analysis.</p

Hypothalamic NS cultures harbor precursors that can differentiate in neurons and glial cells.

<p>A: Gene expression analysis of some lineage-specific CNS markers by RT-PCR. RNA was isolated from hypothalamic-derived fetal secondary NS and adult hypothalami (HT) used for comparison: <i>NSE</i>, a marker for neurons, <i>GFAP</i>, a marker for astrocytes and <i>Nestin</i>, a marker for undifferentiated/neuroepithelial cells. β-2 microblobulin ( <i>β-2m</i>) was used as housekeeping gene. B: Phase-contrast image showing the typical morphology of differentiating NS cultures after 7DIV. C: Representative fluorescence micrograph after double-labeling immunofluorescent detection of neuronal and astrocyte cells in fetal differentiating cultures (7DIV) by using antibody lineage-specific markers: βTubIII (red: neurons) and GFAP (green: astrocytes). Nuclei are stained in blue by the dye DAPI. D, fluorescence micrograph after immunofluorescent detection of oligodendrocyte cells (showned here in red) in fetal differentiating cultures (7DIV) by using an antibody oligodendrocyte-specific marker: O4 (left side). DAPI-stained nuclei from same micrograph shown on right side. Scale bars: 50 μm.</p

Morphology of hypothalamic-derived NS, phase-contrast images.

<p>A, Dividing cells after 2 days in vitro (2DIV). B, Fetal hypothalamic NS after 7DIV. C, Adult hypothalamic NS after 7DIV. Scale bars: 50 μm.</p