Methodological considerations for ghrelin isoforms assay in clinical evaluation in anorexia nervosa.

The growing interest concerning the role of metabolic sensors in various eating disorders requires the implementation of a strict methodology to collect, store and process blood samples in clinical studies. In particular, measurement of isoforms of the appetite-stimulating hormone, ghrelin, has been challenging in clinical settings. Indeed the acyl ghrelin (AG) isoform is rapidly degraded into desacyl ghrelin (DAG) by blood esterases, thus optimal conditions for the conservation of AG and accurate determination of AG/DAG ratio should be used. Here, we compared different protease inhibitors (Aprotinin, PHMB, AEBSF) during blood collection, increasing delays (0-180 min) before centrifugation, plasma supplementation with various HCl concentrations, storage durations of frozen plasma (8 and 447 days) and immunoenzyme-assay procedures (one-step versus sequential) in healthy subjects. Optimal conditions were obtained by collecting blood with aprotinin and supplementation of plasma with 0.1 N HCl with subsequent freezing for at least 8 days and using one-step assay. Under such conditions, different patterns of secretion of ghrelin isoforms were characterized in patients with restrictive-type anorexia nervosa (AN-R) before and after nutritional recovery. We illustrate the pulsatile variations of ghrelin isoforms according to the time around a meal and hunger rates in 3 patients with AN-R. This study offers a comprehensive comparison of various conditions using selective and specific immunoassays for both ghrelin isoforms in order to optimize assay sensitivity and consistency among procedures. These assay conditions could therefore be widely used to elucidate precisely the role of ghrelin isoforms on eating behavior in physiological and pathological situations.

Immune challenge induces differential corticosterone and interleukin-6 responsiveness in rats bred for extremes in anxiety-related behavior.

Disturbances in mood such as anxiety and depression are often associated with altered hypothalamo-pituitary-adrenal (HPA) axis reactivity, but also with changes in cytokine production, such as interleukin-6 (IL-6), an essential immune factor produced by macrophages and lymphocytes during inflammatory processes. The reciprocal relationship between the HPA axis and the immune system is now well established. In order to understand better the endocrine reactivity of anxious individuals faced with an immune challenge, a model of innate anxiety-related behavior, HAB and LAB rats (HABs, high and LABs, low anxiety-related behavior) was used in this study. We sought to determine whether injection of lipopolysaccharide (LPS) induced a differential HPA axis reactivity and plasma IL-6 release in HABs and LABs. After LPS injection, the plasma adrenal corticotrophic hormone increase did not differ between HABs and LABs, whereas a larger increase in plasma corticosterone levels occurred in HABs than in LABs at 2 h after injection. Moreover, basal IL-6 levels were lower in HABs than in LABs, leading to a higher IL-6 2 h/basal ratio in HABs. In conclusion, we propose for the first time a link between the endocrine and immune systems of HABs and LABs and suggest that IL-6 could be a neuroendocrine correlate of trait anxiety in HABs.

Effect of hindlimb unloading on motor activity in adult rats: impact of prenatal stress.

Environmental changes that occur in daily life or, in particular, in situations like actual or simulated microgravity require neuronal adaptation of sensory and motor functions. Such conditions can exert long-lasting disturbances on an individual's adaptive ability. Additionally, prenatal stress also leads to behavioral and physiological abnormalities in adulthood. Therefore, the aims of the present study were (a) to evaluate in adult rats the behavioral motor adaptation that follows 14 days of exposure to simulated microgravity (hindlimb unloading) and (b) to determine whether restraint prenatal stress influences this motor adaptation. For this purpose, the authors assessed rats' motor reactivity to novelty, their skilled walking on a ladder, and their swimming performance. Results showed that unloading severely impaired motor activity and skilled walking. By contrast, it had no effect on swimming performance. Moreover, results demonstrated for the first time that restraint prenatal stress exacerbates the effects of unloading. These results are consistent with the role of a steady prenatal environment in allowing an adequate development and maturation of sensorimotor systems to generate adapted responses to environmental challenges during adulthood.

Genes involved in obesity: Adipocytes, brain and microflora.

The incidence of obesity and related metabolic disorders such as cardiovascular diseases and type 2 diabetes, are reaching worldwide epidemic proportions. It results from an imbalance between caloric intake and energy expenditure leading to excess energy storage, mostly due to genetic and environmental factors such as diet, food components and/or way of life. It is known since long that this balance is maintained to equilibrium by multiple mechanisms allowing the brain to sense the nutritional status of the body and adapt behavioral and metabolic responses to changes in fuel availability. In this review, we summarize selected aspects of the regulation of energy homeostasis, prevalently highlighting the complex relationships existing between the white adipose tissue, the central nervous system, the endogenous microbiota, and nutrition. We first describe how both the formation and functionality of adipose cells are strongly modulated by the diet before summarizing where and how the central nervous system integrates peripheral signals from the adipose tissue and/or the gastro-intestinal tract. Finally, after a short description of the intestinal commensal flora, rangingfrom its composition to its importance in immune surveillance, we enlarge the discussion on how nutrition modified this perfectly well-balanced ecosystem.

Hypodynamia--hypokinesia induced variations in expression of fos protein in structures related to somatosensory system in the rat.

There have been many reports describing modifications of the sensory and motor cortex following various types of disuse. Hypodynamia--hypokinesia is characterized by the absence of weight-bearing and by a decrease in motor activity. We have shown a reorganization of the cortical cartography after hypodynamia--hypokinesia. In order to give an anatomical account for this cortical plasticity, we set out to determine whether cerebral and spinal structures exhibited variations of their neuronal activation. For this purpose, immunocytochemical detection of Fos protein was performed in the rat brain and spinal cord. Following stimulation of the sciatic nerve, Fos protein was detected in the primary and secondary somatosensory cortex in control rats and in rats submitted to an episode of 14 days of hypodynamia--hypokinesia. Results showed that the stimulation of the sciatic nerve induced an increase in the number of Fos-immunoreactive neurons in all these structures. Moreover, after hypodynamia--hypokinesia, the number of Fos-immunoreactive neurons was increased in the primary and secondary somatosensory cortex and in the spinal cord. These results provide evidence for a higher activation of cortical cells after hypodynamia--hypokinesia in comparison to controls. These data support the hypothesis that hypodynamia--hypokinesia contributes to the development of functional plasticity.

Activation of ventrolateral medullary neurons projecting to spinal autonomic areas after chemical stimulation of the central nucleus of amygdala: a neuroanatomical study in the rat.

Several studies have shown that the central nucleus of amygdala is involved in cardiovascular regulation. The control of this function may be mediated by activation of the ventrolateral medulla neurons that project to preganglionic neurons located in the intermediolateral nucleus of the spinal cord. The aim of the present study was to examine whether stimulation of the central nucleus of amygdala activated ventrolateral medulla neurons projecting to the intermediolateral nucleus. For this purpose, the injection of a retrograde tracer, the cholera toxin b subunit (CTb), into the intermediolateral nucleus of the T2 segment was combined with immunohistochemical detection of Fos protein following chemical stimulation of the central nucleus of amygdala. Results showed that retrogradely labeled neurons were found throughout the ventrolateral medulla. Moreover, chemical stimulation of the central nucleus of amygdala induced: (1) a decrease of arterial blood pressure; (2) an expression of Fos protein mainly in sub-populations of neurons located in the intermediate and caudal parts of the ventrolateral medulla; (3) a significantly higher number of double labeled neurons (CTb-immunoreactive/Fos-immunoreactive) in the rostral part of the ventrolateral medulla than in the other parts of this region. These results show that the central nucleus of amygdala influences the activity of brainstem neurons projecting to the intermediolateral nucleus. Data were discussed in terms of descending amygdalofugal pathways involved in the hypotension.

Cortical control of somato-cardiovascular integration: neuroanatomical studies.

This paper will discuss experiments dedicated to the exploration of pathways linking the sensorimotor cortex (SMC) and the main bulbar nuclei involved in cardiovascular control: the nucleus tractus solitarius (NTS), the dorsal nucleus of the vagus (DMV) and the rostral ventrolateral medulla (RVLM). Results obtained through neurofunctional and neuroanatomical methods are presented in order to bring new answers to relevant points concerning somato-cardiovascular integration: firstly to show the ability of the SMC to influence neurons in bulbar cardiovascular nuclei, and secondly to identify pathways that transmit such influences. The neurofunctional approach, based on the identification of Fos-like immunoreactive neurons, indicated that the SMC has functional connections with cardiovascular bulbar nuclei. The neuroanatomical approach, which employed retrograde and anterograde axonal tracing methods, provided evidence of direct projections from the SMC to NTS/DMV and RVLM. Furthermore, experiments showed clearly that corticospinal neurons sent collaterals to bulbar cardiovascular nuclei, especially to the RVLM. Direct cortical projections to the NTS/DMV and the RVLM provide the anatomical basis for cortical influences on the baroreceptor reflex and sympathetic vasomotor mechanisms for blood pressure control, and support the hypothesis of cortical commands coupling somatic and cardiovascular outputs for action.

Mu opioid receptor mRNA expression in neuronal nitric oxide synthase-immunopositive preoptic area neurons.

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.

Double immunocytochemistry for the detection of Fos protein in retrogradely identified neurons using cholera toxin B subunit.

The focus of this paper was to describe a method combining the neuroanatomical technique of retrograde transport of cholera toxin B subunit (CTB) with the technique of Fos functional labeling. This method allowed us to evaluate whether neurons identified by retrograde tracing were activated following chemical stimulation of another brain area. We have used this method at the light microscopic level to determine whether the stimulation of the rostral ventrolateral medulla activated retrogradely labeled adrenal sympathetic preganglionic neurons in the spinal cord. CTB-containing neurons, Fos immunoreactive neurons and double labeled neurons were observed in spinal autonomic areas. These results suggest that the rostral ventrolateral medulla exerts a descending activation upon identified adrenal preganglionic neurons. The method described in this protocol can be applied for other brain areas in order to establish if a given structure can activate an identified population of neurons linked with a particular target of central or peripheral nervous system.

Expression of Fos protein in adrenal preganglionic neurons following chemical stimulation of the rostral ventrolateral medulla of the rat.

The ventrolateral medulla is known to be involved in the regulation of arterial blood pressure, especially via its connections with sympathetic preganglionic neurons (SPNs) mainly located in the intermediolateral nucleus of the spinal cord. It has been shown that stimulation of the rostral part of the ventrolateral medulla (RVLM) elicits a release of catecholamines from the adrenal medulla. The aim of the present study was to demonstrate the existence of a functional pathway between the RVLM and adrenal SPNs using the combination of a retrograde tract tracing technique (cholera toxin B subunit) with the immunohistochemical detection of Fos protein following the chemical stimulation of RVLM. The data obtained showed that: (1) chemical stimulation of the RVLM induced Fos immunoreactivity in the intermediolateral nucleus and particularly in SPNs projecting to the adrenal medulla; (2) along the thoracic segments T2-T12, 26.1% of retrogradely identified adrenal SPNs were Fos-immunoreactive with the greatest percentage (30.9%) in the T8 segment. These results favored a functional control of the RVLM on adrenal SPNs which may contribute to a substantial activation of the cardiovascular system via the release of adrenal catecholamines.

Immunocytochemical detection of fos protein combined with anterograde tract-tracing using biotinylated dextran.

The present report deals with an axonal tract-tracing procedure in rat enabling visualization of anterogradely transported biotinylated dextran amine (BDA) combined with immnunocytochemical detection of Fos protein following electrical stimulation of the brain. This method allows us to evaluate whether a given structure, receiving both injection of BDA and electrical stimulation, elicits neuronal activation in another part of the brain via direct or indirect projections. We have used the method at the light microscopic level to determine the connectivity of the sensorimotor cortex in the rat. In various parts of the forebrain and brainstem, BDA-labeled fibers originating from the cortex were observed in close apposition to Fos-like immunoreactive cells (FLI) activated by stimulation. This result suggests a direct (probably monosynaptic) projection. On the contrary, FLI neurons were observed in areas devoid of direct afferents, indicating a cascade of activations. The method described in this protocol is applicable for functional anatomy purposes elsewhere within the central nervous system. It constitutes a preliminary step in identifying the validity of a pathway before examination of the reality of the monosynaptic relationship at the electron microscopic level.

GHRP-6-induced changes in electrical activity of single cells in the arcuate, ventromedial and periventricular nucleus neurones [correction of nuclei] of a hypothalamic slice preparation in vitro.

Previously, we demonstrated that systemic injection of the growth hormone secretagogue, growth hormone-releasing peptide (GHRP)-6, selectively activated cells in the hypothalamic arcuate nucleus, as reflected by increased electrical activity and induction of the immediate early gene c-fos. The growth hormone secretagogue receptor distribution is not confined to the arcuate nucleus, suggesting that additional sites of action may exist. In the present study we characterized the electrophysiological responses of cells in the arcuate nucleus, ventromedial nucleus and periventricular nucleus in an in-vitro hypothalamic slice preparation, following bath application of GHRP-6. Additionally, since central somatostatin administration has been shown to attenuate the induction of the c-fos gene by GHRP-6, we sought to determine whether the arcuate cells activated by GHRP-6 are also somatostatin-sensitive. Male Wistar rats (100-150 g body weight (BW)) were anaesthetized (urethane; 1.2 g/kg BW) and the brains removed. Coronal sections (400 microm thickness) were cut through a block of hypothalamus and were transferred to a slice chamber perfused with artificial cerebrospinal fluid. Forty-one arcuate nucleus cells were tested with bath application of 15 microm GHRP-6 for 10 min, 16 of which were tested subsequently (>30 min later) with application of 10 microM somatostatin. Following GHRP-6 administration, 19 cells (46. 3%) showed a significant increase in firing rate during the 15-min period after GHRP-6 application (P<0.001), 17 cells (41.5%) did not respond and the remaining five cells (12.2%) were significantly inhibited. Six of the eight arcuate nucleus cells that were excited by GHRP-6 were significantly inhibited by somatostatin. By contrast, five of the six arcuate nucleus cells that were unresponsive to GHRP-6 were also unresponsive to somatostatin. In the ventromedial nucleus, of 19 cells tested, eight cells (42.1%) were excited by GHRP-6, eight cells (42.1%) were unresponsive and the remaining three cells (15.8%) were significantly inhibited. Of 19 cells recorded in the periventricular nucleus, 13 (68.4%) were unresponsive to GHRP-6 and six (31.6%) were significantly inhibited. Thus, electrophysiological studies in vitro suggest that: (1) neurones in the hypothalamic arcuate nucleus, ventromedial nucleus and periventricular nucleus show changes in electrical activity in response to GHRP-6; and (2) the arcuate nucleus cells excited by GHRP-6 are also subject to inhibitory control by somatostatin.

Induction of c-Fos-like protein in bulbar catecholaminergic neurones by electrical stimulation of the sensorimotor cortex in the rat.

The sensorimotor cortex (SMC) establishes a functional connectivity with the nucleus tractus solitarius (NTS) and the rostral ventrolateral medulla (RVLM). These bulbar nuclei are known to contain catecholaminergic neurones involved in the cardiovascular control. The aim of the present study was to establish the proportion of catecholaminergic neurones activated by electrical stimulation of SMC. For this purpose, double immunocytochemical procedures were used to reveal the distribution of Fos protein and tyrosine hydroxylase (TH). The results showed that, in the NTS, 7% of the neurones immunoreactive for TH expressed Fos-protein, versus 34% in the RVLM. These data provide evidence that the SMC activated preferentially catecholaminergic neurones of the RVLM which are known to be involved in cardiovascular control via spinal preganglionic neurones.

Distribution of cortical fibers and fos immunoreactive neurons in ventrolateral medulla and in nucleus tractus solitarius following the motor cortex stimulation in the rat.

The present study demonstrates that the motor cortex (MC) stimulation induces expression of Fos-like immunoreactivity (FLI) in the rostro-caudal parts of ventrolateral medulla (VLM) and nucleus tractus solitarius (NTS). The coupling of biotinylated dextran (BD) injections with the MC stimulation also permits to identify cortical labeled fibers in the vicinity of FLI neurons in the VLM. Results suggest that the MC is involved in a direct and an indirect modulation of bulbar cardiovascular nuclei.

Attenuation of the growth hormone secretagogue induction of Fos protein in the rat arcuate nucleus by central somatostatin action.

We set out to determine whether the central action of growth hormone (GH) secretagogues to induce Fos protein expression in the arcuate nucleus is influenced by central somatostatin action. Conscious male rats were injected i.v. with 100 micrograms sandostatin (octreotide, a long-acting somatostatin analogue) or saline, 10 min before an i.v. injection of either 50 micrograms GH-releasing peptide (GHRP-6), 50 micrograms MK-0677 (a non-peptide GH secretagogue) or saline. In a separate study, conscious male rats were injected i.c.v. with either 2 micrograms sandostatin or artificial cerebrospinal fluid (aCSF) vehicle 20 min before an i.v. injection of 50 micrograms GHRP-6. In all studies, rats were anaesthetized 90 min following GH secretagogue injection, perfused with fixative and the brains processed for the immunocytochemical detection of Fos protein. The number of Fos-positive nuclei detected in the arcuate nucleus of the i.v. sandostatin/i.v. GHRP-6 treated rats (28 +/- 5 nuclei/section) and the i.v. sandostatin/i.v. MK-0677-injected rats (8 +/- 2 nuclei/section) was significantly less than the i.v. saline/i.v. GHRP-6-treated group (56 +/- 5 nuclei/section) and the i.v. saline/ i.v. MK-0677-treated group (20 +/- 2 nuclei/section) respectively. Intracerebroventricular sandostatin injection attenuated the GHRP-6-induced Fos response, from 53 +/- 6 nuclei/section in the i.c.v. aCSF/i.v. GHRP-6 group, to 39 +/- 5 nuclei/section in the i.c.v. sandostatin/i.v. GHRP-6 group. Thus, the central action of GH secretagogues to induce Fos protein expression in the arcuate nucleus appears to be subject to central inhibitory control by somatostatin.

Expression of c-fos in bulbar nuclei involved in cardiovascular control following the electrical stimulation of sensorimotor cortex in the rat.

Previous studies have shown that electrical stimulation of the sensorimotor cortex (SMC) induces responses of the autonomic nervous system such as variations in heart rate and arterial pressure. Neuroanatomical studies have shown the existence of monosynaptic projections from the SMC to the nucleus tractus solitarius (NTS), the rostral ventrolateral medulla (RVLM) and the dorsal nucleus of the vagus nerve (DNV), which are bulbar nuclei involved in cardiovascular control. The aim of the present study was to establish whether there exists a functional connectivity between the SMC and these nuclei. Electrical stimulation applied to the SMC of 7 rats for 1 h induced the expression of c-fos-protein-like immunoreactivity in the nucleus of some neurons in NTS, RVLM and DNV. These data support the view that the SMC has functional connections with bulbar neurons involved in cardiovascular control.

Retrogradely labelled neurosecretory neurones of the rat hypothalamic arcuate nucleus express Fos protein following systemic injection of GH-releasing peptide-6.

Previously, we demonstrated that the synthetic hexapeptide GH-releasing peptide (GHRP-6) activates a subpopulation of arcuate neurones, as reflected by increased electrical activation and by the detection of Fos protein in cell nuclei. Here we set out to determine (1) what proportion of the cells activated by GHRP-6 are neurosecretory neurones and (2) whether the cells activated by GHRP-6 contain tyrosine hydroxylase (TH; a marker of dopaminergic cells in this region) or beta-endorphin. In the first study, adult male rats were injected i.v. with the retrograde tracer, Fluorogold, to detect cells which project outside the blood-brain barrier (and are therefore likely to be neurosecretory neurones). Three days later the conscious rats were injected i.v. with 50 micrograms GHRP-6 and the brains processed for the immunocytochemical detection of Fos protein. Between 68% and 82% of the arcuate neurones expressing Fos protein following GHRP-6 injection were retrogradely labelled with Fluorogold. In the second study, conscious male rats, bearing a chronically implanted jugular catheter, were killed 90 min following an i.v. injection of 50 micrograms GHRP-6 and the brains were processed for the double immunocytochemical detection of Fos protein and either TH or beta-endorphin. Less than 7% (mean +/- S.E.M. = 6.7 +/- 2.6% nuclei/section per rat) of the arcuate neurones expressing Fos protein following GHRP-6 injection were TH-containing cells. Of 143 beta-endorphin-containing arcuate cells detected only four cells were identified as containing Fos protein. Thus, the majority of arcuate neurones activated by GHRP-6 (1) project outside the blood-brain barrier (and are therefore likely to be neuro-secretory neurones) and (2) were not identified as TH- or beta-endorphin-containing cells.

Distribution of NADPHdiaphorase and calbindin-D28k neurons in the lateral septal area of the guinea pig, with special reference to the enkephalinergic hypothalamo-septal tract.

Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPHd) histochemical techniques were used to identify neurons synthesizing nitric oxide in the lateral septum of the guinea pig. Double immunocytochemical procedures were used to detect neurons immunoreactive for calbindin-D28k and enkephalinergic fibers which project to the lateral septum. The present data demonstrate that (1) the neurons containing NADPH diaphorase and the neurons immunoreactive for calbindin-D28k are observed in discrete regions of the lateral septum; (2) these populations overlap in various areas of the lateral septum including its dorsal and mediolateral parts; (3) NADPH diaphorase and calbindin-D28k are colocalized in neurons located in the overlapping areas; (4) neurons identified by the presence of calbindin-D28k, NADPH diaphorase or both substances, are surrounded by enkephalinergic fibers. These observations indicate the chemical heterogeneity of the lateral septum and suggest that the enkephalinergic hypothalamo-septal tract does not preferentially contact a subpopulation of neurons.

GH-deficient dw/dw rats and lit/lit mice show increased Fos expression in the hypothalamic arcuate nucleus following systemic injection of GH-releasing peptide-6.

In the rat, the synthetic GH secretagogue GH-releasing peptide (GHRP-6) acts centrally to activate a subpopulation of arcuate neurones as reflected by increased electrical activation and by the detection of Fos protein in cell nuclei. Since GHRP-6 also induces GH secretion via a direct action on the pituitary, we set out to determine whether the central actions of GHRP-6 are mediated by GH itself. First, we demonstrated that peripherally administered GHRP-6 induces Fos expression in the arcuate nucleus of GH-deficient animals (dw/dw rats and lit/lit mice). Secondly, in dw/dw rats, neither intracerebroventricular injection of 15 micrograms recombinant bovine GH nor 1 microgram recombinant human IGF-I resulted in an increase in the number of cells expressing Fos protein in the arcuate nucleus (or in any other hypothalamic structure studied). These results support our hypothesis that GHRP-6 has a central site and mechanism of action and provide evidence to suggest that the activation of arcuate neurones by GHRP-6 is not mediated by a central action of GH or IGF-I. Furthermore, since the lit/lit mouse pituitary does not release GH following GHRP-6 administration, our finding that the central actions of GHRP-6 remain intact in these animals suggests the possible existence of two subpopulations of putative GHRP-6 receptors.