Effects of intracerebroventricularly administered nesfatin-1 on slow wave sleep and passive wake (PW) vigilance stages.

<p>The time spent and the number of episodes in light slow wave sleep (SWS1, <b>A</b> and <b>D</b>, respectively), deep slow wave sleep (SWS2, <b>B</b> and <b>E</b>, respectively) as well as in PW (<b>C</b> and <b>F</b>, respectively), per hour in the 2^nd–6^th hours of passive (light) phase. Data are presented as mean ± SEM, n = 6 per group, p*<0.05, p**<0.01.</p

Stress-related changes and energy balance of the experimental animals.

<p>Data show significantly increased levels of CRH mRNA in the hypothalamic paraventricular nucleus (<b>A</b>) and decreased body weights (<b>B</b>) both in the SP and LP kept animals, compared to HC, without difference between the above mentioned groups. <b>C</b>. Cumulative food intake shows no alterations. HC: home cage control, sacrificed with the animals kept on platforms, HCR: home cage control “rebound”, sacrificed at the same time as rebound groups, SP: small pot, SPR: small pot plus sleep rebound, LP: large pot, LPR: large pot plus sleep rebound groups. Data are shown as mean ± SEM, n = 10–11, p*<0.05, p**<0.01 <i>vs</i>. HC.</p

Effect of rebound sleep on the activity of nesfatin1/NUCB2 (nesfatin) - positive cell population.

<p>Fos (green) and nesfatin (red) double fluorescent immunostainings showing the three investigated areas arranged in columns (left: zona incerta, middle: perifornical area, right: lateral hypothalamic area) in rapid eye movement sleep (REMS) - deprived - sleep rebound (<b>A–C</b>), REMS - deprived without rebound (<b>D–F</b>) and home cage kept (<b>G–I</b>) animals. c: capsula interna, f: fornix, scale: 100 µm.</p

Participation of melanin-concentrating hormone (MCH)-positive and MCH - negative nesfatin-1/NUCB2 (nesfatin) neurons in the sleep - wake cycle.

<p><b>A–C</b>. Illustrative pictures of the lateral hypothalamic area demonstrating the results of the triple fluorescent immunostainings for nesfatin (red), MCH (blue) and Fos (green) in a home cage kept (<b>A</b>), a rapid eye movement sleep (REMS) - deprived (<b>B</b>) and a REMS - deprived - sleep rebound (<b>C</b>) animal. Nesfatin/MCH double-positive neurons are pink (arrows), MCH - negative nesfatin neurons are red (arrowheads). Activated nesfatin/MCH neurons show white nuclei, activated nesfatin - positive, but MCH - negative neurons show yellow nuclei. Note that majority of the MCH - positive nesfatin neurons are activated (Fos - positive, arrows) by rebound, while only a few of the MCH - negative neurons showed Fos - positivity (arrowhead). Scale bar: 100 µm. <b>D</b>. Distribution of MCH - positive (N⁺/M⁺) and MCH - negative (N⁺/M^¯) neurons within the nesfatin producing cell population and percentage of activated (Fos - positive) cells after REMS deprivation followed by rebound. Data are shown as mean ± SEM, n = 5.</p

Effects of intracerebroventricularly administered nesfatin-1 on rapid eye movement sleep (REMS) and intermediate stage of sleep (IS).

<p><b>A,B</b>. The time spent in REMS and IS per hour, respectively in the 2^nd–6^th hours of passive (light) phase. <b>C,D</b>. The number and -E,F- the average duration of REMS and IS episodes per hour, respectively. Data are presented as mean ± SEM, n = 6 per group, p*<0.05, p**<0.01.</p

Nesfatin-1/NUCB2 expression as controlled by rapid eye movement sleep stage. A

<p>. Autoradiographic images of coronal sections through the middle portion of the hypothalamus showing the area of interest hybridized against nesfatin-1/NUCB2 mRNA in control animals. The upper and the lower panels show two different rostro - caudal levels. Distance from the bregma is indicated at bottom left in millimeters. Asterix: fornix, LH: lateral hypothalamic area, ZI: zona incerta, scale: 1 mm. <b>B</b>,<b>C</b>. Nesfatin-1/NUCB2 mRNA and protein levels in the different experimental groups determined by quantitative ISH and ELISA measurements, respectively. HC: home cage control, sacrificed with the animals kept on platforms, HCR: home cage control “rebound”, sacrificed at the same time as rebound groups, SP: small pot, SPR: small pot plus sleep rebound, LP: large pot, LPR: large pot plus sleep rebound groups. p*<0.05 <i>vs.</i> all other groups, p# <0.05 <i>vs.</i> SP group, n = 5–9 for <b>B</b> and n = 4–7 for <b>C</b>.</p

Bayesian posterior probabilities of relevance of <i>5-HTTLPR</i> for the multivariate phenotype.

<p>RLE was grouped into three categories: low = 0–1, medium = 2, high = 3 or more number of recent negative life events (in the past year). CHA (childhood adversity) was divided into two categories (based on the original three): low = 0–3, medium or high = 4 or more scores. Multivariate phenotype (more accurately describes depression related psychiatric state than one phenotype measure alone) encompasses lifetime depression, BSI depression score and BSI anxiety score. Results are displayed according to CHA and age, in groups differentially exposed to RLE. <b>3A and 3B.</b> Results demonstrate moderate Bayesian probability of relevance of <i>5-HTTLPR</i> in both age groups and CHA groups in those who had 3 or more RLE. <b>3C.</b> In the younger age group (≤30) <i>5-HTTLPR</i> was strongly relevant in those who had medium or high CHA and even moderate number of RLE. <b>3D.</b> In the older age group (>30) <i>5-HTTLPR</i> was strongly relevant in those who had 3 or more RLE, irrespective of CHA.</p

Summary of the genetic association and interaction results using PLINK.

<p>BSI: Brief Symptom Inventory; BSI-DEP: BSI depression score; BSI-ANX: BSI anxiety score; CHA: childhood adversity; DEP: lifetime depression; Pperm: permutated p values; RLE: recent negative life events (in the last year). Additive genetic models were calculated, where <i>5-HTTLPR</i> s allele represents the minor allele. Results are displayed in different groups: total population (all); those up to 30 years (≤30); and those above 30 years (>30). Regression equations (linear regression and beta for BSI-DEP and BSI-ANX scores, and logistic regression and odds ratio for DEP) always involve gender and age as covariates. In case of interaction models, main effect of the respective life event (RLE or CHA) was also covariate in the equation, besides its interaction with <i>5-HTTLPR</i>. And in case of the third model BSA-ANX was also a covariate. Permutated p values were calculated for the nominal p<0.05 results using PLINK—mperm 1000 for main 5HTTLPR effect on anxiety and using the “glmperm” R-package (<a href="http://cran.r-project.org/web/packages/glmperm/index.html" target="_blank">http://cran.r-project.org/web/packages/glmperm/index.html</a>, with 1000 permutations) for the interaction effects on DEP and BSI-DEP.</p><p>The three categories of RLE were: low = 0–1, medium = 2, high = 3 or more number of recent negative life events. The three categories of CHA were: low = 0–3, medium = 4–6, high = 7 or more scores.</p><p>Italics represent trends, and bold represents significant findings.</p><p>Summary of the genetic association and interaction results using PLINK.</p

<i>5-HTTLPR</i>xRLE interaction, with PLINK (left column) and Bayesian (right column) analyses.

<p>LR+: likelihood ratio of emergence of the disease; BSI: Brief Symptom Inventory; BSI-DEP: BSI depression score; BSI-ANX: BSI anxiety score; DEP: lifetime depression; RLE: recent negative life events (in the last year). The three categories of RLE are: low = 0–1, medium = 2, high = 3 or more. Numbers in groups: low RLE: ss = 292, sl = 746, ll = 480; medium RLE: ss = 82, sl = 207, ll = 144; high RLE: ss = 51, sl = 146, ll = 134. Standard errors of means are displayed in case of continuous variables (left column). Right column figures display outlines of posterior distributions of Bayesian Odds Ratios of <i>5-HTTLPR</i> ss versus ll genotype with respect to DEP, BSI-DEP (severe vs. low), and BSI-ANX (severe vs. low). Subsets according to RLE categories (low, medium and high) were analyzed individually. Curve flatness refers to the number of possible models, each with a different odds ratio. An odds ratio greater than one represents a risk for the given phenotype. <b>1A.</b> Logistic regression analysis showed that having the more s alleles increased the risk of DEP with increasing number of RLE. <b>1B.</b> Regarding DEP there is a clear difference between subjects with low RLE (with a Bayesian Odds Ratio close to 1) and subjects with medium or high RLE (where the effect of ss genotype is stronger). <b>1C.</b> As in case of DEP: having the more s alleles also increased BSI-DEP with increasing number of RLE, using linear regression analysis. <b>1D.</b> As in case of DEP: effect of ss genotype on BSI-DEP is negligible in the low RLE group, but higher in the medium, and especially high in the high RLE group. <b>1E.</b> In contrast to depression phenotypes: linear regression analysis showed that carrying the more s alleles increased BSI-ANX without interaction with RLE. <b>1F.</b> In contrast to depression phenotypes, ss genotype represents a risk for BSI-ANX irrespective of RLE group.</p