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 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

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