Нейроендокринний супровід поліваріантних ефектів біоактивної води Нафтуся на рівень хронічного стресу у жінок з різним оваріальним статусом

Проанализированы изменения нейроэндокринных показателен, сопутствующие поливариантным эффектам биоактивной воды Нафтуся курорта Трускавец на уровень хронического стресса у женщин детородного возраста с различным овариальным статусом. Обнаружена значительная (R=0,59) каноническая корреляционная связь между динамикой нейро-гормонального индекса стресса, с одной стороны, и вегетативной реактивности, лютеинизирующего гормона, тиреотропного гормона, тироксина и прогестерона - с другой стороны.The changes in neuroendocrine parameters, concomitant multivariate effects of bioactive water Naftussya spa Truskavets to the level of chronic stress in women of childbearing age with different ovarian status. A significant (R=0,59) canonical correlation between the dynamics of the neuro-hormonal index of stress, on the one hand, and autonomic reactivity, luteinizing hormone, thyroid-stimulating hormone, thyroxine and progesterone - the other side

Amygdala 14-3-3ζ as a Novel Modulator of Escalating Alcohol Intake in Mice

Alcoholism is a devastating brain disorder that affects millions of people worldwide. The development of alcoholism is caused by alcohol-induced maladaptive changes in neural circuits involved in emotions, motivation, and decision-making. Because of its involvement in these processes, the amygdala is thought to be a key neural structure involved in alcohol addiction. However, the molecular mechanisms that govern the development of alcoholism are incompletely understood. We have previously shown that in a limited access choice paradigm, C57BL/6J mice progressively escalate their alcohol intake and display important behavioral characteristic of alcohol addiction, in that they become insensitive to quinine-induced adulteration of alcohol. This study used the limited access choice paradigm to study gene expression changes in the amygdala during the escalation to high alcohol consumption in C57BL/6J mice. Microarray analysis revealed that changes in gene expression occurred predominantly after one week, i.e. during the initial escalation of alcohol intake. One gene that stood out from our analysis was the adapter protein 14-3-3ζ, which was up-regulated during the transition from low to high alcohol intake. Independent qPCR analysis confirmed the up-regulation of amygdala 14-3-3ζ during the escalation of alcohol intake. Subsequently, we found that local knockdown of 14-3-3ζ in the amygdala, using RNA interference, dramatically augmented alcohol intake. In addition, knockdown of amygdala 14-3-3ζ promoted the development of inflexible alcohol drinking, as apparent from insensitivity to quinine adulteration of alcohol. This study identifies amygdala 14-3-3ζ as a novel key modulator that is engaged during escalation of alcohol use

Alcohol intake and preference for C57BL/6J mice that consumed alcohol for 1, 2 or 4 weeks.

<p><b><i>A</i></b> The mice show a progressive increase in alcohol intake during the first two weeks of the experiment, stabilizing thereafter. <b><i>B</i></b> Preference for alcohol was high throughout the experiment. <b><i>C</i></b> RNA for microarray analysis was isolated from 0.6 mm diameter punches (<b>○</b>) from serial sections through the CeA. <b><i>D</i></b> The majority of the gene expression changes occurred after 1 week of alcohol consumption; the bar graph shows the total numbers of up- (black) and down-regulated (grey) genes for each time-point while the Venn Diagram shows the total number of up- and down-regulated genes (marked by arrows) for each time-point and overlap between the experimental groups. <b><i>E–F</i></b> Analysis of the microarray data using the <i>Short Time Series Expression Miner (STEM</i>), revealed significant gene enrichment particularly during the early stages of escalation of alcohol intake (week 1 and/or week 2). When considering all possible time profiles for gene expression after 1 wk, 2 wk and 4 wk of alcohol consumption, relative to data from naïve control mice (exemplified by <b><i>E</i></b>), 6 time profiles showed significant gene enrichment: they represented more genes than expected based on chance (<b><i>F</i></b>). The commonality between these 6 significant time profiles is that they all show an initial change in gene expression, normalizing thereafter to baseline levels.</p

Design and validation of 14-3-3ζ shRNA constructs

<p><b><i>A</i></b> 14-3-3ζ specific shRNA sequences (1222 and 1854 bp) were cloned into pLentiLox3.7 vectors and <b><i>B</i></b> western blot analysis revealed that both constructs effectively reduced 14-3-3ζ protein levels in Neuro2A cells, the 1854 construct being most effective <i>in vitro. </i><b><i>C</i></b><i> In situ</i> hybridization confirmed effective knockdown of 14-3-3ζ in the CeA after infection with the 1854 shRNA expressing lentivirus. The top panels show 14-3-3ζ expression in the CeA after infection with the control or the 1854 shRNA expressing lentivirus. The bottom panels show GFP mRNA and therefore the infection site in adjacent sections. 14-3-3ζ mRNA is completely absent in the area that is infected with the 1854 shRNA expressing lentivirus.</p

Gene Enrichment analysis in STEM.

<p>Gene Ontology analysis for the significant time profiles 45 and 47 in STEM revealed significant enrichment of genes involved in transport, ion transport, ligand-gated ion channel activity, synaptic transmission, cytoplasm and protein transport. Highlighted are the selected <b>8 top candidate genes</b>.</p

qPCR data for mice that consumed alcohol for 1 week or 2 weeks.

<p>qPCR confirmed significant up-regulation of 4 out of 8 candidate genes when compared to naïve control mice: Gria3, 14-3-3 zeta, Gabrb3 and Prkacb. *P < 0.05, **P < 0.01 from naïve mice by Tukey HSD multiple comparisons.</p