Characterization and Expression Dynamics of Key Genes Involved in the Gilthead Sea Bream (<Emphasis Type="Italic">Sparus aurata</Emphasis>) Cortisol Stress Response during Early Ontogeny

The present study identified and characterized six key genes involved in the hypothalamic-pituitary-interrenal (HPI) axis of gilthead sea bream (Sparus aurata), a commercially important European aquaculture species. The key genes involved in the HPI axis for which gene structure and synteny analysis was carried out, comprised of two functional forms of glucocorticoid receptors (GR), as well as three forms of pro-opiomelanocortin (POMC) genes and one form of mineralocorticoid receptor (MR) gene. To explore their functional roles during development but also in the stress response, the expression profiles of gr1, gr2, mr, pomc_aI, pomc_aII, and pomc_β were examined during early ontogeny and after an acute stress challenge. The acute stress challenge was applied at the stage of full formation of all fins, where whole body cortisol was also measured. Both the cortisol and the molecular data implied that sea bream larvae at the stage of the full formation of all fins at 45 dph are capable of a response to stress of a similar profile as observed in adult fish.     

The 70-kDa heat shock protein response in two intertidal sculpins,Oligocottus maculosus and O. snyderi: relationship of hsp70 and thermal tolerance

The role that hsp70 plays in influencing thermal tolerance of a whole animal is not clearly understood. We explored this question by examining liver hsp70 response in the tidepool sculpin (Oligocottus maculosus) and fluffy sculpin (O. snyderi), which have distinct distribution patterns in the intertidal zone. The tidepool sculpin is in upper and lower tidepools, while the fluffy sculpin is exclusively in lower tidepools during a low tide. We conducted experiments in order to investigate: (1) habitat water temperatures; (2) upper thermal tolerance limits; (3) the cellular hsp70 response to changes in water temperature in nature; (4) induction temperatures for hepatic hsp70 and hsp70 mRNA; and (5) effects of long-term heat stress on liver hsp70 levels, in these sculpins. Accordingly, we found: (1) the tidepool sculpin was exposed to a wider temperature range in nature; (2) the tidepool sculpin had higher lethal and induction temperatures for hsp70; (3) the liver hsp70 level of the tidepool sculpin was less sensitive to changes in water temperatures; and (4) the tidepool sculpin had higher constitutive hsp70 levels in nature, compared with the fluffy sculpin. From these results, we proposed that the less thermally sensitive tidepool sculpin may enhance its thermal tolerance by having a large pool of cellular hsp70, thus allowing it to inhabit the upper intertidal zone with relatively large and unpredictable fluctuations in environmental variables.     

Chronic Stress Decreases Availability of Heat Shock Proteins to Glucocorticoid Receptor in Response to Novel Acute Stress in Wistar Rat Hypothalamus

Chronic psychosocial isolation (CPSI) is known to cause several maladaptive changes in the limbic brain structures, which regulate the hypothalamic–pituitary–adrenal (HPA) axis activity. In this study, we focused our investigation on CPSI effects in the hypothalamus (HT) since it is a major driver of HPA axis activity. We also investigated whether the exposure to CPSI could alter the response to subsequent acute stress (30-min immobilization). In the HT, we followed cytosolic and nuclear levels of the glucocorticoid receptor (GR), as a mediator of HPA axis feedback inhibition, and its chaperones, the heat shock proteins (HSPs), hsp70 and hsp90. The CPSI did not cause any changes in either GR or HSPs levels. However, we observed increase of the GR and hsp70 in both HT cellular compartments as a response of naïve rats to acute stress, whereas the response of CPSI rats to acute stress was associated with elevation of the GR in the cytosol and decrease of HSPs in the nucleus. Thus, our data indicated reduced availability of HSPs to GR in both cytosol and nucleus of the HT under acute stress of CPSI animals, and therefore, pointed out to potentially negative effects of CPSI on GR function in the HT.     

Down modulation of N-myc, heat-shock protein 70, and nucleolin during the differentiation of human neuroblastoma cells.

Cultured human neuroblastoma (GOTO) cells were induced to differentiate by dibutyryl cyclic AMP (Bt2cAMP) and/or retinoic acid (RA). A combination of Bt2cAMP (1 mM) and RA (1 microM) yielded the most significant networks of neurites after 3 to 4 days, this being associated with the reduction of N-myc mRNA levels. Next, we examined several cellular genes that were possibly linked with changes in N-myc gene expression under these conditions. Among the genes examined, both nucleolin and a major heat-shock protein (hsp70) mRNAs showed changes concomitant with those in N-myc mRNA levels when induced by Bt2cAMP and RA. Dibutyryl cAMP alone induced several short cellular processes and caused a marked decrease in N-myc mRNA within 2 days. RA alone induced a few long and straight neurites along the longitudinal axis of individual cells and a significant decrease in growth rate but showed neither network formation nor a decrease in N-myc gene expression. These results indicate differential effects of Bt2cAMP and RA on the regulatory mechanisms of both cell proliferation and differentiation and also indicate a possible association of expression of N-myc gene with those of hsp70 and nucleolin genes.     

Novel zebrafish behavioral assay to identify modifiers of the rapid,nongenomic stress response

When vertebrates face acute stressors, their bodies rapidly undergo a repertoire of physiological and behavioral adaptations, which is termed the stress response. Rapid changes in heart rate and blood glucose levels occur via the interaction of glucocorticoids and their cognate receptors following hypothalamic‐pituitary‐adrenal axis activation. These physiological changes are observed within minutes of encountering a stressor and the rapid time domain rules out genomic responses that require gene expression changes. Although behavioral changes corresponding to physiological changes are commonly observed, it is not clearly understood to what extent hypothalamic‐pituitary‐adrenal axis activation dictates adaptive behavior. We hypothesized that rapid locomotor response to acute stressors in zebrafish requires hypothalamic‐pituitary‐interrenal (HPI) axis activation. In teleost fish, interrenal cells are functionally homologous to the adrenocortical layer. We derived eight frameshift mutants in genes involved in HPI axis function: two mutants in exon 2 of mc2r (adrenocorticotropic hormone receptor), five in exon 2 or 5 of nr3c1 (glucocorticoid receptor [GR]) and two in exon 2 of nr3c2 (mineralocorticoid receptor [MR]). Exposing larval zebrafish to mild environmental stressors, acute changes in salinity or light illumination, results in a rapid locomotor response. We show that this locomotor response requires a functioning HPI axis via the action of mc2r and the canonical GR encoded by nr3c1 gene, but not MR (nr3c2). Our rapid behavioral assay paradigm based on HPI axis biology can be used to screen for genetic and environmental modifiers of the hypothalamic‐pituitary‐adrenal axis and to investigate the effects of corticosteroids and their cognate receptor interactions on behavior.     

Counter-Regulatory Response to a Fall in Circulating Fatty Acid Levels in Rainbow Trout. Possible Involvement of the Hypothalamus-Pituitary-Interrenal Axis

We hypothesize that a decrease in circulating levels of fatty acid (FA) in rainbow trout Oncorhynchus mykiss would result in the inhibition of putative hypothalamic FA sensing systems with concomitant changes in the expression of orexigenic and anorexigenic factors ultimately leading to a stimulation of food intake. To assess this hypothesis, we lowered circulating FA levels treating fish with SDZ WAG 994 (SDZ), a selective A1 adenosine receptor agonist that inhibits lipolysis. In additional groups, we also evaluated if the presence of intralipid was able to counteract changes induced by SDZ treatment, and the possible involvement of the hypothalamus-pituitary-interrenal (HPI) axis by treating fish with SDZ in the presence of metyrapone, which decreases cortisol synthesis in fish. The decrease in circulating levels of FA in rainbow trout induced a clear increase in food intake that was associated with the decrease of the anorexigenic potential in hypothalamus (decreased POMC-A1 and CART mRNA abundance), and with changes in several parameters related to putative FA-sensing mechanisms in hypothalamus. Intralipid treatment counteracted these changes. SDZ treatment also induced increased cortisol levels and the activation of different components of the HPI axis whereas these changes disappeared in the presence of intralipid or metyrapone. These results suggest that the HPI axis is involved in a counter-regulatory response in rainbow trout to restore FA levels in plasma.