Recruitment of Corticotropin-Releasing Hormone (CRH) Neurons in Categorically Distinct Stress Reactions in the Mouse Brain.

Corticotropin-releasing hormone (CRH) neurons in the paraventricular hypothalamic nucleus (PVH) are in the position to integrate stress-related information and initiate adaptive neuroendocrine-, autonomic-, metabolic- and behavioral responses. In addition to hypophyseotropic cells, CRH is widely expressed in the CNS, however its involvement in the organization of the stress response is not fully understood. In these experiments, we took advantage of recently available Crh-IRES-Cre;Ai9 mouse line to study the recruitment of hypothalamic and extrahypothalamic CRH neurons in categorically distinct, acute stress reactions. A total of 95 brain regions in the adult male mouse brain have been identified as containing putative CRH neurons with significant expression of tdTomato marker gene. With comparison of CRH mRNA and tdTomato distribution, we found match and mismatch areas. Reporter mice were then exposed to restraint, ether, high salt, lipopolysaccharide and predator odor stress and neuronal activation was revealed by FOS immunocytochemistry. In addition to a core stress system, stressor-specific areas have been revealed to display activity marker FOS. Finally, activation of CRH neurons was detected by colocalization of FOS in tdTomato expressing cells. All stressors resulted in profound activation of CRH neurons in the hypothalamic paraventricular nucleus; however, a differential activation of pattern was observed in CRH neurons in extrahypothalamic regions. This comprehensive description of stress-related CRH neurons in the mouse brain provides a starting point for a systematic functional analysis of the brain stress system and its relation to stress-induced psychopathologies.

Wild Strawberry, Blackberry, and Blueberry Leaf Extracts Alleviate Starch-Induced Hyperglycemia in Prediabetic and Diabetic Mice.

Intestinal α-glucosidase and α-amylase break down nutritional poly- and oligosaccharides to monosaccharides and their activity significantly contributes to postprandial hyperglycemia. Competitive inhibitors of these enzymes, such as acarbose, are effective antidiabetic drugs, but have unpleasant side effects. In our ethnopharmacology inspired investigations, we found that wild strawberry (Fragaria vesca), blackberry (Rubus fruticosus), and European blueberry (Vaccinium myrtillus) leaf extracts inhibit α-glucosidase and α-amylase enzyme activity in vitro and are effective in preventing postprandial hyperglycemia in vivo. Toxicology tests on H9c2 rat embryonic cardiac muscle cells demonstrated that berry leaf extracts have no cytotoxic effects. Oral administration of these leaf extracts alone or as a mixture to normal (control), obese, prediabetic, and streptozotocin-induced diabetic mice attenuated the starch-induced rise of blood glucose levels. The efficiency was similar to that of acarbose on blood glucose. These results highlight berry leaf extracts as candidates for testing in clinical trials in order to assess the clinical significance of their effects on glycemic control.

Enhanced innate fear and altered stress axis regulation in VGluT3 knockout mice.

Glutamatergic neurons, characterized by vesicular glutamate transporters (VGluT1-3) provide the main excitation in the brain. Their disturbances have been linked to various brain disorders, which could be also modeled by the contextual fear test in rodents. We aimed to characterize the participation of VGluT3 in the development of contextual fear through its contribution to hypothalamic-pituitary-adrenocortical axis (HPA) regulation using knockout (KO) mice. Contextual fear conditioning was induced by foot shock and mice were examined 1 and 7 d later in the same environment comparing wild type with KO. Foot shock increased the immobility time without context specificity. Additionally, foot shock reduced open arm time in the elevated plus maze (EPM) test, and distance traveled in the open field (OF) test, representing the generalization of fear. Moreover, KO mice spent more time with freezing during the contextual fear test, less time in the open arm of the EPM, and traveled a smaller distance in the OF, with less entries into the central area. However, there was no foot shock and genotype interaction suggesting that VGluT3 does not influence the fear conditioning, rather determines anxiety-like characteristic of the mice. The resting hypothalamic CRH mRNA was higher in KO mice with reduced stressor-induced corticosterone elevations. Immunohistochemistry revealed the presence of VGluT3 positive fibers in the paraventricular nucleus of hypothalamus, but not on the hypophysis. As a summary, we confirmed the involvement of VGluT3 in innate fear, but not in the development of fear memory and generalization, with a significant contribution to HPA alterations. Highlights VGluT3 KO mice show innate fear without significant influence on fear memory and generalization. A putative background is the higher resting CRH mRNA level in their PVN and reduced stress-reactivity.

Xenoestrogens Ethinyl Estradiol and Zearalenone Cause Precocious Puberty in Female Rats via Central Kisspeptin Signaling.

Xenoestrogens from synthetic or natural origin represent an increasing risk of disrupted endocrine functions including the physiological activity of the hypothalamo-pituitary-gonad axis. Ethinyl estradiol (EE2) is a synthetic estrogen used in contraceptive pills, whereas zearalenone (ZEA) is a natural mycoestrogen found with increasing prevalence in various cereal crops. Both EE2 and ZEA are agonists of estrogen receptor-α and accelerate puberty. However, the neuroendocrine mechanisms that are responsible for this effect remain unknown. Immature female Wistar rats were treated with EE2 (10 µg/kg), ZEA (10 mg/kg), or vehicle for 10 days starting from postnatal day 18. As a marker of puberty, the vaginal opening was recorded and neuropeptide and related transcription factor mRNA levels were measured by quantitative real time PCR and in situ hybridization histochemistry. Both ZEA and EE2 accelerated the vaginal opening, increased the uterine weight and the number of antral follicles in the ovary, and resulted in the increased central expression of gnrh. These changes occurred in parallel with an earlier increase of kiss1 mRNA in the anteroventral and rostral periventricular hypothalamus and an increased kisspeptin (KP) fiber density and KP-GnRH appositions in the preoptic area. These changes are compatible with a mechanism in which xenoestrogens overstimulate the developmentally unprepared reproductive system, which results in an advanced vaginal opening and an enlargement of the uterus at the periphery. Within the hypothalamus, ZEA and EE2 directly activate anteroventral and periventricular KP neurons to stimulate GnRH mRNA. However, GnRH and gonadotropin release and ovulation are disrupted due to xenoestrogen-mediated inhibitory KP signaling in the arcuate nucleus.

Comparison of stress-induced changes in adults and pups: is aldosterone the main adrenocortical stress hormone during the perinatal period in rats?

Positive developmental impact of low stress-induced glucocorticoid levels in early development has been recognized for a long time, while possible involvement of mineralocorticoids in the stress response during the perinatal period has been neglected. The present study aimed at verifying the hypothesis that balance between stress-induced glucocorticoid and mineralocorticoid levels is changing during postnatal development. Hormone responses to two different stressors (insulin-induced hypoglycaemia and immune challenge induced by bacterial lipopolysaccharid) measured in 10-day-old rats were compared to those in adults. In pups corticosterone responses to both stressors were significantly lower than in adults, which corresponded well with the stress hyporesponsive period. Importantly, stress-induced elevations in aldosterone concentration were significantly higher in pups compared both to corticosterone elevations and to those in adulthood with comparable adrenocorticotropin concentrations in the two age groups. Greater importance of mineralocorticoids compared to glucocorticoids in postnatal period is further supported by changes in gene expression and protein levels of gluco- (GR) and mineralocorticoid receptors (MR) and selected enzymes measured by quantitative PCR and immunohystochemistry in the hypothalamus, hippocampus, prefrontal cortex, liver and kidney. Gene expression of 11beta-hydroxysteroid dehydrogenase 2 (11ß-HSD2), an enzyme enabling preferential effects of aldosterone on mineralocorticoid receptors, was higher in 10-day-old pups compared to adult animals. On the contrary, the expression and protein levels of GR, MR and 11ß-HSD1 were decreased. Presented results clearly show higher stress-induced release of aldosterone in pups compared to adults and strongly suggest greater importance of mineralocorticoids compared to glucocorticoids in stress during the postnatal period.

Differential Changes in Expression of Stress- and Metabolic-Related Neuropeptides in the Rat Hypothalamus during Morphine Dependence and Withdrawal.

Chronic morphine treatment and naloxone precipitated morphine withdrawal activates stress-related brain circuit and results in significant changes in food intake, body weight gain and energy metabolism. The present study aimed to reveal hypothalamic mechanisms underlying these effects. Adult male rats were made dependent on morphine by subcutaneous implantation of constant release drug pellets. Pair feeding revealed significantly smaller weight loss of morphine treated rats compared to placebo implanted animals whose food consumption was limited to that eaten by morphine implanted pairs. These results suggest reduced energy expenditure of morphine-treated animals. Chronic morphine exposure or pair feeding did not significantly affect hypothalamic expression of selected stress- and metabolic related neuropeptides - corticotropin-releasing hormone (CRH), urocortin 2 (UCN2) and proopiomelanocortin (POMC) compared to placebo implanted and pair fed animals. Naloxone precipitated morphine withdrawal resulted in a dramatic weight loss starting as early as 15-30 min after naloxone injection and increased adrenocorticotrophic hormone, prolactin and corticosterone plasma levels in morphine dependent rats. Using real-time quantitative PCR to monitor the time course of relative expression of neuropeptide mRNAs in the hypothalamus we found elevated CRH and UCN2 mRNA and dramatically reduced POMC expression. Neuropeptide Y (NPY) and arginine vasopressin (AVP) mRNA levels were transiently increased during opiate withdrawal. These data highlight that morphine withdrawal differentially affects expression of stress- and metabolic-related neuropeptides in the rat hypothalamus, while relative mRNA levels of these neuropeptides remain unchanged either in rats chronically treated with morphine or in their pair-fed controls.

Changes in metabolic-related variables during chronic morphine treatment.

To reveal neuroendocrine/neurochemical changes that are responsible for the robust metabolic alterations seen during chronic morphine treatment we followed hormonal-, transcriptional- and behavioral changes during chronic morphine administration in adult male Wistar rats. Animals were implanted with increasing amount of slow release morphine tablets for 8 days. Morphine treated animals gain significantly less weight than placebo implanted controls. This weight loss is due to the dramatic decrease in the food intake and caloric efficiency in the first days of drug administration and to the lasting disregulated feeding pattern. Changes in feeding behavior included increase of diurnal and decrease of nocturnal feeding frequency in morphine treated rats. Significantly less leptin and insulin plasma levels were detected in morphine implanted animals than in placebo implanted controls, while adiponectin and ACTH concentration remain unchanged. Morphine treated rats display an increase of FosB/Delta FosB immunoreactivity at brain sites that have been implicated regulation of food intake and energy expenditure, including hypothalamic arcuate, paraventricular and ventromedial nuclei and in the lateral hypothalamic area as well as in the caudal brainstem. However, morphine-induced long-term metabolic alterations were not accompanied with any significant changes in the expression of anorexigenic neuropeptides POMC and CART in the hypothalamus and in the brainstem. The disregulated feeding pattern was not reflected in changes of orexin transcription, however, a compensatory upregulation was revealed in hypothalamic NPY expression.

Differential regulation of hypothalamic neuropeptide Y hnRNA and mRNA during psychological stress and insulin-induced hypoglycemia.

Many signals reflecting energy balance and stress are integrated at the hypothalamic orexigenic NPY neurons. To determine transcriptional changes of the NPY gene in response to stress, we followed the time course and compared the expression of heteronuclear (hn)- and messenger (m)RNA levels by in situ hybridization histochemistry and by real time PCR in mice following insulin-induced hypoglycemia and restraint. Hypoglycemia in fasted mice resulted in a rapid increase of NPY hnRNA that peaked at 1h, declined thereafter by 2-4h after insulin injection and run parallel to that of NPY mRNA. Throughout the time course examined, NPY expressing cells in the medial basal hypothalamus remained overwhelmingly localized to the arcuate nucleus. Following restraint NPY mRNA slightly increased, however hnRNA levels decreased up to 2h, suggesting increased stability of mature NPY mRNA. These results highlight rapid changes and differential regulation of NPY expression in response to metabolic and stress challenges.