Serotonergic mechanisms on breathing modulation in the rat locus coeruleus

The locus coeruleus (LC) is a noradrenergic nucleus that plays an important role in the ventilatory response to hypercapnia. This nucleus is densely innervated by serotonergic fibers and contains high density of serotonin (5-HT) receptors, including 5-HT(1A) and 5-HT(2). We assessed the possible modulation of respiratory response to hypercapnia by 5-HT, through 5-HT(1A) and 5-HT(2) receptors, in the LC. To this end, we determined the concentrations of 5-HT and its metabolite 5-hydroxyindole-3-acetic acid (5-HIAA) in the LC after hypercapnic exposure. Pulmonary ventilation (V(E), plethysmograph) was measured before and after unilateral microinjection (100 nL) of WAY-100635 (5-HT(1A) antagonist, 5.6 and 56 mM), 8-OHDPAT (5-HT(1A/7) agonist, 7 and 15 mM), Ketanserin (5-HT(2A) antagonist, 3.7 and 37 mM), or (+/-)-2,5-dimethoxy-4-iodoamphetaminehydrochloride (DOI; 5-HT(2A) agonist, 6.7 and 67 mM) into the LC, followed by a 60-min period of 7% CO(2) exposure. Hypercapnia increased 5-HTIAA levels and 5-HIAA/5-HT ratio within the LC. WAY-100635 and 8-OHDPAT intra-LC decreased the hypercapnic ventilatory response due to a lower tidal volume. Ketanserin increased CO(2) drive to breathing and DOI caused the opposite response, both acting on tidal volume. The current results provide evidence of increased 5-HT release during hypercapnia in the LC and that 5-HT presents an inhibitory modulation of the stimulatory role of LC on hypercapnic ventilatory response, acting through postsynaptic 5-HT(2A) receptors in this nucleus. In addition, hypercapnic responses seem to be also regulated by presynaptic 5-HT(1A) receptors in the LC.

Effects of estrogen receptor alpha and beta gene deletion on estrogenic induction of progesterone receptors in the locus coeruleus in female mice

Locus coeruleus (LC) is involved in the LHRH regulation by gonadal steroids. We investigated the expression of progesterone and estrogen receptors (PR; ER) in LC neurons of ER alpha (alpha ERKO) or ER beta (beta ERKO) knockout mice, and their wild-type (alpha WT and beta WT). Immunocytochemical studies showed that LC expresses PR and both ERs, although ER beta was more abundant. Estradiol benzoate (EB) decreased ER alpha-positive cells in WT and beta ERKO mice, and progesterone caused a further reduction, whereas none of the steroids influenced ER beta expression. ER beta deletion increased ER alpha while ER alpha deletion did not alter ER beta expression. In both WT mice, EB increased PR expression, which was diminished by progesterone. These steroid effects were also observed in alpha ERKO animals but to a lesser extent, suggesting that ER alpha is partially responsible for the estrogenic induction of PR in LC. Steroid effects on PR in beta ERKO mice were similar to those in the alpha ERKO but to a lesser extent, probably because PR expression was already high in the oil-treated group. This expression seems to be specific of LC neurons, since it was not observed in other areas studied, the preoptic area and ventromedial nucleus of hypothalamus. These findings show that LC in mice expresses alpha ER, beta ER, and PR, and that a balance between them may be critical for the physiological control of reproductive function.

Modulatory role of locus coeruleus and estradiol on the stress response of female rats

The activity of the hypothalamic-pituitary-adrenal axis is modulated by the norepinephrinergic system and, in females, also by the ovarian hormones. We investigated the role of ovarian steroids and the locus coeruleus (LC) on stress-induced corticosterone secretion in female rats. Ovariectomized rats without hormonal replacement (OVX) or treated with estradiol (OVE) or estradiol plus progesterone (OVEP) were subjected to jugular cannulation. Immediately after that, each hormonal treatment group was subjected to LC lesion or sham surgery or no brain surgery. After 24 h, blood samples of all 9 groups were collected before and after ether inhalation. Other four groups (OVX control, sham and lesioned, and OVE) were perfused for glucocorticoid receptor (GR) immunocytochemistry in hippocampal CA1 neurons and paraventricular nucleus (PVN). Estradiol replacement decreased while LC lesions increased stress-induced corticosterone secretion. The effect of LC lesion was potentiated with the removal of ovarian steroids. Since GR expression of lesioned animals decreased in the hippocampus, but not in PVN, we suggest that the effect of LC lesion on corticosterone secretion could be due to a reduction in the efficiency of the negative feedback system in the CA1 neurons. However, this mechanism is not involved in the estradiol modulation on corticosteroid secretion, as no change in GR expression was observed in estradiol-treated animals.

Locus coeruleus mediates cold stress-induced polycystic ovary in rats

Previous reports about the rat ovary have shown that cold stress promotes ovarian morphological alterations related to a polycystic ovary (PCO) condition through activation of the ovarian sympathetic nerves. Because the noradrenergic nucleus locus coeruleus (LC) is activated by cold stress and synaptically connected to the preganglionic cell bodies of the ovarian sympathetic pathway, this study aimed to evaluate the LC`s role in cold stress-induced PCO in rats. Ovarian morphology and endocrine and sympathetic functions were evaluated after 8 wk of chronic intermittent cold stress (4 C, 3 h/d) in rats with or without LC lesion. The effect of acute and chronic cold stress upon the LC neuron activity was confirmed by Fos protein expression in tyrosine hydroxylase-immunoreactive neurons. Cold stress induced the formation of follicular cysts, type III follicles, and follicles with hyperthecosis alongside increased plasma estradiol and testosterone levels, irregular estrous cyclicity, and reduced ovulation. Considering estradiol release in vitro, cold stress potentiated the ovarian response to human chorionic gonadotropin. Ovarian norepinephrine (NE) was not altered after 8 wk of stress. However, LC lesion reduced NE activity in the ovary of cold-stressed rats, but not in controls, and prevented all the cold stress effects evaluated. Cold stress increased the number of Fos/tyrosine hydroxylase-immunoreactive neurons in the LC, but this effect was more pronounced for acute stress as compared with chronic stress. These results show that cold stress promotes PCO in rats, which apparently depends on ovarian NE activity that, under this condition, is regulated by the noradrenergic nucleus LC.

Role of locus coeruleus heme oxygenase-carbon monoxide-cGMP pathway during hypothermic response to restraint

Central heme oxigenase-carbon monoxide (HO-CO) pathway has been shown to play a pyretic role in the thermoregulatory response to restraint. However, the specific site in the central nervous system where CO may act modulating this response remains unclear. LC is rich not only in sGC but also in heme oxygenase (HO; the enzyme that catalyses the metabolism of heme to CO, along with biliverdin and free iron). Therefore, the possible role of the HO-CO-cGMP pathway in the restraint-induced-hypothermia by LC neurons was investigated. Body temperature dropped about 0.7 degrees C during restraint. ZnDPBG (a HO inhibitor; 5 nmol, intra-LC) prevented the hypothermic response during restraint. Conversely, induction of the HO pathway in the LC with heme-lysinate (7.6 nmol, intra-LC) intensified the hypothermic response to restraint, and this effect was prevented by pretreatment with ODQ (a sGC inhibitor; given intracerebroventricularly, 1.3 nmol). Taken together, these data suggest that CO in the LC produced by the HO pathway and acting via cGMP is implicated in thermal responses to restraint. (C) 2007 Elsevier Inc. All rights reserved.

IBD sharing around the PPARG locus is not increased in dizygotic twins or their mothers

We observe no evidence of linkage to the region around the PPARG locus in several samples of DZ twins who have been genotyped at multiple markers on chromosome 3 (Fig. 1). Among 199 Australian DZ twins ascertained for a history of wheezing2, mean identity by descent (IBD) sharing at the position of PPARG is 0.463 (99% bootstrapped confidence interval=0.412−0.516). We obtained a similar result with 232 pairs of Australian adolescent DZ twins taking part in a longitudinal study of naevus development3 (0.444, 0.390−0.499), and a set of 125 Australian adult DZ twin pairs assessed for anxiety4 (0.508, 0.435−0.580). A Dutch scan of 160 DZ twin pairs5 obtained slightly more encouraging results (0.553, 0.482−0.587, peak maximum lod score (MLS)=0.57). Pooling all these samples gives 0.477 (0.454−0.512) at the position of PPARG. The test for heterogeneity of sharing between studies was not significant (P=0.10). In the combined dataset, the peak IBD sharing (MLS=0.70) is 50 cM closer to the centromere than PPARG. Finally, in a sample of 203 Australian and New Zealand sister pairs where each had given birth to DZ twins6, sharing across the region is also not increased (0.433). We do not replicate linkage in the populations we study to survival of a twin pregnancy or polyovulation.

Familial hyperaldosteronism

Primary aldosteronism (PAL) may be as much as ten times more common than has been traditionally thought, with most patients normokalemic. The study of familial varieties has facilitated a fuller appreciation of the nature and diversity of its clinical, biochemical, morphological and molecular aspects. In familial hyperaldosteronism type I (FH-I), glucocorticoid-remediable PAL is caused by inheritance of an ACTH-regulated, hybrid CYP11B1/CYP11B2 gene. Genetic testing has greatly facilitated diagnosis. Hypertension severity varies widely, demonstrating relationships with gender, affected parent's gender, urinary kallikrein level, degree of biochemical disturbance and hybrid gene crossover point position. Analyses of aldosterone/PRA/cortisol 'day-curves' have revealed that (1) the hybrid gene dominates over wild type CYP11B2 in terms of aldosterone regulation and (2) correction of hypertension in FH-I requires only partial suppression of ACTH, and much smaller glucocorticoid doses than those previously recommended. Familial hyperaldosteronism type II is not glucocorticoid-remediable, and is clinically, biochemically and morphologically indistinguishable from apparently sporadic PAL. In one informative family available for linkage analysis, FH-II does not segregate with either the CYP11B2, AT1 or MEN1 genes, but a genome-wide search has revealed linkage with a locus in chromosome 7. As has already occurred in FH-I, elucidation of causative mutations is likely to facilitate earlier detection of PAL and other curable or specifically treatable forms of hypertension. (C) 2001 Elsevier Science Ltd. All rights reserved.

The human melanocortin-1 receptor locus: analysis of transcription unit, locus polymorphism and haplotype evolution

The complete sequence of the MCIR locus has been assembled, the coding region of the gene is intronless and placed within a 12 kb region flanked by the NULP1 and TUBB4 genes. The immediate promoter region has an E-box site with homology to the M-box consensus known to bind the microphthalmia transcription factor (MITF), however, promoter deletion analysis and transactivation studies have failed to show activation through this element by MITF. Polymorphism within the coding region, immediate 5' promoter region and a variable number tandem repeat (VNTR) minisatellite within the locus have been examined in a collection of Caucasian families and African individuals. Haplotype analysis shows linkage disequilibrium between the VNTR and MCIR coding region red hair variant alleles which can be used to estimate the age of these missense changes. Assuming a mean VNTR mutation rate of 1% and a star phylogeny, we estimate the Arg151Cys variant arose 7500 years before the present day, suggesting these variants may have arisen in the Caucasian population more recently than previously thought. (C) 2001 Published by Elsevier Science B.V.

DAF yields a cloned marker linked to the soybean (Glycine max) supernodulation nts-1 locus

We report a further characterization of the genomic region containing the soybean supernodulation gene NTS-1. We performed a search for new markers linked to NTS-1 by combining DNA amplification fingerprinting (DAF) and bulked segregant analysis (BSA). The search resulted in one cloned polymorphism (B44-456) linked in trans, 8.5cM from the locus. Southern hybridization showed duplication of the B44-456 sequence in the soybean genome. Additionally, a DNA database search revealed one Arabidopsis thaliana genomic clone from chromosome I possessing 62% homology to the B44-456 marker. A relatively low number of polymorphisms were identified by several PCR marker technologies for this soybean genomic region, providing an additional support for its highly conserved and/or duplicated organization.

Transcripts of the MHM region on the chicken Z chromosome accumulate as non-coding RNA in the nucleus of female cells adjacent to the DMRT1 locus

The male hypermethylated (MHM) region, located near the middle of the short arm of the Z chromosome of chickens, consists of approximately 210 tandem repeats of a BamHI 2.2-kb sequence unit. Cytosines of the CpG dinucleotides of this region are extensively methylated on the two Z chromosomes in the male but much less methylated on the single Z chromosome in the female. The state of methylation of the MHM region is established after fertilization by about the 1-day embryonic stage. The MHM region is transcribed only in the female from the particular strand into heterogeneous, high molecular-mass, non-coding RNA, which is accumulated at the site of transcription, adjacent to the DMRT1 locus, in the nucleus. The transcriptional silence of the MHM region in the male is most likely caused by the CpG methylation, since treatment of the male embryonic fibroblasts with 5-azacytidine results in hypo-methylation and active transcription of this region. In ZZW triploid chickens, MHM regions are hypomethylated and transcribed on the two Z chromosomes, whereas MHM regions are hypermethylated and transcriptionally inactive on the three Z chromosomes in ZZZ triploid chickens, suggesting a possible role of the W chromosome on the state of the MHM region.

Reciprocal relationship between methylation status and loss of heterozygosity at the p14 (ARF) locus in Australian and South African hepatocellular carcinomas

Chromosome 9p21, a locus comprising the tumor suppressor genes (TSG) p16(INK4) (a) and p14(ARF) , is a common region of loss of heterozygosity (LOH) in hepatocellular carcinoma (HCC). p14(ARF) shares exon 2 with p16 in a different reading frame. p14 binds to MDM2 resulting in a stabilization of functional p53 . This study examined the roles of p14, p16 and p53 in hepatocarcinogenesis, in 37 Australian and 24 South African patients. LOH at 9p21 and 17p13.1, p14 and p16 mutation analysis, p14 and p16 promoter methylation and p14, p16 and p53 protein expression was examined. LOH at 9p21 was detected more frequently in South African HCC (P = 0.04). Comparable rates of p53 LOH were observed in Australian and South African HCC (10/22, 45%vs 13/22, 59%, respectively). Hypermethylation of the p14 promoter was more prevalent in Australian HCC than in South African HCC (17/37, 46%vs 7/24, 29%, respectively). In Australian HCC the prevalence of p14 methylation increased with age (P = 0.03). p16 promoter methylation was observed in 12/37 (32%) and 6/24 (25%) in Australian and South African HCC, respectively. Loss of p16 protein expression was detected in 14/36 Australian HCC whereas p53 protein expression was detected in 9/36. Significantly, a reciprocal relationship between 9p21 LOH and p14 promoter hypermethylation was observed (P less than or equal to0.05 ). No significant association between p14 and p53 was seen in this study. The reciprocal relationship identified indicates different pathways of tumorigenesis and likely reflects different etiologies of HCC in the two countries. (C) 2002 Blackwell Science Asia Pty Ltd.