Sex differences in serotonergic but not gamma-aminobutyric acidergic (GABA) projections to the rat ventromedial nucleus of the hypothalamus.

Hormonal conditions that elicit lordosis in female rats are ineffective in males, suggesting that this behavior is actively suppressed in males. Previous studies theorize that serotonergic and gamma-aminobutyric acidergic (GABA) inputs to the ventrolateral division of the ventromedial nucleus of the hypothalamus (VMNvl) may contribute to lordosis inhibition in males. Using triple-label immunofluorescent techniques, the present studies explored potential sex differences in the density of these projections within three hypothalamic sites: the VMNvl, the arcuate nucleus (ARC), and the dorsomedial nucleus of the hypothalamus. Antibodies directed against HuC/D, estrogen receptor (ER)-alpha and either serotonin (5-HT) or the gamma-aminobutyric acid synthetic enzyme glutamic acid decarboxylase-65 were used to compare the densities of glutamic acid decarboxylase (GAD)-65- and 5-HT-containing fibers in each brain area, the percentage of VMNvl HuC/D immunoreactive (ir) neurons that contained ERalpha, and the percentage of HuC/D and ERalpha double-labeled cells receiving apparent contacts from 5-HT fibers between adult, gonadectomized male and female rats. The densities of VMNvl and ARC 5-HT immunolabeled fibers were significantly higher in the males, and the percentage of VMNvl HuC/D-ir neurons containing ERalpha was significantly higher in the females. The percentage of HuC/D-ir cells contacted by 5-HT fibers was significantly higher in the males, compared with the females, but there was no sex difference in the proportion of those cells receiving contacts that were ERalpha-ir. Neonatal administration of estradiol but not genistein masculinized 5-HT content in the adult female VMNvl, but the percentage of HuC/D-ir cells colabeled with ERalpha was not significantly affected by treatment. A similar, but not statistically significant, pattern was observed in the ARC. These findings suggest that the development of serotonergic inputs to the male VMNvl is orchestrated by neonatal estradiol exposure. The hormone-dependent organization of these 5-HT projection patterns may be an important developmental mechanism accounting for sex-specific behaviors in adulthood.

Differential disruption of nuclear volume and neuronal phenotype in the preoptic area by neonatal exposure to genistein and bisphenol-A.

Changes in the volumes of sexually dimorphic brain nuclei are often used as a biomarker for developmental disruption by endocrine-active compounds (EACs). However, these gross, morphological analyses do not reliably predict disruption of cell phenotype or neuronal function. In the present experiments, we used a more comprehensive approach to assess whether postnatal exposure to the EACs genistein (GEN) or bisphenol-A (BIS) affected the development of two sexually dimorphic brain regions in male rats: the anteroventral periventricular nucleus of the hypothalamus (AVPV) and the sexually dimorphic nucleus of the preoptic area (SDN). In addition to nuclear volumes, we also measured the number of immunopositive calbindin neurons in the SDN and the activational patterns of gonadotropin-releasing hormone (GnRH) neurons, a neuronal population that is functionally linked to the AVPV. In rats, exposure of the neonatal male brain to endogenous estrogen, aromatized from testicular testosterone, is essential for the proper sexual differentiation of these endpoints. Thus, we hypothesized that exposure to BIS and GEN during this critical period could disrupt brain sexual differentiation. Animals were given four subcutaneous injections of sesame oil (control), 250 microg GEN, or 250 microg BIS at 12 h intervals over postnatal days (PND) 1 and 2, gonadectomized on PND 85, and treated sequentially with estrogen and progesterone to stimulate Fos expression in GnRH neurons, a marker for their activation. A cohort of age-matched ovariectomized (OVX) females that were given the same hormone treatment in adulthood served as a positive control group. SDN volume was unchanged by treatment, but the number of calbindin neurons in the SDN was significantly increased by both BIS and GEN. GEN, but not BIS, demasculinized male AVPV volume, but patterns of GnRH neuronal activation were not affected by either compound. These results suggest that acute exposure to EACs during a critical developmental period can independently alter nuclear volumes of sexually dimorphic nuclei and their phenotypic profiles in a region specific manner.

Neonatal genistein or bisphenol-A exposure alters sexual differentiation of the AVPV.

There is growing concern that naturally occurring and chemically manufactured endocrine-active compounds (EACs) may disrupt hormone-dependent events during central nervous system development. We examined whether postnatal exposure to the phytoestrogen genistein (GEN) or the plastics component bisphenol-A (BIS) affected sexual differentiation of the anteroventral periventricular nucleus of the hypothalamus (AVPV) in rats. The AVPV is sexually differentiated in rodents. The female AVPV is larger than the male AVPV and contains a higher number of cells expressing tyrosine hydroxylase (TH). Sexual differentiation of the AVPV results from exposure of the male nervous system to estrogen aromatized from testicular testosterone secreted in the first few days after birth. Thus, we hypothesized that exposure to EACs during this critical period could alter the sexually dimorphic expression of TH and the overall expression of estrogen receptor alpha (ERalpha) in the AVPV. Animals were given 4 subcutaneous injections of sesame oil (control), 50 microg 17beta-estradiol (E2), 250 microg GEN, or 250 microg BIS at 12-h intervals over postnatal days (PND) 1 and 2 and sacrificed on PND 19. E2 treatment masculinized TH immunoreactivity (TH-ir) in the female AVPV while exposure to GEN or BIS demasculinized TH-ir in the male AVPV. In addition, we identified a population of neurons co-expressing TH and ERalpha located primarily in the medial region of the AVPV. Normally, females have nearly three times as many double-labeled cells as males, but their numbers were defeminized by E2, GEN or BIS treatment. These results suggest that acute exposure to EACs during a critical developmental period alters AVPV development.

Sequestration of p27 within the cytoplasm of cardiac myocytes in chronic ischemic heart disease: pathogenic implications for ischemic cardiomyopathy.

A number of recent studies have suggested that cardiac myocytes, previously considered post-mitotic, re-enter the cell cycle and possess the ability to proliferate with certain pathogenic stimuli. To study this further, we examined cellular proliferation in myocardial tissue from subjects with chronic ischemic heart disease-associated myocardial infarction and subsequent congestive heart failure. We found striking increases in cytoplasmic phospho-p27, a well-known mitotic regulator, compared to controls by both immunocytochemical and immunoblot analyses. However, we found no evidence for cardiac myocyte proliferation in either disease or control subjects using both mitotic counting (no mitotic figures were observed) and Ki-67 immunocytochemistry, which demonstrated a 0% proliferation index. That increased cytoplasmic phospho-p27 is not accompanied by division prompts us to speculate that ectopic cell cycle activation occurs in the face of minimal to absent myocyte proliferation per se. Based on these findings, and the parallel findings in post-mitotic neurons in neurodegenerative disease, we suggest that cell-cycle activation in ischemic heart disease is a deleterious event that perpetuates disease pathogenesis culminating in myocardial failure.

Chitin-like polysaccharides in Alzheimer's disease brains.

The role of polysaccharides in the pathogenesis of Alzheimer disease (AD) is unclear. However, in light of studies indicating impaired glucose utilization in AD and increased activation of the hexosamine pathway that is seen with hyperglycemia, in the brains of patients with AD, aberrantly high levels of glucosamine may result in synthesis of glucosamine polymers such as chitin, a highly insoluble polymer of N-acetyl glucosamine, linearized by beta1-4 linkages. To examine this further, we studied brain tissue at autopsy from subjects with sporadic and familial AD using calcofluor histochemistry. Calcofluor excites on exposure to ultraviolet light and exhibits a high affinity for chitin in vivo by interacting with beta1-4 linkages. Amyloid plaques and blood vessels affected by amyloid angiopathy showed bright fluorescence. Moreover, treatment with chitinase, followed by beta-N-acetyl glucosaminidase showed a decrease in calcofluor fluorescence. Since chitin is a highly insoluble molecule and a substrate for glycan-protein interactions, chitin-like polysaccharides within the brain could facilitate nucleation of amyloid proteins in various amyloidoses including AD.