Differing effects of copper,zinc superoxide dismutase overexpression on neurotoxicity elicited by nitric oxide, reactive oxygen species, and excitotoxins.

Overexpression of Cu,Zn superoxide dismutase (SOD1) reduces ischemic injury in some stroke models but exacerbates injury in a neonatal stroke model and in other settings. The current study used a SOD1 transgenic (SOD1-Tg) murine cortical culture system, derived from the same mouse strain previously used for the stroke models, to identify conditions that determine whether SOD1 overexpression in neurons is protective or detrimental. The nitric oxide (NO) donors S-nitroso-N-acetylpenicillamine, spermine-NONOate, and diethylamine-NONOate produced less death in SOD1-Tg neurons than in wild-type neurons (p < 0.01). Also, NO produced markedly less 3-nitrotyosine in SOD1-Tg cells. In contrast, the superoxide generator menadione produced significantly greater death and nearly twice as much 2'7'-dichlorofluorescein fluorescence in SOD1-Tg neurons than in wild-type neurons, suggesting increased peroxide formation in the SOD1-Tg cells. No significant difference was observed in the vulnerability of the two cell types to H2O2, the product of the SOD reaction. Overexpression of SOD1 also had no effect on neuronal vulnerability to glutamate, N-methyl-D-aspartate, or kainate. These observations suggest that SOD1 overexpression can reduce neuronal death under conditions where peroxynitrite formation is a significant factor, but may exacerbate neuronal death under conditions of rapid intracellular superoxide formation or impaired H2O2 disposal.

Glutamate induces rapid upregulation of astrocyte glutamate transport and cell-surface expression of GLAST.

Glutamate transporters clear glutamate from the extracellular space by high-affinity binding and uptake. Factors that regulate glutamate transporter expression and activity can thereby influence excitatory neurotransmission. Transporter function in GABAergic and other systems has been shown to be regulated by transporter substrates. Here, glutamate regulation of glutamate transport was studied using primary murine astrocyte cultures that express the GLAST (EAAT1) and GLT-1 (EAAT2) transporter subtypes. Glutamate was found to stimulate glutamate transport capacity (V(max)) in a dose- and time-dependent manner. The maximal increase was 100%, with an ED(50) of 40 microM glutamate and with onset beginning approximately 15 min after onset of glutamate exposure. The uptake stimulation was reproduced by D-aspartate, which is also a transporter substrate, but not by nontransported glutamate receptor agonists. Moreover, glutamate incubation did not stimulate transport when performed in a sodium-free medium, suggesting that the stimulatory effect of glutamate is triggered by increased transporter activity rather than receptor activation. Treatment with the actin-disrupting agents cytochalasin B or cytochalasin D prevented the glutamate-induced increase in glutamate uptake. Biotinylation labeling of membrane surface proteins showed that glutamate incubation produced an increase in GLAST expression at the astrocyte cell surface. These results suggest that cell-surface expression of GLAST can be rapidly regulated by glutamate through a process triggered by GLAST activity and involving the actin cytoskeleton. This feedback loop provides a mechanism by which changes in extracellular glutamate concentrations could rapidly modulate astrocyte glutamate transport capacity.

Astrocyte energetics, function, and death under conditions of incomplete ischemia: a mechanism of glial death in the penumbra.

Cerebral artery occlusion produces regions of incomplete ischemia (the ischemic penumbra), which, in the absence of reflow, undergo progressive metabolic deterioration culminating in infarction. The factors causing infarction are not yet established, but progression to cell death is preceded by progressive acidosis, decreasing glucose utilization, and ATP depletion. To identify potential mechanisms of glial death in the ischemic penumbra, astrocytes in culture were subjected to conditions that occur during incomplete ischemia: hypoxia, acidosis, and raised extracellular K+. Neither acidosis (to pH 6.2) nor chemical hypoxia (5 mM azide) alone produced significant astrocyte death or marked ATP depletion. By contrast, hypoxia combined with acidosis caused near-complete ATP depletion by 3.5 h and 70% cell death after 7 h. Glycolytic rate increased during hypoxia alone but decreased during hypoxia with acidosis. Since glycolysis is the sole source of ATP production during hypoxia, acidosis inhibition of glycolysis is a likely cause of the far greater ATP depletion resulting from hypoxia with acidosis. Glutamate uptake was reduced during hypoxia and further reduced during hypoxia with acidosis, consistent with the changes in astrocyte ATP. Glutamate uptake, ATP levels, and glycolytic rate each exhibited reductions that were progressive over 3 h of hypoxia with acidosis, and these changes were accompanied by progressive intracellular acidosis. Since ATP depletion leads to acidosis, and acidosis inhibits glycolysis, these findings suggest a regenerative cycle initiated by the combination of hypoxia with acidosis. This cycle could result in progressive metabolic decline and cell death in the ischemic penumbra.

Neuronal regulation of glutamate transporter subtype expression in astrocytes.

GLT-1, GLAST, and EAAC1 are high-affinity, Na(+)-dependent glutamate transporters identified in rat forebrain. The expression of these transporter subtypes was characterized in three preparations: undifferentiated rat cortical astrocyte cultures, astrocytes cocultured with cortical neurons, and astrocyte cultures differentiated with dibutyryl cyclic AMP (dBcAMP). The undifferentiated astrocyte monocultures expressed only the GLAST subtype. Astrocytes cocultured with neurons developed a stellate morphology and expressed both GLAST and GLT-1; neurons expressed only the EAAC1 transporter, and rare microglia in these cultures expressed GLT-1. Treatment of astrocyte cultures with dBcAMP induced expression of GLT-1 and increased expression of GLAST. These effects of dBcAMP on transporter expression were qualitatively similar to those resulting from coculture with neurons, but immunocytochemistry showed the pattern of transporter expression to be more complex in the coculture preparations. Compared with astrocytes expressing only GLAST, the dBcAMP-treated cultures expressing both GLAST and GLT-1 showed an increase in glutamate uptake Vmax, but no change in the glutamate K(m) and no increased sensitivity to inhibition by dihydrokainate. Pyrrolidine-2,4-dicarboxylic acid and threo-beta-hydroxyaspartic acid caused relatively less inhibition of transport in cultures expressing both GLAST and GLT-1, suggesting a weaker effect at GLT-1 than at GLAST. These studies show that astrocyte expression of glutamate transporter subtypes is influenced by neurons, and that dBcAMP can partially mimic this influence. Manipulation of transporter expression in astrocyte cultures may permit identification of factors regulating the expression and function of GLAST and GLT-1 in their native cell type.

Increased lectin binding capacity of trophoblastic cells of late day 5 rat blastocysts.

The binding of lectins to the trophoblast of rat blastocysts has been studied using quantitative ultrastructural cytochemistry. Rat blastocysts from early, mid and late d 5 of gestation were stained using biotinylated lectins (Phytolacca americana [Phy am], fucose binding protein [FBP] and soybean agglutinin [SBA]) and a sensitive avidin-ferritin cytochemical method. Electron micrographs of ferritin particles along the membrane were processed to produce images for which grey scale levels could be established and the ferritin particles automatically counted. The ferritin:membrane ratio was then calculated. Increased binding with Phy am (which detects short chain oligosaccharides) was found after midday of d 5, i.e. after hatching. Binding of FBP and SBA did not alter during the period studied. The increased concentration of oligosaccharides on the blastocyst surface membrane after hatching may have important implications for blastocyst attachment to the endometrium.

Morphometric evidence of changes in the vasculature of the uterine tube of mice induced by the 2-cell embryo on the second day of pregnancy.

Some evidence suggests that the uterine tube plays an active role in supporting the development of the preimplantation embryo. To determine whether there were morphological and/or ultrastructural changes in the uterine tube as a consequence of pregnancy, the region of the uterine tube containing 2-cell embryos or oocytes was examined in untreated d 2 pregnant and pseudopregnant mice. The general morphology of the uterine tube was assessed by light and electron microscopy. Qualitative assessment of uterine tube morphology suggested that although the epithelium and muscular layers of the oviduct were similar in pregnancy and pseudopregnancy there were differences in the vasculature between the 2 groups. In transverse sections of the uterine tube, cross-sectional vascular surface area was assessed morphometrically. There was a statistically significant decrease (P < 0.001) in the cross-sectional vascular surface of the oviduct in pregnant mice compared with pseudopregnant mice. This reduction was apparently due to (1) a decrease in the median cross-sectional surface area of small intramural blood vessels, and (2) an apparent collapse of many subepithelial capillaries. Pregnant mice also had fewer fenestrated capillaries whereas such vessels were common in pseudopregnancy. Activated, aggregated platelets were only observed in the capillaries and venules of pregnant, untreated mice. Some animals were treated with 10 micrograms WEB 2086/day on d 1 and d 2, a platelet-activating factor receptor antagonist. This treatment reversed the reduced vascular surface cross-sectional area found in early pregnancy, but had no effect on vascular measurements in pseudopregnancy.

The relationship between life events during adolescence and affect and personality functioning.

Bowlby's concept of the internal working model is useful in conceptualizing how a child develops a sense of self and security through the availability of significant relationships. The lack of secure attachments may lead to dysphoric moods and poor personality functioning. We have shown that a proportion of teenagers develop dysphoria as well as personality disturbance during adolescence. A study sample of 59 youths was examined at early, middle and late adolescence. Ratings were made of affect, attitudes and personality functions. At the same time, information was obtained about family status and functioning. Results showed that certain family and life events were more strongly correlated with changes in affect in early adolescence. These affects were both internalized and externalized. Middle adolescence appeared to be quiescent, without any correlations with affect or personality problems. In late adolescence, there were correlations between family changes and personality dysfunction as well as dysphoria. This shows that family changes which threaten the availability of significant attachment figures are linked to emotional and behavioural disturbance in early adolescence and personality and emotional disturbance in late adolescence.

Dose-dependent effects of exogenous gonadotrophins on the endometrium of the rat.

We compared the serum levels of oestrogen and progesterone and the endometrial morphology of normal pregnant rats at 5,5 days' gestation with those of pregnant rats given either low (10 IU) or high (20 IU) doses of two gonadotrophins: follicle-stimulating hormone (FSH) and human chorionic gonadotrophin (HCG). Evidence of ovarian hyperstimulation was observed in the high- but not the low-dose group; both treatment regimens caused significant changes in the endometrial surface, epithelial height, the microvillous border, the glycocalyx, the subepithelial stromal cells and the mitotic activity of the surface epithelial and stromal connective tissue cells. The effects of the high-dose treatment were more severe than those of the low-dose treatment. The serum oestradiol and progesterone levels of the treated groups were not significantly different from those of the control group. The changes in the endometrium after both treatment regimens may interfere with normal trophoblastic-endometrial interactions and could influence the maintenance of pregnancy. This investigation demonstrated that even low doses of gonadotrophins, which do not cause obvious ovarian stimulation, affect uterine morphology. The findings have important implications for in vitro fertilisation and embryo transfer programmes.

Consistency and change in personality characteristics and affect from middle to late adolescence.

Non clinical adolescents in a longitudinal study were examined at ages 16 and 18 to see whether or not there were changes in personality functioning, attitudes and affect over time, and whether or not these were related to each other. Personality functioning remained fairly consistent at both ages, with marked personality problems occurring in about one out of seven. As a group, the older adolescents demonstrated more anxiety and depression, as well as changes in certain attitudes such as more curiosity and interest in people. For individual adolescents it was found that there was considerable predictability of affect and attitudes at age 18 from the presentation at age 16. Furthermore, a relative increase in the amount of anger in middle adolescence was a predictor of personality problems in late adolescence.