Gene transfer of calbindin D28k cDNA via herpes simplex virus amplicon vector decreases cytoplasmic calcium ion response and enhances neuronal survival following glutamatergic challenge but not following cyanide.

Excitatory amino acid overstimulation of neurons can lead to a marked rise in cytoplasmic Ca2+ concentration ([Ca2+])i) and be followed by neuron death from hours to days later. If the rise in [Ca2+]i is prevented, either by removing Ca2+ from the extracellular environment or by placing Ca2+ chelators in the cytosol of the stimulated cells, the neurotoxicity associated with excitotoxins can be ameliorated. We have recently shown that neurons infected with a herpes simplex virus amplicon vector expressing cDNA for calbindin D28k responded to hypoglycemia with decreased [Ca2+]i and increased survival relative to controls. We now report that vector-infected neurons respond to glutamatergic insults with lower [Ca2+]i than controls and with increased survival. Infected neurons exposed to sodium cyanide did not respond with lower [Ca2+]i than controls, nor did they demonstrate increased survival postinsult. We examine these results in light of our earlier report and in the context of the potential of vectors like this for neuronal gene therapy.

Energy dependency of glucocorticoid exacerbation of gp120 neurotoxicity.

The HIV envelope glycoprotein, gp120, a well documented neurotoxin, may be involved in AIDS-related dementia complex. gp120 works through an NMDA receptor- and calcium-dependent mechanism to damage neurons. We have previously demonstrated that both natural and synthetic glucocorticoids (GCs) exacerbate gp120-induced neurotoxicity and calcium mobilization in hippocampal mixed cultures. GCs, steroid hormones secreted during stress, are now shown to work in conjunction with gp120 to decrease ATP levels and to work synergistically with gp120 to decrease the mitochondrial potential in hippocampal cultures. Furthermore, energy supplementation blocked the ability of GCs to worsen gp120's effects on neuronal survival and calcium mobilization. A GC-induced reduction in glucose transport in hippocampal neurons, as previously documented, may contribute to this energetic dependency. These results may have clinical significance, considering the common treatment of severe cases of Pneumocystis carinii pneumonia, typical of HIV infection, with large doses of synthetic GCs.

Glucocorticoids may alter antioxidant enzyme capacity in the brain: baseline studies.

Glucocorticoids (GCs), the adrenal steroids secreted during stress, have been shown to increase the vulnerability of hippocampal neurons to metabolic insults, potentially by altering the neuronal defense capacity against oxidative damage. These experiments assessed the effect of long term in vivo GC supplementation on basal activity of the antioxidant enzymes copper/zinc superoxide dismutase (Cu/Zn SOD), manganese superoxide dismutase (Mn SOD), catalase, and glutathione peroxidase (GSPx). Kinetic enzyme studies were done using brain tissue from the hippocampus, cortex, cerebellum, and also from liver as a peripheral control. Cu/Zn SOD activity was significantly lower in all brain regions of GC-treated rats, but higher in the liver. Mn SOD activity was unaffected by treatment. Catalase in the brain appeared largely unaffected by GC treatment, although liver catalase was significantly decreased. GSPx activity was significantly decreased by GCs at high peroxide levels in all tissues. These results indicate that the presence of GCs may lower the antioxidant capacity of tissues in a region-specific manner, and that the deficit may not appear until the tissue is challenged with supranormal levels of oxidative products (as seen with GSPx).

Glucocorticoids may alter antioxidant enzyme capacity in the brain: kainic acid studies.

Glucocorticoids (GCs) predispose hippocampal neurons to damage during metabolic stressors. One component of hippocampal GC-endangerment may be changes in neuronal defenses against oxidative challenge. Previous experiments showed a decrease in basal levels of copper/zinc superoxide dismutase (Cu/Zn SOD) and glutathione peroxidase (GSPx) in the brain of rats treated with GCs [L. McIntosh, K. Hong, R. Sapolsky, Glucocorticoids may alter antioxidant enzyme capacity in the brain: baseline studies, 1997.]. In this study we administered the excitotoxin kainic acid (KA) to generate reactive oxygen species (ROS) in the brain, and monitored the activity of four antioxidant enzymes over 24 h in GC-free and GC-supplemented rats. We tested the response pattern in three regions of the brain (hippocampus, cortex, cerebellum) and the liver as a peripheral control. In the hippocampus, KA induced Cu/Zn SOD and catalase, but GCs prevented the induction of catalase and maintained the lowered GSPx activity seen previously in the baseline studies. In the cortex, KA induced Cu/Zn SOD, Mn SOD and catalase activity, but there was no significant GC effect. There was no response to KA in the cerebellum, but GCs decreased GSPx activity. In the liver, KA produced a rise in Cu/Zn SOD and catalase activity, and GC-treated animals showed a slower return to baseline. These experiments indicate that the impairment of antioxidant enzyme defenses, particularly the hippocampal peroxidases, could be a component of GC-mediated neuroendangerment.

Potential behavioral modification of glucocorticoid damage to the hippocampus.

Glucocorticoids (GCs), the adrenal steroids secreted during stress, can damage the hippocampus, a principal neural target site for GCs. The extent of cumulative exposure to GCs influences the rate of neuron loss in the aging hippocampus, such that stress can accelerate senescent hippocampal degeneration. Moreover, under circumstances where GC exposure is insufficient to damage neurons, the hormones impair the capacity of neurons to survive neurological insults such as hypoxia-ischemia, seizure, or hypoglycemia. Considerable progress has been made in understanding how GCs endanger hippocampal neurons. The effect is a direct one, in that the endangerment is mediated by GC receptors and occurs in cultured hippocampal neurons. The endangerment is energetic in nature--the insults worsened by GCs represent energetic crises, and the GC endangerment is prevented by supplementation of neurons with energy substrates. As the likely mechanism by which GCs induce an energetic vulnerability, the steroids inhibit glucose transport in hippocampal neurons and glia. As a result of this effect of GCs upon energetics is that neurons are less capable of the costly task of containing the damaging fluxes of glutamate and calcium triggered by the neurological insults. Thus, following such insults, GCs disrupt glutamate removal and elevate synaptic glutamate concentrations, enhance the magnitude and duration of the subsequent mobilization of free cytosolic calcium, and exacerbate the magnitude of calcium-dependent degenerative events. Thus, stress has the capacity to damage the hippocampus and exacerbate the toxicity of some common neurological disorders; nevertheless, some behavioral interventions are known to cause sustained diminution of GC concentrations, and thus have the potential to protect the hippocampus from these deleterious effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Corticosterone exacerbates kainate-induced alterations in hippocampal tau immunoreactivity and spectrin proteolysis in vivo.

Aberrant elevations in intracellular calcium levels, promoted by the excitatory amino acid glutamate, may be a final common mediator of the neuronal damage that occurs in hypoxic-ischemic and seizure disorders. Glutamate and altered neuronal calcium homeostasis have also been proposed to play roles in more chronic neurodegenerative disorders, including Alzheimer's disease. Any extrinsic factors that may augment calcium levels during such disorders may significantly exacerbate the resulting damage. Glucocorticoids (GCs), the adrenal steroid hormones released during stress, may represent one such extrinsic factor. GCs can exacerbate hippocampal damage induced by excitotoxic seizures and hypoxia-ischemia, and we have observed recently that GCs elevate intracellular calcium levels in hippocampal neurons. We now report that the excitotoxin kainic acid (KA) can elicit antigenic changes in the microtubule-associated protein tau similar to those seen in the neurofibrillary tangles of Alzheimer's disease. KA induced a transient increase in the immunoreactivity of hippocampal CA3 neurons towards antibodies that recognize aberrant forms of tau (5E2 and Alz-50). The tau immunoreactivity appeared within 3 h of KA injection, preceded extensive neuronal damage, and subsequently disappeared as neurons degenerated. KA also caused spectrin breakdown, indicating the involvement of calcium-dependent proteases. Physiological concentrations of corticosterone (the species-typical GC of rats) enhanced the neuronal damage induced by KA and, critically, enhanced the intensity of tau immunoreactivity and spectrin breakdown. Moreover, the GC enhancement of spectrin proteolysis was prevented by energy supplementation, supporting the hypothesis that GC disruption of calcium homeostasis in the hippocampus is energetic in nature. Taken together, these findings demonstrate that neurofibrillary tangle-like alterations in tau, and spectrin breakdown, can be induced by excitatory amino acids and exacerbated by GCs in vivo.

Patterns of adrenocorticotropin secretagog release with hypoglycemia, novelty, and restraint after colchicine blockade of axonal transport.

Colchicine blockade of axonal transport from the paraventricular nucleus to the median eminence was used to indirectly infer hypothalamic ACTH secretagog release in awake rats. Median eminence contents of CRF, arginine vasopressin (AVP) and oxytocin (OT) were determined by RIA after insulin-induced hypoglycemia, restraint, and novelty. Insulin decreased circulating glucose concentrations and increased ACTH and corticosterone values. Median eminence CRF and AVP content declined but OT content did not. Both novelty and restraint stressors increased circulating ACTH and corticosterone concentrations. Secretagog measurements indicated decreases in OT content without concomitant decreases in either CRF or AVP with both stressors. These results indicate that: 1) colchicine blockade of axonal transport is useful in studying patterns of secretagog release in animals undergoing psychological stressors; 2) in contrast to physical stressors, OT appears to be a major component of the hypothalamic-pituitary-adrenal response to psychological stress; 3) the patterns of secretagog release differ with regards to physical and psychological stressors.

Corticosterone enhances kainic acid-induced calcium elevation in cultured hippocampal neurons.

Corticosterone, a steroid secreted during stress, increases hippocampal neuronal vulnerability to excitotoxins, hypoxia-ischemia, and antimetabolites. Energy supplementation and N-methyl-D-aspartate receptor antagonists prevent this corticosterone-enhanced neurotoxicity. Because neuronal calcium regulation is energy dependent and a large calcium influx accompanies N-methyl-D-aspartate receptor activation, we investigated whether corticosterone exacerbates the elevation of hippocampal neuronal calcium induced by the glutamatergic excitotoxin kainic acid. Corticosterone caused a 23-fold increase in the magnitude of the calcium response to kainic acid, a sevenfold increase in the peak magnitude of the calcium response, and a twofold increase in calcium recovery time. This corticosterone effect may be energetic in nature as corticosterone decreases hippocampal neuronal glucose transport. Glucose supplementation reduced the corticosterone effect on the magnitude and peak magnitude of the calcium response to kainic acid. Glucose reduction, by the approximate magnitude by which corticosterone inhibits glucose transport, mimicked the corticosterone effect on the peak magnitude of the calcium response to kainic acid. Thus, corticosterone increases calcium after kainic acid exposure in hippocampal neurons in an energy-dependent manner. Elevated calcium is strongly implicated in stimulating neurotoxic cascades during other energetic insults and may be the mechanism for the corticosterone-induced hippocampal neuronal vulnerability and toxicity.

Behavioral, endocrine, and immunological correlates of immigration by an aggressive male into a natural primate group.

A very aggressive young adult male entered one of three long-term study groups of yellow baboons. Papio cynocephalus, approximately 3 weeks after an immobilization project began. The immigrant male's rate of agonistic encounters was appreciably higher than average, and these interactions disproportionately involved adult females as targets. Basal cortisol concentrations were higher and total lymphocyte counts lower for individuals immobilized during the immigration situation than for other individuals; these effects were greater for females than for males. Among animals whose endocrine data were obtained during the immigration period, some were specific targets of the immigrant male's aggression and others were not. Lymphocyte counts were significantly lower for those individuals who were victims of the male's aggression than for noninvolved individuals; a nonsignificant tendency toward higher basal cortisol concentrations for victims was observed as well. The immigrant male himself had a high basal cortisol concentration, a low lymphocyte count, and a testosterone concentration that was triple the average for adult males and almost double the second highest value in the population.

Glucocorticoids inhibit glucose transport and glutamate uptake in hippocampal astrocytes: implications for glucocorticoid neurotoxicity.

Glucocorticoids (GCs), the adrenal steroid hormones secreted during stress, can damage the hippocampus and impair its capacity to survive coincident neurological insults. This GC endangerment of the hippocampus is energetic in nature, as it can be prevented when neurons are supplemented with additional energy substrates. This energetic endangerment might arise from the ability of GCs to inhibit glucose transport into both hippocampal neurons and astrocytes. The present study explores the GC inhibition in astrocytes. (1) GCs inhibited glucose transport approximately 15-30% in both primary and secondary hippocampal astrocyte cultures. (2) The parameters of inhibition agreed with the mechanisms of GC inhibition of glucose transport in peripheral tissues: A minimum of 4 h of GC exposure were required, and the effect was steroid specific (i.e., it was not triggered by estrogen, progesterone, or testosterone) and tissue specific (i.e., it was not triggered by GCs in cerebellar or cortical cultures). (3) Similar GC treatment caused a decrease in astrocyte survival during hypoglycemia and a decrease in the affinity of glutamate uptake. This latter observation suggests that GCs might impair the ability of astrocytes to aid neurons during times of neurologic crisis (i.e., by impairing their ability to remove damaging glutamate from the synapse).

Glucocorticoid endangerment of the hippocampus: tissue, steroid and receptor specificity.

Glucocorticoids (GCs) can damage the hippocampus when exposure is prolonged, as well as impair the capacity of hippocampal neurons to survive various neurological insults. We have recently demonstrated that GCs impair the capacity of primary hippocampal cultures to survive many of these same insults. Using this culture system, we have characterized the features with which the GC corticosterone (CORT) impairs the capacity of these cells to survive the excitotoxin kainic acid. The GC endangerment seems to be mediated by the type II, but not type I corticosteroid receptor. As evidence for type II involvement, endangerment of cells was caused by CORT concentrations in the kilodalton range for the type II receptor, and also by the type II ligand dexamethasone; moreover, the endangerment was blocked by a type II antagonist. In contrast, a type I antagonist was not protective. Cultures contained both type I and II receptors. The effect was GC-specific, as cultures were endangered by CORT, cortisol and dexamethasone, but not by non-GC steroids. GCs did not exacerbate kainic acid damage in cerebeller or hypothalamic cultures, despite the presence of corticosteroid receptors. This agrees with the in vivo data showing that the GC exacerbation of neurological insults is either exclusive to or predominant in the hippocampus.

Thyroid hormones influence the development of hippocampal glucocorticoid receptors in the rat: a mechanism for the effects of postnatal handling on the development of the adrenocortical stress response.

The role of thyroid hormones on the development of intracellular glucocorticoid receptor concentrations was examined in the hippocampus, hypothalamus, and pituitary of the rat. Adult animals, administered triiodothyronine (T3; 1.0 micrograms/g body weight) on days 1, 2, and 4 of life or thyroxine (T4; 2.5 micrograms/g body weight) on days 1 and 2 of life, had significantly elevated glucocorticoid receptor concentrations in the hippocampus, but not in hypothalamus or pituitary. Adult animals treated with propylthiouracil (PTU; 0.2% in the mother's food), a thyroid hormone synthesis inhibitor, for the first 2 weeks of life showed decreased glucocorticoid receptor concentrations in hippocampus, but not in hypothalamus or pituitary. We then examined whether thyroid hormones might mediate the effects of early stimulation on the development of hippocampal glucocorticoid receptor concentrations. Animals that were handled for 15 min daily (Ha) for the first 2 weeks of life showed increased hippocampal glucocorticoid receptor concentrations as adults compared to nonhandled (NHa) controls. PTU administration blocked the effects of handling, such that Ha/PTU animals showed hippocampal glucocorticoid receptor concentrations that were indistinguishable from those of NHa animals. In contrast, corticosterone administration over the first 2 weeks of life had no effect on adult hippocampal glucocorticoid receptor concentrations. These data suggest that thyroid hormones mediate, in part at least, the development of glucocorticoid receptor concentrations in the hippocampus and that this effect occurs independently of their effects on corticosterone titers.

Early postnatal handling alters glucocorticoid receptor concentrations in selected brain regions.

Norway rat pups were either handled (H) or undisturbed (nonhandled, NH) in the period between birth and weaning on Day 21. Following weaning, half of the animals in each group were housed socially (Soc), and half were housed in isolation (Isol). At 120-150 days of age, all animals were sacrificed, and the following regions were dissected and frozen at -70 degrees C until the time of assay: frontal cortex, hippocampus, hypothalamus, amygdala, septum, and pituitary. [3H]Dexamethasone (3H Dex) binding in each region was examined by an in vitro, cytosol, receptor assay. 3H Dex binding was significantly higher in the hippocampus of both H-Soc and H-Isol than in NH groups. In the frontal cortex, 3H Dex binding was higher in the H-Soc animals than in the H-Isol and NH-Isol animals. There were no significant handling or housing effects found in the amygdala, hypothalamus, septum, or pituitary. Thus, early postnatal handling appears to influence the development of the glucocorticoid receptor system in the hippocampus and frontal cortex. These results are discussed as providing a possible mechanism for some of the previously reported effects of early handling on the development of the pituitary-adrenal response to stress.

Down-regulation of neural corticosterone receptors by corticosterone and dexamethasone.

Stress or elevated corticosterone titers can reduce the concentration of corticosterone receptors in the brain. We demonstrate that corticosterone and the related glucocorticoid, dexamethasone, induce different anatomical patterns of such 'down-regulation'. Corticosterone administration reduces receptor number in the hippocampus, particularly the CA1 and CA2 pyramidal cell fields, but nowhere else in the brain or pituitary. In contrast, equivalent dosages of dexamethasone down-regulate pituitary, amygdaloid and hypothalamic corticosterone receptor numbers. These different anatomical profiles of sensitivity to down-regulation appear due to differential access of the two steroids to the receptor pools.

The adrenocortical stress-response in the aged male rat: impairment of recovery from stress.

To test if the adrenocortical axis of the rat loses sensitivity to negative feedback control during aging, we examined corticosterone secretion under basal, stressed and post-stress conditions in young and in aged Fisher male rats. We find no age-related change in the speed or magnitude of the stress response or in the capacity to manifest a corticosterone response following chronic stress. However, we do observe in aging rats an elevation of basal corticosterone and an impaired capacity to adapt to and recover from stress. This latter finding is illustrated by longer latencies relative to young rats, in the return of serum corticosterone concentrations to basal values during sustained exposure to mild cold or following the end of immobilization stress. All of these deficits reflect an increased rate of corticosterone secretion during physiologically inappropriate circumstances. Such observations support the concept that there is an age-related loss of sensitivity of the brain and pituitary to the inhibitory effects of high circulating levels of corticosterone on ACTH release.