Heinz H. Osterburg

Messenger RNA for glial fibrillary acidic protein is decreased in rat brain following acute and chronic corticosterone treatment

RNA coding for a 50 kDa polypeptide decreased by 50% in 5 brain regions after corticosterone (CORT) treatment (40 mg/kg for 3 days). By hybrid selection and in vitro translation, the 50 kDa polypeptide is identified as glial fibrillary acidic protein (GFAP). Hippocampal GFAP mRNA (2.9 kb) decreases in a dose-dependent manner in response to CORT by RNA blot hybridization using a mouse GFAP cRNA probe; a similar decrease in response to the glucocorticoid agonist, RU 28362, is consistent with a type II glucocorticoid receptor-mediated effect. GFAP mRNA is decreased in both hippocampus and cortex following acute (1-3 days) and chronic (3 days to 3 months) CORT treatment. GFAP gene expression is disinhibited in the rat hippocampus by 7 days post adrenalectomy but not by 3 days. Finally, two clones (CR46 and CR59) that were isolated from a rat hippocampal cDNA library by differential hybridization, show decreased RNA abundance in CORT-treated rats compared to controls. A partial DNA sequence derived from the two clones exhibits 94% nucleotide identity and 96% derived amino acid identity with mouse GFAP mRNA. These results indicate that GFAP mRNA is under negative regulation by glucocorticoids and suggests that glucocorticoids may be used to inhibit GFAP gene expression in vivo in order to assess the role of GFAP in temporal aspects of central nervous system damage.

Slow changes of tyrosine hydroxylase gene expression in dopaminergic brain neurons after neurotoxin lesioning: a model for neuron aging

Slow neuron regression develops during the adult phase of life in select brain systems of mammals. We describe a model in adult rats that resolves several phases in a slow atrophic process that differentially influences levels of mRNA and protein for tyrosine hydroxylase (TH). Responses of striatal dopaminergic markers to 6-hydroxydopamine (6-OHDA) lesions in rats indicated that the striatal terminals maintained TH protein, despite greater than 3-fold loss of TH mRNA in the substantia nigra pars compacta (SNC) cell bodies whose axons project to the striatum. The loss of TH mRNA/cell was progressive up to 9 months, whereas SNC cell body shrinkage stabilized by 3 months post-lesioning. Consideration of possible mechanisms in protein turnover motivated a search for PEST motifs in the TH of rats and other vertebrates that could be a point of regulation by altering the rate of TH protein turnover.

Elevated Density of [3H]Imipramine Binding in Aged Human Brain

Abstract: Aging was associated with an increase in the density of specific binding sites for [3H]imipramine in postmortem specimens of human hypothalamus, frontal cortex, and parietal cortex. In general, [3H]imipramine binding was not affected by factors considered difficult to control in postmortem studies, i.e., time from death to autopsy and cause of death. The in vitro regulation of [3H]imipramine binding by sodium was impaired with age in hypothalamic homogenates. In vitro regulation of [3H]imipramine binding by chloride was intact. Determination of the concentrations of 5‐hydroxytryptamine (serotonin) and 5‐hydroxyindoleacetic acid in hypothalamus and frontal cortex indicated no apparent age‐related changes in indole metabolism. The age‐related increase in brain [3H]imipramine binding and impairment in the in vitro regulation of binding by ions are similar to changes observed previously in aged mouse brain. The increase in brain antidepressant binding sites is discussed in relationship to other indices of brain serotonergic function in aging and to the relationship of [3H]imipramine binding and depression.

Age-dependent increases of DNA adducts (I-compounds) in human and rat brain DNA

Brain DNA from 20 humans ranging in age from neonatal to 100 years was analyzed by the nuclease P1-enhanced version of the 32P-postlabeling assay for bulky covalently modified nucleotides. A reproducible pattern of three 32P-labeled spots was obtained by thin-layer chromatography followed by autoradiography. Two of these spots increased with age (Mann-Whitney U-test; P < 0.001; comparison of ages < or = 60 years and ages > 60 years). Thus, these spots met the definition of I-compounds. Rat brain DNA exhibited the same two I-spots, whose intensities also increased with animal age (1, 4, and 10 months). In humans, considerable individual variation of brain I-compound levels was observed, especially at ages > 60 years, presumably reflecting environmental, life-style, or genetic factors. This variation was not noted for brain DNA of laboratory rats. Thus, human brain DNA undergoes progressive covalent modifications with aging.

Brain Poly(A)RNA During Aging: Stability of Yield and Sequence Complexity in Two Rat Strains

To evaluate whether major age‐related changes occur during aging in the brain, we determined the sequence complexity of total poly(A)RNA and polysomal poly(A)RNA of brains from Sprague‐Dawley and Fischer 344 male rats, aged 2‐32 months. RNA complexity, determined by RNA‐driven hybridization reactions with nonrepetitive DNA, was 6‐7 × 108 nucleotides for total poly(A)RNA, whereas polysomal poly(A)RNA had a sequence complexity of 2.3‐2.8 × 108 nucleotides. Sprague‐Dawley rats had polysomal poly(A)RNA with a yield per gram of brain and a complexity that was 20% greater than Fischer 344 rats.

Studies on ovarian-hypothalamic-pituitary interactions during reproductive aging in C57BL/6J mice

Abstract Studies on the reproductive senescence of C57BL/6J female mice from this and other laboratories are reviewed. Although the ovary ages irreversibly as monitored by the loss of primary oocytes and follicles, at ages (9–12 mo) when estrous cycles lengthen and fertility decreases strikingly, normal numbers of ova are still produced. Analysis of hormone output reveals impairments in the spontaneous and experimentally induced LH surges of aging mice which imply altered transduction of the estradiol signal triggering the release of LHRH. The loci responsible for this failure to transduce the estradiol signal are unknown, but could involve hypothalamic monoaminergic transmission as well as the LHRH neurons. Morphologic studies on C57BL/6J mice and other rodents indicate degenerative changes in arcuate neuronal processes with concommitant glial hyperactivity and reduced density of LHRH containing fibres. Recent evidence indicates that exposure to estradiol is one cause of the hypothalamic changes associated with reproductive senescence. Age changes in the regulation of the LH surge are delayed by long-term ovariectomy, and accelerated by short-term exposure to high levels of estradiol. These results are paralleled by morphologic changes in the arcuate nucleus. Taken together, the available data suggest that there is a cumulative impact of steroids on hypothalamic loci critical to the LH surge which is independent of chronologic age.

Methylation of the glial fibrillary acidic protein gene shows novel biphasic changes during brain development

The gene for glial fibrillary acidic protein (GFAP) was analyzed in the rat for developmental changes in methylation of cytosine at CpG sequences as a correlate of the onset of GFAP mRNA expression and for the effect of methylation on GFAP promoter activity. The methylation of nine CpG sites in the GFAP promoter and ten sites in exon 1 was analyzed in F344 rats by a quantitative application of ligation‐mediated polymerase chain reaction. Whole rat brain poly(A) + RNA showed an exponential increase of GFAP mRNA after embryo day 14 that reached stable adult levels by postnatal day 10. During development, only the seven CpG sites in the far‐upstream promoter showed large changes in methylation; these sites constitute the brain‐specific domain of methylation described in adult rats (Teter et al: J Neurosci Res 39:680, 1994). These seven CpG sites showed a cycle of demethylation during the onset of GFAP transcription in the embryo (between embryonic day 14 and postnatal day 10) followed by remethylation at later postnatal ages when GFAP mRNA remains prevalent. The minimum levels of methylation across these CpG sites displayed a gradient with the lowest minima at the 3′ sites. This demethylation/remethylation cycle is a novel phenomenon in DNA methylation during perinatal development. The demethylation/remethylation cycle during development was also shown by the opposite‐strand cytosines. Two cytosines in this region that are conserved in rat and mouse also undergo the same demethylation/remethylation cycle in the mouse GFAP gene during development, implying evolutionary conservation and functional significance. As a further test of functional significance, a Luciferase reporter gene assay was evaluated in primary cultured astrocytes; the activity of the GFAP promoter was reduced when it was methylated at one or all CpG sites. Therefore, the GFAP promoter may be activated in rodent development by transient demethylation of a conserved brain‐specific methylation domain.

Hypothalamic monoamines and their catabolites in relation to the estradiol-induced luteinizing hormone surge

Monoamines and non-conjugated catabolites (serotonin (5-HT), 5-hydroxyindole acetic acid (5-HIAA), 3,4-dihydroxyphenyl-acetic acid (DOPAC), homovanillic acid (HVA), 4-hydroxy-3-methoxyphenylethyleneglycol (MHPG), norepinephrine (NE), and dopamine (DA] were measured in the medial basal hypothalamus (MBH) and preoptic area (POA) of ovariectomized (OVX) and OVX estradiol (E2)-treated rats using high-performance liquid chromatography with electrochemical detection. These E2 treatments were sufficient to induce an LH surge. The use of MHPG/NE ratios as estimates of NE release was validated in the rat hypothalamus by the major decreases of MHPG after injection of the alpha 2-adrenergic agonist, clonidine, and by MHPG increases after the alpha 2-antagonist, yohimbine. The ratio, MHPG/NE, decreased between morning and afternoon in the MBH but not in the POA; there were no differences between OVX and E2-treated rats. Previous studies using a variety of methods indicate that NE turnover increases during LH surges. The present data suggest that unconjugated MHPG is not a sensitive measure of NE release in the rat hypothalamus, but can detect the large changes produced by stimulating or inhibiting the alpha 2-adrenergic autoreceptor. The ratios of DOPAC/DA and 5-HIAA/5-HT in the MBH decreased consistently between morning and afternoon in OVX rats, with or without E2 treatment. This suggests that the release of DA and 5-HT decreases during the day regardless of steroidal milieu.

Aging and haloperidol-induced dopamine turnover in the nigro-striatal pathway of C57BL/6J mice

The responsiveness of male C57BL/6J mice to acute haloperidol (2.5 mg/kg, IP) was studied throughout the average adult lifespan (4, 8, 12, 21, 28 months) by effects on dopamine (DA) turnover, as estimated by DA loss after alpha-methyl-p-tyrosine (AMPT). Previously, striatal receptors for spiroperidol (a related butyrophenone) decreased progressively after 3 months to a loss of 40% by 28 months [29]. Haloperidol treatment (2 hours) accelerated striatal DA turnover similarly in all age groups, by about 100%. The haloperidol-induced accumulation of 3,4-dihydroxyphenylacetic acid (DOPAC) was not significantly impaired in AMPT treated mice with age. These results suggest that the age-related loss of butyrophenone binding sites may not limit acute compensatory responses to blockade of the remaining sites by a large dose of haloperidol. Regional differences in DA metabolism were detected between the substantia nigra (cell bodies and dendrites) and striatum (axonal terminals). Dopamine turnover and DOPAC levels were less in striatum than in substantia nigra; each region had a characteristic DOPAC/DA ratio (nigra, 38%; striatum, 7%); and, DOPAC levels did not precisely covary with DA turnover between regions. The constancy of DOPAC/DA ratios in controls and after AMPT treatment, in all ages suggests that a constant fraction of DA continued to be released and catabolized to DOPAC despite major decrease of DA after blockade of synthesis of DA.

New mRNA probes for hippocampal responses to entorhinal cortex lesions in the adult male rat: A preliminary report

Three new mRNA responses were found in the hippocampus of the adult male rat after entorhinal cortex lesions (ECL) that induce synaptic reorganization. Hippocampus cDNA libraries were screened by a subtractive hybridization strategy designed to detect ECL-induced mRNAs. Partial sequencing showed clones with similarities to mouse vimentin, ferritin, and polypeptide 7B-2. A sequence similar to mouse SNAP-25 sequence was also detected. Using a mouse SNAP-25 probe, rat SNAP-25 mRNA increased in the hippocampus after ECL.