Effects of ageing on the content in sulfur-containing amino acids in rat brain

Summary Concentrations of the sulfur-containing amino acids methionine, homocysteic acid, cysteic acid and taurine were measured in brain structures of young and old Wistar rats in an attempt to etablish a possible link between the increase in oxidative stress with ageing and changes in tissue levels of these amino acids. Contrary to data reported by others, in all brain structures of young and old rats homocysteic acid levels could not be quantified. Compared with young rats, in old animals taurine and methionine concentrations significantly decreased in striatum and cortex; decreased taurine levels were also found in nucleus accumbens and cerebellum and lower concentrations of methionine were found in midbrain, hippocampus and pons-medulla. Cysteic acid levels either did not change or significantly increased in cortex and hippocampus. These results are discussed taking into account the biosynthesis of sulfur-containing amino acids in rat brain and the decrease in glutathione in relation to oxidative stress with ageing.Changes in aspartic acid, glutamic acid, serine, glutamine, glycine and GABA concentrations with ageing were also determined in the same brain structures and were in good agreement with those previously reportedAbbreviations H ₂O₂ hydrogen peroxide - MAO monoamine oxidase - GSHP glutathione peroxidase - PAPS 3-phosphoadenosine-5-phosphosulfate - OPA O-phthaldialdehyde - HPLC high performance liquid chromatography - Asp aspartic acid - CA cysteic acid - CSA cysteine sulfinic acid - Cys cysteine - GABA -aminobutyric acid - Gln glutamine - Glu glutamic acid - Gly glycine - HCA homocysteic acid - Met methionine - Ser serine - Tau taurine     

The presence of glutathione in primary neuronal and astroglial cultures from rat cerebral cortex and brain stem

Summary The concentration of the tripeptide glutathione (GSH) was measured in primary cultures of neurons and astroglial cells from rat cerebral cortex and brain stem. The concentration of GSH was found to be approximately 20 nmol/ mg protein in the neuronal culture from the cerebral cortex and ca. 40 nmol/ mg protein in the neuronal brain stem cultures. A GSH concentration of approximately 20 nmol/mg was observed in the astrocyte cultures from both brain regions. The possibility to increase the GSH concentration was tested by incubating the cultures in the presence of the GSH precursor -glutamylcysteine (-GC). In the cultured astrocytes -GC produced a dose-dependent increase in GSH. A similar increase was observed in the neuronal cultures, but this effect failed to reach statistical significance.     

Specificity in the Development of Vertical Connections in Cat Striate Cortex

The main efferent axons of pyramidal cells in layer 2/3 in the adult cat striate cortex make collateral connections specifically within layer 2/3 and layer 5 and avoid the intervening layer 4. Intracellular dye injections in vitro were used to determine how, during early postnatal development, this precise pattern of laminar connections was achieved. These investigations revealed that the pattern of collateral outgrowth was specific from the very earliest time that axons began sprouting collaterals. During the first postnatal week, sprouts were seen exclusively within layers 2/3 and 5; no evidence for a transient connection to layer 4 was observed. Furthermore, collaterals emerged simultaneously within layers 2/3 and 5, despite the large difference in the postmigratory ages of the two layers. By the end of the second postnatal week, the adult number of collaterals was achieved. Further elaboration of the local arbors occurred by repeated branching of already existing collaterals, rather than by addition of new collaterals to the main axon. These results demonstrate that the formation of local connections between cortical layers is highly specific, in contrast to the development of clustered horizontal connections by these same cells within layers 2/3 and 5, which involves extensive remodelling of local connections.     

An Organotypic Spinal Cord - Dorsal Root Ganglion - Skeletal Muscle Coculture of Embryonic Rat. II. Functional Evidence for the Formation of Spinal Reflex Arcs In Vitro

Electrical properties of motoneurons, muscle fibres and dorsal root ganglion (DRG) cells were studied in an organotypic coculture of embryonic rat spinal cord, dorsal root ganglia and skeletal muscle. The motoneurons were identified by their morphology and position in culture. Their size and input conductance were significantly larger than those of spinal interneurons. Intracellular current injection evoked action potentials in all motoneurons, but only evoked stable repetitive firing patterns in some. Excitability was correlated to somatic size and the rate of spontaneous excitatory input. It is suggested that the somatic growth and the increase in excitability is regulated by the excitatory afferents. The motoneurons showed spontaneous excitatory and inhibitory postsynaptic potentials and action potentials which disappeared with the application of various agents known to inhibit excitability or excitatory synaptic transmission. Excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs respectively) were distinguished by their shape, reversal potential and pharmacology. IPSPs could be depolarizing or hyperpolarizing in different cells. A higher percentage of cells with hyperpolarizing IPSPs was found in older cultures and in the presence of skeletal muscle, suggesting a reversal of the polarity of IPSPs with development. The spontaneous muscle contractions observed in the cultures could be due either to innervation, spontaneous oscillations of the membrane potential, or electrical coupling between neighbouring fibres. A small percentage of DRG cells showed spontaneous action potentials, all of which were found in cultures with spontaneous muscle contractions. The electrical stimulation of DRG afferents evoked mono- and polysynaptic EPSPs in motoneurons, endplate potentials and muscle contractions. The stimulation of the ventral horns evoked endplate potentials and muscle contractions via mono- or polysynaptic pathways. Together these results indicate that appropriate and functional contacts were established in the culture between myotubes and DRG cells, between DRG cells and motoneurons, and between motoneurons and muscle fibres.     

Differential Distribution of Tau Proteins in Developing Cat Cerebral Cortex and Corpus Callosum

During the postnatal development of cat visual cortex and corpus callosum the molecular composition of tau proteins varied with age. In both structures, they changed between postnatal days 19 and 39 from a set of two juvenile forms to a set of at least two adult variants with higher molecular weights. During the first postnatal week, tau proteins were detectable with TAU-1 antibody in axons of corpus callosum and visual cortex, and in some perikarya and dendrites in the visual cortex. At later ages, tau proteins were located exclusively within axons in all cortical layers and in the corpus callosum. Dephosphorylation of postnatal day 11 cortical tissue by alkaline phosphatase strongly increased tau protein immunoreactivity on Western blots and in numerous perikarya and dendrites in all cortical layers, in sections, suggesting that some tau forms had been unmasked. During postnatal development the intensity of this phosphate-dependent somatodendritic staining decreased, but remained in a few neurons in cortical layers II and III. On blots, the immunoreactivity of adult tau to TAU-1 was only marginally increased by dephosphorylation. Other tau antibodies (TAU-2, B19 and BR133) recognized two juvenile and two adult cat tau proteins on blots, and localized tau in axons or perikarya and dendrites in tissue untreated with alkaline phosphatase. Tau proteins in mature tissue were soluble and not associated with detergent-resistant structures. Furthermore, dephosphorylation by alkaline phosphatase resulted in the appearance of more tau proteins in soluble fractions. Therefore tau proteins seem to alter their degree of phosphorylation during development. This could affect microtubule stability as well as influence axonal and dendritic differentiation.     

Brain Spectrins 240/235 and 240/235E: Differential Expression During Development of Chicken Dorsal Root Ganglia in vivo and in vitro

Brain spectrin, a membrane-related cytoskeletal protein, exists as two isoforms. Brain spectrin 240/235 is localized preferentially in the perikaryon and axon of neuronal cells and brain spectrin 240/235E is found essentially in the neuronal soma and dendrites and in glia (Riederer et al., 1986, J. Cell Biol., 102, 2088 - 2097). The sensory neurons in dorsal root ganglia, devoid of any dendrites, make a good tool to investigate such differential expression of spectrin isoforms. In this study expression and localization of both brain spectrin isoforms were analysed during early chicken dorsal root ganglia development in vivo and in culture. Both isoforms appeared at embryonic day 6. Brain spectrin 240/235 exhibited a transient increase during embryonic development and was first expressed in ventrolateral neurons. In ganglion cells in situ and in culture this spectrin type showed a somato - axonal distribution pattern. In contrast, brain spectrin 240/235E slightly increased between E6 and E15 and remained practically unchanged. It was localized mainly in smaller neurons of the mediodorsal area as punctate staining in the cytoplasm, was restricted exclusively to the ganglion cell perikarya and was absent from axons both in situ and in culture. This study suggests that brain spectrin 240/235 may contribute towards outgrowth, elongation and maintenance of axonal processes and that brain spectrin 240/235E seems to be exclusively involved in the stabilization of the cytoarchitecture of cell bodies in a selected population of ganglion cells.     

Characterization and distribution of [3H]ohmefentanyl binding sites in the human brain

Binding properties and localization of [3H]ohmefentanyl, a new ligand for mu opioid receptors, were investigated on normal human brain sections. Binding assays performed at the level of the basal ganglia revealed: (1) a steady-state binding reached after 60 min incubation at room temperature, (2) the presence, in saturation experiments, of an apparent single class of binding sites with a Kd = 1.68 +/- 0.45 nM and a Bmax = 162 +/- 9 fmol/mg protein, (3) an order of potency to inhibit [3H]ohmefentanyl binding as follows: ohmefentanyl greater than [D-Ala2, MePhe4, Gly-ol5] enkephalin (DAGO) greater than ethylketocyclazocine (EKC) much greater than Tyr-D-Ser(OtBu)-Gly-Phe-Leu-Thr(OtBu) (BUBU) and U-50,488H. Quantitative autoradiography showed an heterogeneous distribution of [3H]ohmefentanyl binding sites with the highest densities in amygdala, medical geniculate body, thalamus, and caudate nucleus. Binding characteristics and anatomical distribution also show that [3H]ohmefentanyl may bind to a small proportion of additional sites called "DAGO-inaccessible [3H]ohmefentanyl specific binding sites." [3H]Ohmefentanyl binding to these sites can be partly inhibited by sigma ligands such as 1,3-di-o-tolylguanidine (DTG) and haloperidol. However, unlabeled DAGO inhibited more than 80% of [3H]ohmefentanyl specific binding in most of the human brain regions studied, suggesting that the major population of sites labeled by [3H]ohmefentanyl represented mu opioid receptors.     

脑脓肿切除的显微外科技术

作者采用显微外科技术切除脑脓肿40例,在切除前,先用自制三通装置穿刺脓肿,并用10％甲醛溶液及庆大霉素溶液冲洗脓腔,然后在手术显微镜下切除脓壁。随访3～5年,治愈率98.0％(38例),复发1例,死亡2例。     

Correlated, simultaneous, multiple-wavelength optical monitoring in vivo of localized cerebrocortical NADH and brain microvessel hemoglobin oxygen saturation

Current forms of brain monitoring, such as electroencephalography (EEG), have had limited clinical utility. The EEG records spontaneous cerebrocortical activity and thus is an indirect indicator of metabolic demand and, to a lesser extent, an indicator of mismatch of supply versus demand. Ischemia modulates EEG activity in ways that can usually be detected, but EEG patterns can be similarly modulated by many other factors, including temperature and pharmacologic manipulation. This in vivo study in physiologically monitored animals evaluated the use of correlated optical spectroscopy, performed with an instrument having a fiberoptic light-guide bundle in contact with the cerebral cortex, for the simultaneous monitoring of cerebrovascular oxygen availability and intracellular oxygen delivery. A highly specific monitor of cerebral intracellular oxygen supply, the cerebrocortical intramitochondrial NADH redox state, was monitored in vivo with a fluorescence technique. Absorption spectroscopy was used concurrently to monitor hemoglobin content (blood volume) and oxygen saturation in the microcirculation. Correlated changes in optical signals from cerebrocortical NADH and hemoglobin were studied in a swine model (n=7) of nitrogen hypoxia. Measurements were made at four wavelengths with a time-division, multiplexed fluorometer/reflectometer. Because the NADH fluorescence signal at 450 nm is affected by local changes in blood volume, a corrected fluorescence signal is usually calculated. In previous studies, where only two wave lengths have been measured, attempts at correction were based on reflectance at the excitation wavelength (366 nm). We compared estimators of changes in microcirculatory blood volume using reflection at two wavelengths: 366 nm and 585 nm, the wavelengths for maximum and isobestic absorption. The results of the studies were as follows: (1) during transient hypoxia, NADH and local hemoglobin saturation signals changed in concert with arterial pulse oximetry, with changes in NADH lagging behind changes in saturation by an average of 5.3 seconds; (2) after hypocapnic ventilation to a mean Paco ₂ of 20.2 ± 0.8 mm Hg, NADH increased by 11.5 ± 8.7% (as compared with maximal change during anoxia), local hemoglobin saturation decreased by 7.7 ± 6.4%, and local blood volume decreased by 12.5 ± 13%, while arterial SpO₂ was unchanged; (3) our two measures of local blood volume were closely correlated during carbon dioxide perturbations, but poorly correlated during hypoxic perturbation; and (4) NADH fluorescence provided a more rapid, sensitive indicator of oxygen deprivation than did the EEG. During transient hypoxia, EEG changes occurred 57.4 ± 10.4 seconds after the onset of decline in local hemoglobin saturation, after NADH had completed 50% of its maximal increase.This work was supported in part by research grants from the NIH (GM34767), the Academic Senate of the University of California, and the UCSF Anesthesia Research Foundation.     

Evidence of a preferential role of brain serotonin in the mechanisms leading to naloxone-precipitated compulsive jumping in morphine-dependent rats

Various drugs acting on brain serotonin or catecholamines were administered concurrently with morphine during the development of dependence or before naloxoneprecipitated withdrawal syndrome. Of the various drugs only cyproheptadine, a serotonin antagonist, and piribedil, a dopamine agonist, reduced the frequency of jumping (but not of diarrhea or ptosis) when administered with morphine during development of dependence. When administered before naloxone, d-fenfluramine, a serotonin releaser, markedly reduced jumping, but not diarrhea and ptosis, and clonidine blocked these latter signs without affecting the frequency of jumping. Of the other drugs examined only phenoxybenzamine reduced diarrhea in morphine-abstinent rats. It is suggested that serotonin is involved in the mechanisms which lead to compulsive jumping during naloxoneprecipitated withdrawal, whereas adrenergic sites on which clonidine acts are mainly involved in the expression of signs, such as ptosis and diarrhea. No clear evidence was obtained of a role for dopamine in the withdrawal signs studied.