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Purification of phospholipase C from rat cerebral cortex.

Phospholipase C from rat cerebral cortex was purified to homogeneity by use of DEAE Bio-Gel A agarose, hydroxyapatite, and heparin agarose chromatography. The purified phospholipase C (PLC) was purified 622.4-fold and its molecular weight is estimated to be 97,500. We obtained a final specific activity of 3.112 mumol of phosphatidylinositol hydrolyzed/min/mg of protein. It is specific for inositol phospholipids. The purified enzyme has an apparent optimum pH 7.0. Calcium is required for its activity. Western blotting analysis showed that two proteins were recognized by anti-PLC antiserum.

Animals↗

Phosphorylation of ribosomal proteins in rat cerebral cortex in vitro.

Incubation of cerebral cortical tissue from immature rats in the presence of [32P]orthophosphate resulted in similar rates of incorporation of radioactivity into the proteins of free and membrane-bound ribosomes. Incorporation of label into ribosomal proteins of both species continued actively for at least 3 hours. Since recovery of membrane-bound ribosomes from rat cerebral cortex is quite low, further analyses of the radioactive phosphoproteins were restricted to the free ribosome population. A significant fraction of the radioactivity which was precipitated with trichloroacetic acid was not removed by heating in trichloroacetic acid at 90 degrees or extracted with organic solvents and therefore was presumed to be covalently bound to protein. The radioactive phosphoryl groups present in the ribosomal proteins were mainly in ester linkages since they were readily removed by exposure to 1 N NaOH, relatively unaltered by 1N HCl, and unaffected by hydroxylamine. This conclusion was supported by the isolation of labeled o-phosphoserine and o-phosphothreonine residues from hydrolysates of ribosomal proteins. A significant fraction of the labeled phosphoproteins in the purified ribosomes appeared to be bound tightly to the ribosome structure since only 40% of the radioactivity could be removed by extraction of these ribosomes with 1 M KCl. Phosphorylation of proteins of cerebral monoribosomes was more rapid than the same process in polyribosomes from the same source. Eight radioactive phosphoprotein bands could be detected by electrophoresis of proteins obtained from unfractionated cerebral ribosomes on unidimensional polyacrylamide gels containing sodium dodecyl sulfate. The protein nature of these materials was confirmed by pronase digestion. Proteins of subribosomal particles isolated from the total free ribosomal population were labeled differentially. When dissociation was carried out in the presence of EDTA, the small subunit contained four radioactive phosphoprotein bands, whereas the large subunit contained five. Three of the radioactive phosphoprotein components of the small subunit were removed when dissociation of cerebral ribosomes which were previously washed with high salt media was carried out in the presence of puromycin and high salt. However, only the largest labeled phosphoprotein band of the large subunit was removed by this procedure. This component exhibited the same electrophoretic mobility as one of the radioactive phosphoprotein bands which was removed from the small subunit by high salt treatment..

Animals↗

The relationship between cellular nucleic acids in the developing rat cerebral cortex.

1. In the rat cerebral cortex net DNA synthesis ceases when the animal has reached about 25g. body weight (18 days of age). There is then little further change in the DNA content per cortex. 2. Nuclear and transfer RNA follow a similar pattern to DNA. 3. Microsomal and ribosomal RNA content increases up to 25g. body weight but then declines. The decrease in ribosomal and microsomal RNA content is associated with a change in RNA base composition. 4. Incorporation of [(14)C]orotic acid into nuclear RNA proceeds at a similar rate in 4-day-old and adult animals. However, there is a lag period of about 60min. in the young animals during which incorporation into the ribosome fractions proceeds slowly. In the adult animals the lag period is not seen.

Adenine↗

[Microglia and pericytes of the human cerebral cortex].

Biopsies of normal human cerebral cortex were fixed by aldehydes and their ultrastructure was studied. Two main types of vessels are observed, capillaries and metarterioles, and around them two types of pericytes are described: a microgliocyte-like pericyte which is either surrounded by a nervous basal membrane, or laying outside this membrane; endothelial-like pericytes which are frequently observed inside the metarterioles perivascular space and contain many lysosomes. Endothelial-like pericytes have probably a metabolic function and microgliocyte-like pericytes are more involved in a role of mechanic regulation of the microcirculation. This hypothesis is supported by the observation of a microgliocyte interposed between two endothelial processes and protuding into the vascular lumen.

Cerebral Cortex↗

Age-related modification of enzyme activities in synaptosomes isolated from rat cerebral cortex.

In synaptosomes from rat cerebral cortex, the potential catalytic activity of some enzymes related to energy metabolism--namely, phosphofructokinase and citrate synthase--is not affected by aging. In contrast, the maximum velocity (Vmax) of cytochrome oxidase and of pyruvate dehydrogenase decreases in aged rats. A marked increase is found in the Vmax of glucose 6-phosphate dehydrogenase in aged rats and could be related to the availability of NADPH for antiperoxidative processes. Pretreatments of experimental animals with certain drugs were done to investigate the plasticity of enzyme proteins during aging. Papaverine, which acts on macrocirculation, is ineffective, but delta-yohimbine acting on microcirculation and metabolism and almitrine acting on oxygen availability both could interfere with the potential activity of some enzymes. However, their influence differs with the age of the rats.

Aging↗

Vibrissaeless mutant rats with a modular representation of innervated sinus hair follicles in the cerebral cortex.

Specialized areas in the cerebral cortex are essential to mediate the various sensory modalities and are crucial to their recovery in disease. We recently observed that prenatal photoreceptor cues are not indispensable for the development of the elaborate modular organization of the primate primary visual (striate) cortex (Kuljis, R. O. and P. Rakic. 1990. Proc. Natl. Acad. Sci. USA 87: 5303-5306). By contrast, the elegant experiments of Woolsey, Van der Loos, and collaborators (Van der Loos, H., and T. A. Woolsey. 1973. Science 179: 395-398; Van der Loos, H. and J. Dörfl. 1978. Neurosci Lett. 7: 23-30; Woolsey, T. A. 1967. John Hopkins Med. J. 121: 91-112; Woolsey, T. A. and H. Van der Loos. 1970. Brain Res. 17: 205-242) indicate that postnatal vibrissal receptor input is necessary for the development of modular organization in the posteromedial barrel subfield (PMBSF) of the rodent somatosensory cortex. The present report is part of a series of studies designed to address the variables that result in seemingly different results in these two models. Here, I address the role of pre- and postnatal tactile experience in the development of the rat homologue of the mouse PMBSF using mutants that lack vibrissae. Mutants exhibit cytoarchitectonic units in layer IV similar to those in controls, as revealed by NissI stains and histochemistry for succinate dehydrogenase and cytochrome oxidase. Sections from flat mounts of the vibrissal pad reveal that all mutants contain vibrissal follicles with stumps of sinus hairs in a geometric array and number similar to that in controls, and that the follicles are innervated heavily by fascicles of fibers from the infraorbital nerve.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Cerebral cortex perfusion rates in dementia.

Cerebral cortex perfusion rates (CPR) were measured in 11 patients with dementia. In six patients the dementia appeared to be due to a primary neuronal degeneration and in the remainder it appeared to be secondary to vascular disease. In the context of blood flow measurement, it is suggested that the terms primary and secondary dementia are preferable to the terms presenile and senile dementia, because the latter are classified by age alone and the terms have no aetiological or pathological significance. The perfusion rates in patients with primary dementia were within normal limits for their age, whereas those in patients with secondary dementia were significantly reduced. This difference is likely to apply only if the measurements are made early in the course of the disease.

Adult↗

Progressive acquisition of cortical phenotypes as a mechanism for specifying the developing cerebral cortex.

The formation of the cerebral cortex requires the assembly of a complex array of neurons that exhibit specific patterns of laminar distribution, connectivity, and macromolecular expression. The problem of how various areas become specified relies to a large extent on the ability to assay certain characteristics of a particular cortical area during development. Certain phenotypic features appear to be acquired early in histogenesis, whereas others have a relatively late onset of expression. This pattern of progressive acquisition of traits suggests that each feature may be regulated by a different set of genomic-environmental interactions. This would lead to an early framing of cortical regions through the commitment of cells to area-specific phenotypes, but allow modifications of later developing traits through changes in certain environmental features.

Animals↗

Zfp312 is required for subcortical axonal projections and dendritic morphology of deep-layer pyramidal neurons of the cerebral cortex.

Pyramidal neurons of the cerebral cortex display marked layer- and subtype-specific differences in their axonal projections and dendritic morphologies. Here we show that transcription factor Zfp312 is selectively expressed by layer V and VI subcortical projection pyramidal neurons and their progenitor cells. Knocking down Zfp312 with small interfering RNAs dramatically reduced the number of subcortical axonal projections from deep-layer pyramidal neurons and altered their dendritic morphology. In contrast, misexpression of Zfp312 in cortically projecting pyramidal neurons of layers II and III induced the expression of Tbr1, a transcription factor enriched in deep-layer neurons, and the formation of ectopic subcortical axonal projections. Thus, our results indicate that transcription factor Zfp312 plays a critical role in layer- and neuronal subtype-specific patterning of cortical axonal projections and dendritic morphologies.

Animals↗

Emx1, emx2 and pax6 in specification, regionalization and arealization of the cerebral cortex.

Three basic aspects of cerebral cortex development - specification of cortical versus ganglionic identity, regionalization of the early cortical primordium and arealization of the developing cortex - were the main subject of our recent investigations. We previously demonstrated that the two homeobox genes Emx2 and Pax6 promote development of caudal-medial and rostral-lateral cortex, respectively, by properly shaping the early cortical protomap and possibly modulating the tangential growth ratio between medial and lateral cortical anlagen. More recently, by analyzing the brains of embryos bearing mutations for Emx2 and Pax6 in different combinations, we found that both genes are necessary and sufficient for a more basic developmental choice, i.e. the specification of neuroblasts in the dorsal telencephalon as cortical versus ganglionic neuroblasts. Finally, we explored the possible roles of the Emx2 paralog, Emx1, in these processes. By looking at embryos mutant for Emx1, Emx2 and Pax6 in various combinations, we did not get any evidence of Emx1 involvement in the process of cortical specification; conversely, this gene appeared to be involved to some extent in the process of regionalization of the cortical primordium along the medial-lateral axis, as a promoter of medial fates.

Animals↗

Optical measurements of oxygen delivery and consumption in gerbil cerebral cortex.

Oxygen metabolism of the cerebral cortex of anesthetized gerbils was monitored by surface fluorescence and reflectance spectrophotometry both in vivo and in specimens freeze-trapped by the surface-freezing technique. Fiber optic light guides were used for optical coupling for determining mitochondrial flavoprotein and pyridine nucleotide fluorescence and for recording dual-wavelength reflectance spectra that indicated the levels of ferrocytochromes aa3 and c + c1 in the cortex and the oxygen saturation of cortical hemoglobin. During anoxic episodes increases in ferrocytochromes aa3 and c + c1 and in reduced pyridine nucleotides and flavoproteins were observed only when the cortical hemoglobin was more than 85% disoxygenated. Indeed the steady-state levels of oxidation-reduction of mitochondrial respiratory-chain components remained constant over a wide range of oxygen delivery, increased reduction being observed only when the fraction of inspired oxygen fell below 6%; these properties of mitochondria in vivo resemble those found in vitro. However, in normoxic animals the absorbance at 605 nm that may arise from ferri- or ferrocytochrome aa3 is larger than would be expected of purified mitochondria and may represent a pool of mitochondria that remains reduced at all levels of tissue oxygenation or a pigment not involved in oxygen metabolism.

Animals↗

Histopathological evaluation of materials implanted in the cerebral cortex.

Histopathological changes of the cerebral cortex in response to small, penetrating metal and non-metal implants were analyzed by means of light and electron microscopy. The needle-shaped implants were left in place during all stages of histological preparation and embedded in plastic together with the cortex. Changes of the brain-implant boundary were classified as non-reactive, reactive, or toxic, according to the reactive cellular constituents. Among the non-reactive materials were several plastics and metals such as aluminum, gold, platinum, and tungsten. The boundary of these implants displayed little or no gliosis and normal neuropile with synapses within 5 micron of the implant's surface. The boundary of reactive materials such as tantalum or silicon dioxide was marked by multinucleate giant cells and a thin layer (10 micron)) of connective tissue. Toxic materials such as iron and copper were separated from the cortical neuropile by a capsule of cellular connective tissue and a zone of astrocytosis. Cobalt, a highly toxic material, produced more extensive changes in the zones of connective tissue and astrocytes. These results indicate that a variety of materials are well tolerated by the brain and could be used in the fabrication of neuroprosthetic devices.

Aluminum↗

Genetics of neuronal migration in the cerebral cortex.

The development of the cerebral cortex requires large-scale movement of neurons from areas of proliferation to areas of differentiation and adult function in the cortex proper, and the patterns of this neuronal migration are surprisingly complex. The migration of neurons is affected by several naturally occurring genetic defects in humans and mice; identification of the genes responsible for some of these conditions has recently yielded new insights into the mechanisms that regulate migration. Other key genes have been identified via the creation of induced mutations that can also cause dramatic disorders of neuronal migration. However, our understanding of the physiological and biochemical links between these genes is still relatively spotty. A number of molecules have also been studied in mice (Reelin, mDab1, and the VLDL and ApoE2 receptors) that appear to represent part of a coherent signaling pathway that regulates migration, because multiple genes cause an indistinguishable phenotype when mutated. On the other hand, two human genes that cause lissencephaly (LIS1, DCX) encode proteins that have recently been implicated as regulators or microtubule dynamics. This article reviews some of the mutant phenotypes in light of the mechanisms of neuronal migration. MRDD Research Reviews 6:34-40, 2000.

Animals↗

Stimulation by acetylcholine of phosphatidylinositol labelling. Subcellular distribution in rat cerebral-cortex slices.

1. Rat cerebral-cortex slices were incubated with (32)P(i), acetylcholine and eserine for periods of 10min and 2h. The specific radioactivity of phosphatidylinositol was elevated during these treatments by 36 and 106% respectively. 2. The specific radioactivities of the phosphatidylinositol in different cell structures were determined after subcellular fractionation. They were highest in the nuclear, microsomal and synaptic-vesicle fractions and lowest in myelin, both in the controls and in the acetylcholine-treated slices. 3. The stimulated labelling of phosphatidylinositol was relatively evenly distributed: no subcellular fraction showed a stimulation markedly higher than that in the homogenate. 4. Studies of the distributions and activities of marker enzymes indicated that the subcellular fractionation achieved was similar to that with fresh tissue. 5. The results are discussed in relation to the previous report that the stimulation is observed throughout the neuronal cell-bodies and in relation to the hypothesis that the labelled phosphatidylinositol produced by stimulation is a component of an acetylcholine-receptor proteolipid localized in the synaptic junction.

Acetylcholine↗

Effects of stimulating the nucleus basalis of Meynert on blood flow and delayed neuronal death following transient ischemia in the rat cerebral cortex.

An increase in cortical cerebral blood flow (CBF), independent of metabolic vasodilation, via the activation of cholinergic neurons originating in the nucleus basalis of Meynert (NBM) in the basal forebrain and projecting to the widespread cortices was recently demonstrated. In the present study, we aimed to clarify whether the increase in CBF following a stimulation of the NBM can improve delayed death of the cortical neurons following transient ischemia in rats. CBF was measured with a laser Doppler flowmeter, and the delayed neuronal death of the cerebral cortex produced by intermittent (every 5 s) occlusions of the unilateral common carotid artery for 60 min was measured histologically in the cortical hemisphere at 3 different coronal levels (6 microm thickness). In control rats without occlusion there were 6000-8000 intact neurons and 9-19 damaged neurons in the cortical hemisphere at each coronal level. During the occlusions, CBF ipsilateral to the occluded artery decreased by 13-32% of the preocclusion level. Five days after the occlusions, the numbers of damaged neurons were increased to 75-181. Repetitive electrical stimulation was delivered to the NBM, ipsilateral to the occluded artery, starting 5 min before the occlusions and finishing around the end of them. The increase in CBF induced by NBM stimulation prevented the occlusion-induced decrease in CBF in all 3 of the cortices. The delayed death of the cortical neurons previously observed after the occlusions was scarcely observable in all the cortices when NBM was stimulated. The present results suggest that NBM-originating vasodilative activation can protect the ischemia-induced delayed death of cortical neurons by preventing a blood flow decrease in widespread cortices.

Animals↗

[Effect of preliminary neurosensitization of rats on the postnatal development of the cerebral cortex of the progeny].

The author investigated the cerebral cortex of 30-day ratlings born of rats preliminarily neurosensitized with a homologous cerebral cortex antigen, and of intact, control rats. In comparison with control, there was a reduction of the width of the sensory-motor region of the cortex, chiefly on account of the lower layers, diminution of the size of cell bodies of large neurons in the layer, inadequate development of the cytoplasm, and retarded accumulation of basophilic substance in it.

Animals↗

Interaction between 5-HT uptake inhibition and activation of 5-HT autoreceptors by exogenous agonists in rat cerebral cortex slices and synaptosomes.

Rat cerebral cortex slices or synaptosomes were labelled with 3H-5-hydroxytryptamine (3H-5-HT) and subsequently superfused. They were depolarized by electrical stimulation (slices) or with high K+ (slices and synaptosomes). Continuous electrical stimulation (2 Hz, 24 mA, 2 ms) and continuous or discontinuous K+ depolarization (15-25 mM) were used. 1. Continuous electrical stimulation or continuous K+-depolarization of slices evoked a steady overflow of tritium that slowly decayed with time. 2. Exposure to increasing concentrations of 5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole succinate (RU 24969) (0.001-0.1 microM) during continuous electrical stimulation produced a concentration-dependent decrease in tritium overflow. Citalopram (1 microM) counteracted the effect of RU 24969. 3. RU 24969 inhibited the evoked 3H-overflow and citalopram reduced the effect of RU 24969 also during continuous depolarization of slices with 20 mM K+. Similar results were obtained by using 5-methoxytryptamine or LSD. 4. In slices 1 microM citalopram increased significantly the tritium overflow evoked by electrical stimulation or by 20 mM K+-depolarization. 5. Increasing the K+ concentration from 20 mM to 25 mM mimicked the effects of 1 microM citalopram both on the RU 24969 activity and on the evoked tritium overflow. 6. RU 24969 (0.001-0.1 microM) decreased in a concentration-dependent way the release of tritium from cortical synaptosomes depolarized with K+ (15-20 mM). The presence of 1 microM citalopram did not modify significantly the effect of the agonist. Citalopram was ineffective also when the serotonin uptake carrier in superfused synaptosomes was activated by tryptamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Characterization of transport systems for the transfer of 3,4-L-dihydroxyphenylalanine into slices of rat cerebral cortex.

1. Slices of rat cerebral cortex incubated aerobically at 37 degrees C in Krebs-Ringer-bicarbonate solution accumulated 3,4-L-dihydroxyphenylalanine (L-DOPA) against its concentration gradient. With 1 mM L-DOPA in the medium, tissue-water/medium concentration ratios of about 6 : 1 are reached, which are modified by the presence of other amino acids in the medium. 2. Kinetic analysis suggested that L-DOPA influx into brain cells occurred by at least two saturable processes, which show apparent Km values in the range of 10(-3) M and 10(-5) M, respectively. 3. Prior incubation of the slices in Na+-free (choline-containing) medium at 37 degrees C depressed their subsequent uptake of L-DOPA in normal Na+-containing medium; this inhibition did not appear when the preincubation was carried out at 0-4 degrees C. Besides this effect of preincubation, most of L-DOPA influx into brain slices was independent of the actual concentration of Na+ in the medium; the two saturable processes described in this article behaved similarly in this respect. 4. Most of L-DOPA uptake by the high-Km process is mediated by an agency that resembles the Na+-independent L system described in Ehrlich cells (Oxender, D. L. and Christensen, H. N. (1963) J. Biol. Chem. 238, 2686-2699), both in its specificity and in its participation in exchange phenomena. A lesser component of uptake by a type A mediation is also suggested as contributing to the high-Km process . 5. The kinetic and specificity properties of the low-Km process of L-DOPA uptake suggest a similarity between its mediation and that of the high-affinity systems for L-tyrosine and L-tryptophan found in brain tissue preparations (Belin, M. F. and Pujol, J. F. (1973) Experientia 29, 411-413; Bauman, A., Bourgoin, S., Benda, P., Glowinski, J. and Hamon, M. (1971 Brain Res. 66, 253-263).

Alanine↗