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Synaptosomes from rat cerebral cortex were studied using the freeze-etching technique. The intra-membranous structure of the pre- and postsynaptic membranes was examined. Particles with an electron-dense spot on their apex are reported from all fracture faces. Most probably these are related to transmembrane channels whose significance in the synaptic transmission is discussed.
Homogenates of rat cerebral cortex contain material corresponding to prostaglandins E(1), E(2), F(1)alpha, and F(2)alpha which are concentrated mainly in the light microsomal and mitochondrial fractions. Only the former fraction exhibits significant ability to synthesize prostaglandins E(1) and F(1)alpha from bis-homo-gamma-linolenic acid. After subfractionation of the crude mitochondrial fraction, prostaglandin E and F material is found mainly in the cholinergic and noncholinergic nerve endings. We conclude that the nerve endings are a storage site, whereas the light microsomes are the site of synthesis.
An efficient, sensitive and rapid analysis of the amino acid neurotransmitters in the cerebral cortex of rats was developed by capillary electrophoresis with laser-induced fluorescence detection and fluorescein isothiocyanate (FITC) derivatization. This method was used to investigate the pharmacological effect of baicalin during cerebral ischemia. Different parameters which influenced derivatization and separation were optimized. The separation of amino acids was carried out in an uncoated fused-silica capillary (57 cm x 75 microm I.D.) with a buffer of 15 mM borate at pH 9.2 and an applied voltage of 17.5 kV. The detection limits for six amino acids were in the range of 2.1 x 10(-11)-6.3 x 10(-10) M. The changes in the level of amino acid neurotransmitters in brain cortex of three experimental rat groups were studied by this capillary electrophoresis-laser-induced fluorescence detection method. The results show that cerebral ischemia can cause a significant elevation in the concentrations of Glu, Asp, GABA, and Gly in cerebral cortex. Baicalin administration can attenuate the elevations of Glu and Asp induced by cerebral ischemia. This research demonstrates that baicalin may act as a neuroprotectant during cerebral ischemia.
The neurons of cerebral cortex are largely autonomous and generate activity that is manifested in trains of microscopic axonal action potentials. The neurons interact by sparse but numerous synaptic connections to generate macroscopic dendritic activity patterns that are observed in electroencephalographic (EEG) waves. The macroscopic patterns are constructed by the populations and they shape the output of cortical neurons in parallel arrays. Sensory cortexes receive sensory information in the form of microscopic action potentials, which induce state transitions in population dynamics. Each state transition transforms sensory information to perceptual meaning. The EEG reflects both kinds of activity. The sensory input is accessed by time ensemble averaging, whereas the perceptual output is found by spatial ensemble averaging. Spatial phase gradients in the EEG are useful for identifying EEG segments in a sequence of state transitions in response to sensory input. The rapidity and flexibility with which they take place give strong reason to postulate that the mechanism for the construction of these sequences of patterns is a dynamical system operating in a chaotic domain.
Membranes from rat cerebral cortex are able to bind S-adenosyl-L-homocysteine (SAH) with a KD of 5 . 10(-7) M and n of 170 pmol/g fresh tissue (i.e. 20 mg protein). The binding is enhanced by Mg2+ and Ca2+ but not K+ and Na+. gamma-Aminobutyric acid, diazepine, noradrenaline and alpha antagonists are without any effect; S-adenosyl-L-methionine, adenosine and adenosine triphosphate inhibit SAH binding. Linkage with an adenosine receptor has not been expressly demonstrated by our method. SAH binding proteins are more abundant in the crude synaptosomal pellet (P2). A similar fixation seems to occur on brain membranes after [3H]SAH administration to rat. The binding might be linked to a methylase activity or an adenosine receptor.
Specimens of human cerebral cortex were obtained during neurosurgical operations and studied by immunocytochemistry and electron microscopy, using antibodies to the metabotropic glutamate receptor subunit mGluR1a and the ionotropic glutamate receptor GluR2/3. A small number of non-pyramidal neuronal cell bodies were labelled for mGluR1a. Double immunolabelling with mGluR1a and GluR2/3 showed that most pyramidal cell bodies were labelled for GluR2/3 but not for mGluR1a. Despite the non-colocalisation of these two receptor subtypes in cell bodies, however, many dendrites and dendritic spines were double-labelled for mGluR1a and GluR2/3 at electron microscopy. As there is evidence that most neurons positive for GluR2/3 are pyramidal cells, this suggests that mGluR1a is present in dendrites of pyramidal neurons, despite absent or low levels of immunoreactivity in their cell bodies.
The developing human cerebral cortex is distinguished by a particularly wide subplate, a transient zone in which crucial cell-cell interactions occur. To further understand the role of the subplate in human brain development, we have studied the immunohistochemical expression of certain neuronal (GAP-43, MAP-2, parvalbumin) and astroglial (vimentin, GFAP) markers in the developing visual cortex from gestational ages of 14 weeks to 9 months post-term. At 14-22 weeks, immunoreactivity to GAP-43, a protein involved in axonal outgrowth, was most prominent in the subplate and marginal zone neuropil and in the fibers of the radiations running near the ventricular zone; at 22-42 weeks, GAP-43 immunoreactive fibers were observed in the maturing cortical plate. Immunoreactivity for the microtubule-associated protein MAP-2 was present in the differentiating cortical plate at 14 weeks, but at 22-42 weeks was most prominent in the somata and dendrites of differentiated neurons, particularly the Cajal-Retzius neurons of the marginal zone, in neurons of the subplate and in those forming cortical layer 5. Parvalbumin immunoreactivity did not appear until 26 weeks, when stained neurons were in a sparse band of cells in layer 6 and upper subplate. Vimentin and GFAP did not stain differentiated neuronal cells. Vimentin immunoreactivity appeared early in neuroepithelial and radial glial cells, decreasing after 35 weeks, with a concomitant increase in GFAP immunoreactivity in radial glial and maturing astrocytic cells. Our results show that despite the greater complexity of the developing human neocortex, molecular markers are expressed in spatial and temporal patterns similar to those observed in non-human primates, carnivores and rodents. These protein markers should prove useful in developmental staging, and in providing a framework in which to examine congenital disorders of cerebral development.
In the adult cerebral cortex, many retrovirally labelled clones are widely dispersed, though the mechanisms of this dispersion are not well understood. Here we investigate the temporal sequence of clonal dispersion after labelling progenitors of rat cortical cells with replication-incompetent retroviruses at early stages of cortical neurogenesis, 14-15 days after conception (E14/15). The location of labelled daughter cells was determined 3, 6 or 10 days later. Labelled sibling cells were radially arrayed three days after infection (E18). In contrast, by six days after infection (E20/21), 43% of cortical clones were dispersed non-radially by at least 500 microns. Four of these widespread clones were dispersed longitudinally by > or = 2 mm, implying sustained rates of dispersion of > 15 microns per hour. Dispersed sibling cells occurred within proliferative zones of the forebrain in 35% of widely dispersed clones, suggesting that some dispersion reflects movement of dividing cells. Some clones dispersed beyond the neocortex into the olfactory bulb. Progenitor cell dispersion represents a previously unrecognized mode of migration by which sibling cells become widely dispersed in the developing forebrain.
Severe neurological symptoms, cerebral edema, and atrophy are common features of the inherited metabolic disorder propionic acidemia. However, the pathomechanisms involved in the neuropathology of this disease are not well established. In this study, we investigate the effects of propionic acid (PA), a metabolite accumulating in this disorder, on cytoskeletal reorganization, on cell viability, and on the in vitro phosphorylation of glial fibrillary acidic protein (GFAP) and vimentin in cultured astrocytes from cerebral cortex of neonatal rats. We observed that the astrocytes changed their usual polygonal morphology when exposed to 5 mM PA for 72 h, leading to the appearance of fusiform or process-bearing cells, without elicit cell death. We also noticed that after 72 h treatment with 5 mM PA cells showed retracted cytoplasm with bipolar processes containing packed GFAP filaments and disorganized actin stress fibers, as revealed by immunocytochemistry. In addition, the morphological alterations were accompanied by increased in vitro 32P incorporation into GFAP and vimentin recovered into the high-salt Triton-insoluble cytoskeletal fraction. In conclusion, our results indicate that PA lead to cytoskeletal reorganization and to increased in vitro phosphorylation of Triton-insoluble GFAP and vimentin. On the basis of our results we could suppose that Triton-insoluble GFAP and vimentin hyperphosphorylation could be implicated in the reorganization of cellular structure and these findings could be involved in the brain damage characteristic of propionic acidemia patients.
When rat cerebral-cortex membranes were labelled with [3H]propylbenzilylcholine mustard ([3H]PrBCM), a single protein of Mr 68,000 was found to carry the atropine-sensitive covalent label. After trypsinolysis of the receptors solubilized in 0.075% SDS, the resulting fragments were submitted to size analysis in combination with wheat-germ agglutinin (WGA)-Sepharose and organomercurial-agarose chromatography. Peptides of Mr 75,000, 50,000, 30,000, 18,000 and 8000 were specifically released from the receptor. All fragments above Mr 8000 were able to bind WGA-Sepharose and therefore the peptide of Mr 18,000 was taken as the upper limit of the distance between the antagonist and the glycan moieties. The limit fragment of Mr 8000 carried chemical groups which were modified by N-ethylmaleimide and reacted with an immobilized organomercurial. About 65-80% of the labelled receptors were adsorbed on concanavalin A-Sepharose with low affinity, generating two further components after sequential application to WGA-Sepharose. About 50% of the receptors were susceptible to neuraminidase treatment, with a concomitant slight modification of the SDS/polyacrylamide-gel-electrophoretic pattern.
Severe neurological symptoms, cerebral edema, and atrophy are common features of the inherited metabolic disorder maple syrup urine disease (MSUD). However, the pathomechanisms involved in the neuropathology of this disease are not well established. In this study, we investigated the effects of the branched-chain keto acids (BCKA) alpha-ketoisocaproic (KIC), alpha-ketoisovaleric (KIV), and alpha-keto-beta-methylvaleric (KMV), which accumulate in MSUD, on astrocyte morphology and cytoskeleton reorganization. Cultured astrocytes from cerebral cortex of neonatal rats were exposed to various concentrations of the BCKA and cell morphology was studied. We observed that these cells changed their usual polygonal morphology when exposed to BCKA, leading to the appearance of fusiform or process-bearing cells. Furthermore, longer exposures to the BCKA elicited cell death at all concentrations studied, attaining massive death at the highest concentrations. Immunocytochemistry with anti-actin or anti-GFAP antibodies revealed that the BCKA induced reorganization of actin and GFAP cytoskeleton. In addition, astrocytes treated with lysophosphatidic acid, an upstream activator of the RhoA GTPase pathway, totally prevented the morphological alterations and cytoskeletal reorganization induced by KIV, indicating that this effect could be mediated by the RhoA signaling pathway. Furthermore, the effects of BCKA on astrocyte morphology were prevented by creatine. In addition, creatine kinase activity was inhibited by KIC and KIV; this inhibition was prevented by creatine, indicating that these keto acids compromise brain energy metabolism. Considering that astroglial cells are critical to brain development and functioning, it is conceivable that alterations of the actin network by BCKA may have important implications in astrocytic function and possibly in the pathogenesis of the neurological dysfunction and brain damage of MSUD patients.
In the developing cerebral cortex of the mouse, binding of antibodies directed against the extracellular matrix glycoprotein fibronectin occurs with a distinct temporal and spatial pattern. On the 10th embryonic day (E10), when the wall of the telencephalic vesicle is made up of only the proliferating cells of the ventricular zone, antifibronectin (aFN) binding is restricted to the blood vessels and pia-arachnoid. Fibronectin-like immunoreactivity first appears in the neuropil as small points of immunofluorescence among the earliest postmitotic neurons that form the preplate (E11-12). A short time later (E12-13), aFN immunoreactivity becomes more diffuse but continues to be restricted to the preplate. As newly arriving neurons form the cortical plate within the preplate (E13-14), aFN binding is present in the marginal zone above the cortical plate and in the subplate below it. Both the marginal zone and the subplate contain early afferents and the cells that were previously part of the preplate. Binding of aFN is transient; by E18-19 it has diminished to the point where it is no longer detectable except in the blood vessels and pia-arachnoid. The transient appearance of fibronectin-like immunostaining in the zones that contain early cortical afferents suggests that fibronectin plays a role in forming the migratory pathway for the growth cones of these axons. In this role it may be acting in concert with other extracellular matrix components such as hyaluronectin, glycosaminoglycans, and laminin, which have been shown to have similar spatial distributions.(ABSTRACT TRUNCATED AT 250 WORDS)
Malformations of the cerebral cortex (MCC) are often associated with severe epilepsy and developmental delay. About 40% of drug-resistant epilepsies are caused by MCC. Classification of MCC is based on embryological brain development, recognising forms that result from faulty neuronal proliferation, neuronal migration and cortical organisation. Hemimegalencephaly, an enlarged dysplastic hemisphere, can present as early onset severe epileptic encephalopathy or as partial epilepsy. In focal cortical dysplasia (FCD), MRI shows focal cortical thickening and simplified gyration. Patients have drug-resistant, often early onset epilepsy. Complete surgical ablation of FCD is accompanied by remission in up to 90% of patients, but may be technically difficult. Tuberous sclerosis (TS) is a multisystemic disorder primarily involving the nervous system; 60% of patients having epilepsy, with 50% having infantile spasms. TS is caused by mutations in the TSC1 and TSC2 genes; 75% of cases are sporadic. TSC1 mutations cause a milder disease. Bilateral periventricular nodular heterotopia (BPNH) consists of confluent and symmetric nodules of grey matter along the lateral ventricles. X-linked BPNH presents with epilepsy in females and prenatal lethality in most males. Most patients have partial epilepsy. Filamin A mutations have been reported in families and sporadic patients. Lissencephaly (LIS smooth brain) is a severe MCC characterised by absent or decreased convolutions. Classical LIS is quite rare and manifests with severe developmental delay, spastic quadriparesis and severe epilepsy. XLIS mutations cause classical lissencephaly in hemizygous males and subcortical band heterotopia in heterozygous females. Thickness of heterotopic band and degree of pachygyria correlate well with phenotype severity. Schizencephaly (cleft brain) has a wide anatomo-clinical spectrum, including partial epilepsy in most patients. Polymicrogyria (excessive number of small and prominent convolutions) has a wide spectrum of clinical manifestations ranging from early onset epileptic encephalopathy to selective impairment of cognitive functions. Bilateral perisylvian polymicrogyria may be familial. Patients present with faciopharingo-glosso-masticatory diplegia and epilepsy, which is severe in about 65% of patients.
Malformations of the cerebral cortex are an important cause of developmental disabilities and epilepsy. Here we review those malformations for which a genetic basis has been elucidated or is suspected and the types of associated epilepsy. Schizencephaly (cleft brain) has a wide anatomo-clinical spectrum, including partial epilepsy in most patients. Familial occurrence is rare. Heterozygous mutations in the EMX2 gene were reported in 13 patients. X-linked bilateral periventricular nodular heterotopia (BPNH) consists of typical BPNH with epilepsy in females and prenatal lethality in males. About 88% of patients have partial epilepsy. Filamin A mutations, all leading to a truncated protein, have been reported in three families and in sporadic patients. The most frequent forms of lissencephaly (agyria-pachygyria) are caused by mutations of LIS1. XLIS mutations cause classical lissencephaly in hemizygous males and subcortical band heterotopia (SBH) in heterozygous females. The thickness of the heterotopic band and the degree of pachygyria correlate with the likelihood of developing Lennox-Gastaut syndrome. Mutations of the coding region of XLIS were found in all reported pedigrees and in 38-91% of sporadic female patients with SBH. With few exceptions, children with LIS1 mutations have isolated lissencephaly, with severe developmental delay and infantile spasms. Autosomal recessive lissencephaly with cerebellar hypoplasia, accompanied by severe developmental delay, seizures, and hypotonia has been associated with mutations of the reelin gene. Fukuyama congenital muscular dystrophy is due to mutations of the fukutin gene and is accompanied by polymicrogyria. Febrile seizures and epilepsy with generalized tonic-convulsions appear in about 50% of children but are usually not severe. Tuberous sclerosis (TS) is caused by mutations in at least two genes, TSC1 and TSC2; 75% of cases are sporadic; 60% of patients have epilepsy, manifested in 50% of them as infantile spasms. TSC1 mutations seem to cause a milder disease with fewer cortical tubers and lower frequency of seizures. Among several syndromes featuring polymicrogyria, bilateral perisylvian polymicrogyria had familial occurrence on several occasions. Genetic heterogeneity is likely, including autosomal recessive, X-linked dominant, X-linked recessive inheritance, and association with 22q11.2 deletions. About 65% of patients have severe epilepsy, often Lennox-Gastaut syndrome.
Homogenates of frontal cerebral cortex of the rat were prepared from microdiscs punched out in areas rich in dopaminergic terminals. Under optimal assay conditions, dopamine (10-4 M) stimulated an adenylate cyclase present in these homogenates by 80-100%. This stimulation reached 200% when microdiscs were punched out from the medial part of the frontal cerebral cortex, adjacent to the forceps minor. Dopamine interacted with an homogeneous population of receptor sites which had an apparent affinity (KD) of 3.8 +/- 0.9 x 10-6 M (N = 4). The dopamine receptor was blocked by fluphenazine and phentolamine but had no affinity for pindolol, propranolol or L-isoproterenol. The affinities of several neuroleptics having different chemical structures were simultaneously determined on striatal and on frontal cerebral cortex dopamine sensitive adenylate cyclases. Fluphenazine was more potent in blocking the striatal than the frontal cerebral cortex dopaminergic receptors. In contrast, in all experiments, haloperidol had an higher affinity for the cerebral frontal cortex than for the striatal dopaminergic receptors. Thus, haloperidol was less effective than fluphenazine in blocking the striatal dopaminergic receptors, and equally potent than fluphenazine in inhibiting the frontal cerebral cortex dopamine sensitive adenylate cyclase. Chlorpromazine, thioridazine and clozapine had the same affinity for the two dopaminergic adenylate cyclase systems. L-isoproterenol interacted with an homogeneous population of beta-adrenergic receptor sites (KD = 3 +/- 2 X 10-7 M; N = 4) coupled with an adenylate cyclase distince from the dopamine sensitive adenylate cyclase. This beta-receptor had no affinity for dopamine or fluphenazine but was blocked by propranolol or pindolol. L-Norepinephrine was shown to stimulate both the dopamine (KD = 1.8 +/- 1 X 10-5 M; N = 4) and the beta-adrenergic (KD = 8 +/- 3 X 10-7 M; N = 4) sensitive adenylate cyclases. Thus, the L-norepinephrine effect was totally blocked in the combined presence of fluphenazine and pindolol.
The GABAA/benzodiazepine receptor complex has been purified from both bovine cerebral cortex and cerebellum by immunoaffinity chromatography on immobilized monoclonal antibody 62-3G1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified receptor from either cerebral cortex or cerebellum revealed 3 main bands corresponding to 51,000, 55,000 and 57,000 Mr silver-stained peptides. In addition, a minor band corresponding to a 53,000 Mr peptide was also found. The differences between the two receptor preparations were: (1) that the main silver-stained 55,000 Mr subunit was present in a relative smaller quantity in cerebellum than in cerebral cortex, and (2) when the membrane-bound receptor was photoaffinity-labeled with [3H]flunitrazepam and subsequently immunoaffinity-purified, two photolabeled peptide bands of 51,000 and 57,000 Mr were found in cerebral cortex while only the 51,000 Mr photolabeled peptide was detected in cerebellum following one-dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Peptide maps of the 57,000 Mr [3H]flunitrazepam photoaffinity-labeled peptide indicated that it was composed of two closely migrating photolabeled peptides of 55,000 Mr and 57,000 Mr. Peptide mapping and deglycosylation experiments using the [3H]flunitrazepam photolabeled receptor suggested that the photolabeled peptides commonly present in cerebellum and cerebral cortex are qualitatively similar if not identical. The results suggest that there are subunits of some type(s) of GABAA R/BZDR complex(es) which are more abundant in cerebral cortex than in cerebellum. Photoaffinity labeling with [3H]muscimol showed similar photolabeled peptides in both cerebral cortex and cerebellum: two main peptides of 54,000 and 57,000 Mr were photolabeled with [3H]muscimol to a similar extent in both receptor preparations.(ABSTRACT TRUNCATED AT 250 WORDS)
During neuronal migration to the developing cerebral cortex, neurons regulate radial glial cell function and radial glial cells, in turn, support neuronal cell migration and differentiation. To study how migrating neurons and radial glial cells influence each others' function in the developing cerebral cortex, we examined the role of glial growth factor (a soluble form of neuregulin), in neuron-radial glial interactions. Here, we show that GGF is expressed by migrating cortical neurons and promotes their migration along radial glial fibers. Concurrently, GGF also promotes the maintenance and elongation of radial glial cells, which are essential for guiding neuronal migration to the cortex. In the absence of GGF signaling via erbB2 receptors, radial glial development is abnormal. Furthermore, GGF's regulation of radial glial development is mediated in part by brain lipid-binding protein (BLBP), a neuronally induced, radial glial molecule, previously shown to be essential for the establishment and maintenance of radial glial fiber system. The ability of GGF to influence both neuronal migration and radial glial development in a mutually dependent manner suggests that it functions as a mediator of interactions between migrating neurons and radial glial cells in the developing cerebral cortex.
The difference in ischemic tolerance between the retina and cerebral cortex may be attributable to a difference in glutamate release during ischemia. Glutamate release in the retina and the cerebral cortex was compared in rats. A dialysis electrode for real-time glutamate measurement was perfused with L-glutamate oxidase, and the current evoked between two voltage-clamped electrodes was detected. Two electrodes were implanted in the retina through the choroid and cerebral cortex in 12 anesthetized rats, each mounted on a stereotaxic frame. Global ischemia was induced by ligation on both carotid arteries and hypotension was induced by blood withdrawal. Under control conditions, the glutamate concentration in the retina was 164 +/- 231 (mean +/- standard deviation) microM, being significantly higher (P < 0.05) than that in the cerebral cortex (83 +/- 105 microM). In 10 of the 12 animals, the glutamate concentration in the retina decreased to a minimum of 134 +/- 149 microM (P < 0.01, compared with the value for the cerebral cortex), but that in the cortex increased to 410 +/- 305 microM (averaged highest value). Immediately after the start of reperfusion, the glutamate concentration in the cortex decreased rapidly to 101 +/- 27 microM, but that in the retina increased gradually to almost the control level (148 +/- 204 microM). In the other two animals, the glutamate concentration remained unchanged. In conclusion, glutamate release in the retina does not proceed as rapidly as that in the cerebral cortex during 20 min of ischemia, and in fact decreases. This opposite trend shown by the two organs may be due to the slow depletion rate of ATP in the retina. This may explain the differing neuronal tolerance to ischemia in these two organs.