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PubMed1979

The isolation and lipid composition of subcellular fractions derived from neuronal perikarya isolated in bulk from rabbit cerebral cortex.

(1) Neuronal perikarya were isolated from rabbit cerebral cortex and were homogenized and separated into a number of subcellular membrane fractions using differential and discontinuous density gradient centrifugation. (2) The efficiency of homogenizing the nerve cell bodies could be greatly increased by the preliminary passage of the cell body fraction through a micropipet tip. (3) Of a number of density media, a discontinuous gradient of metrizamide gave the best resolution of the mitochondrial and lysosomal marker enzymes found in the crude mitochondrial fraction. This yielded a purified mitochondrial fraction and several lighter membrane fractions. (4) The lighter membrane fractions in metrizamide contained a degree of mitochondrial contamination which could be removed by a second gradient spin on 1.2 M sucrose, producing a second mitochondrial fraction and two light membrane fractions. (5) Based on marker analyses, fractions enriched in nuclei, mitochondria, microsomes and plasma membrane/lysosomes were produced. (6) The two mitochondrial fractions showed the highest, and the nuclear fraction the lowest specific phospholipid content. Cholesterol: phospholipid molar ratios showed a gradient of values from a low (0.2) for the mitochondria and nuclei to an intermediate value (0.4) for the microsomes to a high (0.6) for the light membrane fractions. (7) Phospholipid distributions indicated that for the nuclear and mitochondrial fractions phosphatidylinositol was more abundant than sphingomyelin or phosphatidylserine, while for the microsomes and the two light membrane fractions these three phospholipids were present in almost equal amounts.

Acetylglucosaminidase↗

PubMed1978

Brain alpha-adrenergic receptors: comparison of [3H]WB 4101 binding with norepinephrine-stimulated cyclic AMP accumulation in rat cerebral cortex.

The ability of a series of adrenergic agents to displace the binding to brain membranes of [3H]WB 4101, a potent alpha-adrenergic antagonist (WB 4101 = 2-[2-(2,6-dimethoxyphenoxy)ethylaminomethyl]-1,4-benzodioxane hydrochloride), has been compared with the potency of these agents in stimulating or inhibiting the alpha-adrenergic component of cyclic AMP accumulation in rat cerebral cortex slices. [3H]WB 4101 rapidly bound to a high affinity site (KD = 2.7 nM) in membranes from cerebral cortex. Binding came to equilibrium by 2 min at 37 degrees C and was rapidly reversed in the presence of phentolamine. The potencies of adrenergic agents (WB 4101 greater than phentolamine greater than naphazoline) in displacing binding of [3H]WB 4101 were comparable to the potencies of these agents as inhibitors of the alpha-adrenergic component of norepinephrine-stimulated cyclic AMP accumulations. Phenoxybenzamine, clonidine, chlorpromazine and haloperidol were about 10--30 times more potent in inhibiting cyclic AMP accumulation than in displacing [3H]WB 4101 binding. The potency of classical alpha-adrenergic agonists in displacing WB 4101 (epinephrine greater than norepinephrine greater than methoxamine) correlated with the ability of these agonists to increase cyclic AMP levels. Overall a significant correlation (r = 0.87, P less than 0.005) was found between WB 4101 binding and alpha-adrenergically mediated cyclic AMP accumulation in brain. Several ligands bind to specific sites in brain membranes with alpha-adrenergic receptor properties. The identification of these binding sites as receptors depends on a correlation of binding with a known alpha-adrenergic receptor-mediated response in brain. These data demonstrating that WB 4101 correlates with norepinephrine-stimulated cyclic AMP accumulation suggest that WB 4101 may bind to the membrane receptor sites mediating the alpha-adrenergic accumulation of cyclic AMP in rat cerebral cortex.

Adrenergic alpha-Antagonists↗

PubMed1977

Microiontophoretic studies of the effects of cylic nucleotides on excitability of neurones in the rat cerebral cortex.

1. Responses of cerebral cortical neurones to the microiontophoretic application of acetylcholine, noradrenaline, cyclic adenosine 3',5'-monophosphate (cyclic AMP) and cyclic guanosine 3',5'-monophosphate (cyclic GMP) were examined.2. The application of acetylcholine and cyclic GMP to identified pyramidal tract neurones resulted in an increased frequency of firing in a large number of cells. Upon application of both substances to cells which could not be identified as pyramidal tract cells, a reduction in the frequency of spontaneous firing was sometimes observed.3. Careful current controls had no effect on the cells discussed here, indicating that the observed responses were not due to the iontophoretic currents. Also, the electro-osmotic ejection of cyclic GMP (outward current) produced similar changes of cell firing to those which followed iontophoretic application (inward current).4. The microiontophoretic application of atropine resulted in a blockade of acetylcholine responses while leaving responses to cyclic GMP unaffected. This suggests that cyclic GMP was not acting indirectly by releasing acetylcholine from presynaptic endings.5. Ejection of cyclic GMP from solutions containing calcium ions produced responses comparable to those produced by cyclic GMP alone. It is unlikely therefore that cyclic GMP was causing excitation by chelating calcium.6. Applications of noradrenaline and cyclic AMP produced a reduction in the spontaneous discharge rate of most neurones tested.7. Phosphodiesterase inhibitors such as ICI 63,197 caused a potentiation of the noradrenaline responses of pyramidal tract neurones.8. 5'-adenosine monophosphate produced a powerful depression of all cells to which it was applied. This action was blocked by aminophylline, suggesting the effect was mediated through an adenosine receptor. Responses to cyclic AMP were usually not abolished, but were reduced by about 50% in amplitude.9. These results are consistent with the hypothesis that cyclic AMP may mediate some neuronal effects of noradrenaline and cyclic GMP may mediate some effects of acetylcholine. The results are also consistent with the suggestion that the two nucleotides may sometimes mediate opposite cellular responses to humoral stimuli.

Acetylcholine↗

PubMed1979

Early histogenesis in the mouse cerebral cortex: a Golgi study.

The early histogenesis in the cerebral cortex of the mouse was studied with a modification of the rapid Golgi method. Three stages can be distinguished first, a period during which the telencephalon is exclusively populated with neuroepithelial cells (till E12); secondly the transient stage of the primordial cortical organization, characterized by the presence, in the outer part of the neural tube, of a loose network of primitive horizontal nerve cells and third, the appearance of the early cortical plate (E14) which contains closely packed, radial bipolar nerve cells. In addition to some salient morphological features of the various cell types, the technique also reveals the importance of efferent fibers from the cortex during the early cortical development.

Animals↗

PubMed1979

Evidence for the use of a pool of the free arachidonic acid in rat cerebral cortex tissue for prostaglandin F2alpha synthesis in vitro.

To determine if the arachidonic acid which is released post-mortem in rat cerebral cortex is directly available for the in vitro synthesis of prostaglandin F2 alpha, cerebral cortex slices or homogenates were incubated in the presence of [2H8]arachidonic acid. The deuterium to protium ratios in the prostaglandin F2 alpha were compared to those in the free arachidonic acid and other lipids of the tissue after 5 and 60 min of incubation. In the slice, the prostaglandin F2 alpha reached maximum labelling before any of the lipids examined except for a pool of rapidly labelled free arachidonic acid located at the damaged surfaces of the tissue. The prostaglandin F2 alpha recovered in the medium after 60 min of incubation had a deuterium to protium ratio twice that of the prostaglandin F2 alpha found in the tissue. Norepinephrine doubled the labelling of the prostaglandin produced by the slice. The labelling of the prostaglandin F2 alpha in homogenates was 10 times higher than in tissue slices and the deuterium to protium ratio of the total free arachidonic acid was similar to that of prostaglandin F2 alpha. The results indicate that exogenous and endogenous arachidonic acid do not mix appreciably in the intact slice and that the prostaglandin synthetase is present on the damaged surface as well as the intact interior of the slice.

Animals↗

PubMed1976

Guanylate cyclase. Subcellular distribution in cardiac muscle, skeletal muscle, cerebral cortex and liver.

1. Guanylate cyclase of every fraction studied showed an absolute requirement for Mn2+ ions for optimal activity; with Mg2+ or Ca2+ reaction was barely detectable. Triton X-100 stimulated the particulate enzyme much more than the supernatant enzyme and solubilized the particulate-enzyme activity. 2. Substantial amounts of guanylate cyclase were recovered with the washed particulate fractions of cardiac muscle (63-98%), skeletal muscle (77-93%), cerebral cortex (62-88%) and liver (60-75%) of various species. The supernatants of these tissues contained 7-38% of total activities. In frog heart, the bulk of guanylate cyclase was present in the supernatant fluid. 3. Plasma-membrane fractions contained 26, 21, 22 and 40% respectively of the total homogenate guanylate cyclase activities present in skeletal muscle (rabbit), cardiac muscle (guinea pig), liver (rat) and cerebral cortex (rat). In each case, the specific activity of this enzyme in plasma membranes showed a five- to ten-fold enrichment when compared with homogenate specific activity. 4. These results suggest that guanylate cyclase, like adenylate cyclase, and ouabain-sensitive Na+ + K+-dependent ATPase (adenosine triphosphatase), is associated with the surface membranes of cardiac muscle, skeletal muscle, liver and cerebral cortex; however, considerable activities are also present in the supernatant fractions of these tissues which contain very little adenylate cyclase or ouabain-sensitive Na+ + K+-dependent ATPase activities.

Adenylyl Cyclases↗

PubMed1979

Rehabilitation following early malnutrition in the rat: body weight, brain size, and cerebral cortex development.

Sprague-Dawley rats were malnourished by giving their mothers an 8% casein diet starting at day 10 of gestation, while controls were fed a 24% casein diet. Starting at postnatal day 20 (P20), rehabilitation of the malnourished animals was attempted by: (1) feeding both mother and young a 24% casein diet, (2) leaving the pups with their mothers until they were 40 days old, and (3) reducing the litter size from 8 to 4 pups. Observations were made on aldehyde-perfused tissue from animals 20, 40 and 70 days old. The somatosensory cortex from one hemisphere was embedded in Araldite, and that from the other side was processed fro Golgi staining. At 20 days of age the body weight of the malnourished animals was 21% that of the controls, but at 70 days it was no longer different. The anterior-posterior length, the width, and the height of the cerebral hemispheres were also significantly reduced at P20, but the differences had disappeared by P70. The thickness of area 3 of the cerebral cortex was measured in 1 micron sections. It was significantly reduced in the malnourished animals at P20, but at P40, following rehabilitation, the difference was no longer statistically significant. In tangential 1 micron sections the fraction of the volume of tissue occupied by neuropil was measured in layers II through IV. At P20 it was significantly reduced only in the upper half of layers II/III of the malnourished animals; at P40 this difference was no longer present. The mean volume of upper layer II/III cell bodies was estimated and found to be significantly reduced in the experimental animals at P20 but not at P40. In the Golgi preparations, pyramidal cells in upper layer II/III were studied. Their estimated volume, as well as the thickness of their basal dendrites, was significantly reduced in the 20 day malnourished animals, but not in the rehabilitated animals. These results show that animals severely malnourished until 20 days of age can reach normal body weight and attain cerebral hemispheres of normal size when proper nutrition is provided. The effects of malnutrition on the cerebral cortex of these animals are most apparent in upper layer II/III which, during the time of nutritional restriction, is the least developed of the cortical layers. However, when proper nutrition is provided, the cerebral cortex may attain normal morphology.

Animals↗

PubMed1975

Cyclic AMP and cyclic GMP may mediate opposite neuronal responses in the rat cerebral cortex.

Electrophysiologically identified pyramidal tract neurons in the rat cerebral cortex were tested with norepinephrine, acetylcholine, adenosine 3',5'-monophosphate (cyclic AMP), and guanosine 3',5'-monophosphate (cyclic GMP) applied by microiontophoresis. The neurons were usually inhibited by norepinephrine and cyclic AMP, but excited by acetylcholine and cyclic GMP. These opposing responses of pyramidal tract neurons to cyclic AMP and cyclic GMP suggests that these two nucleotides could function as reciprocal intracellular second messengers for norepinephrine and acetylcholine, respectively.

Acetylcholine↗

PubMed1975

The involvement of lysophosphoglycerides in neurotransmitter release; the composition and turnover of phospholipids of synaptic vesicles of guinea-pig cerebral cortex and Torpedo electric organ and the effect of stimulation.

(1) Crude synaptosomal fractions (P2) derived from guinea-pig cerebral cortex were incubated in the presence of 50 mM KCl in a Krebs-glucose medium. Torpedo marmorata electric organs were stimulated electrically in vivo at 5 pulses/sec for 30 min by electrodes placed on the electric lobe. Synaptic vesicles were isolated from each source and the phospholipid compositions analysed and compared with vesicles from unstimulated controls. (2) Lysophosphatidylcholine was the only lysophosphoglyceride demonstrable in the synaptic vesicles from either source and its low levels did not increase as a result of chemical or electircal stimulation. In each case there was a close similarity of the phospholipid distributions in the vesicles taken from control and stimulated samples. (3) Control experiments indicated extensive decreases in the acetylcholine content of the vesicles from the stimulated electric organ and smaller decreases in the acetylcholine content of the synaptic vesicles from stimulated crude synaptosomal fractions. These fractions were found to respire linearly in the presence of 10 mM glucose and the vesicle fractions were shown to have low levels of contaiminating membranes as judged by marker enzyme analyses. (4) Crude synaptosomal fractions from guinea-pig cerebral cortex were incubated in a Krebs-glucose medium with labelled fatty acids and [3H]glucose in the presence or absence of 50 mM KCl. Subsynaptosomal fractionation was carried out and specific radioactivities of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol were determined in fractions D (synaptic vesicles), E (microsomes) and H (disrupted synaptosomes). The release of neurotransmitter did not significantly enhance the labelling of phospholipids in any of the fractions studied as compared with phospholipids from unstimulated fractions. This was found after two incubation times and using [14C]oleate, [14C]arachidonate, [3H]palmitate and [3H]glucose.

Acetylcholine↗

PubMed1979

Temperature coefficients of affinity constants for the binding of antagonists to muscarinic receptors in the rat cerebral cortex.

1 The temperature coefficients of binding of a series of muscarinic antagonists to their receptors in membrane preparations from the rat cerebral cortex has been examined. 2 At 37 degrees C the affinity constants agree with those determined by antagonism of acetylcholine-induced contractions of the guinea-pig ileum. 3 The temperature-dependence of the affinity constants is low; for the antagonists examined, the affinity constants at 0 degrees C differ by less than a factor of 3 from those measured at 37 degrees C. 4 There are qualitative similarities between the estimates of the temperature coefficient of binding of the antagonists to receptors in ileum strips and in the cerebral cortex.

Animals↗

PubMed1979

Bidirectional movement of gamma-aminobutyric acid in cerebral cortex slices incubated in small volumes of medium.

Rat cerebral cortex slices were incubated in small volumes of medium in order to study the bidirectional movements of gamma-aminobutyric acid (GABA) between tissue and medium, and to measure the extent of release of endogenous amino acids of interest as potential neurotransmitters. The apparent uptake, real uptake, and net change of GABA were calculated from the changes in [14C]GABA and endogenous GABA in the medium. The extent of release of endogenous amino acids was dependent upon the amino acid the duration of incubation, and the volume of incubating medium. Substantial amounts of endogenous amino acids may accumulate in the medium under conditions commonly employed in amino acid uptake studies. The accumulation of endogenous GABA may affect the calculation of GABA uptake when this is based on the initial specific activity of exogenous [14C]GABA. The difference between apparent uptake and real uptake was particularly prominent in the presence of the depolarizing agents, potassium or veratridine, which caused a marked increase in GABA release. Exchange or net release of GABA was found when slices were incubated briefly in media containing 1.3 micrometer or 12.5 micrometer exogenous GABA; net uptake accounted for 50% of observed uptake when slices were incubated in media containing 25 micrometer GABA.

Amino Acids↗

PubMed1975

[Energy state of the cerebral cortex of the cat during hyperventilation (author's transl)].

Average Po2 and Pco2, local blood flow and pH values in the cerebral cortex of the cat were measured during passive hyperventilation (arterial Pco2 below 19 mm Hg). At defined intervals tissue samples were taken for metabolite analysis. The object of the study was to correlate the data obtained on the brain surface with metabolic responses. Immediately after the start of hyperventilation blood flow decreased, average cortical tissue pressures of O2 and CO2 fell, and there was a simultaneous rise in cortical pH. At a later stage in the experiment the local blood supply reverted to its resting level. Despite a fivefold rise in tissue lactate level during hyperventilation and a decrease in local O2 pressure on the brain surface to 5-10 mm Hg the degree of phosphorylation of energy rich phosphates was not less than under normal conditions of oxygenation. Our investigations showed no evidence of energy lack in cerebral cortex cells during hyperventilation. Cellular hypoxia and its characteristics are defined. The possible causes of raised tissue lactate levels during hyperventilation despite the lack of evidence of cellular hypoxia are discussed.

Adenosine Diphosphate↗

PubMed1978

Idiopathic Parkinsonism with Lewy-type inclusions in cerebral cortex. A case report.

A case of idiopathic parkinsonism showed specific neuropathological findings, namely, the diffuse appearances of intracytoplasmic inclusions of Lewy type in the cerebral cortex in addition to many Lewy bodies in the pigmented brain stem nuclei. The staining properties and the ultrastructure of the inclusions in the cerebral cortex had a strong resemblance to those of the Lewy bodies in the substantia nigra, though a few electron microscopical differences were observed. Almost all of these 'cortical inclusions' were homogeneous or had an obscure core in their center, and they gave the impression of immature Lewy bodies.

Adult↗

PubMed1979

Sets of neurons in somatic cerebral cortex of the cat and their ontogeny.

The process of set formation is briefly reviewed and five monothetic schemes for classification of neurons in the somatic cerebral cortex are described. Criteria for evaluation of neuronal sets are presented and applied to the five different monothetic classification schemes. Classification by size and distribution of peripheral receptive fields orders existing data on cortical neurons better than classification by possession of an axon in the pyramidal tract, by modality, by lability of receptive field, or by 'lemniscal properties'; however, no monothetic scheme orders all the data. A useful polythetic scheme, using s and m terminology is suggested. The ontogeny of the cerebral cortex is reviewed in detail. It is suggested that sa neurons are Golgi type II neurons while m neurons are Golgi type I neurons. The hypothesis is presented that wide-field or m neurons develop and are recognizable before small-field or sa neurons in ontogeny. Evidence regarding this hypothesis is indirect, often conflicting, but suggestive that the hypothesis may be correct. The idea that m neurons may also be phylogenetically older than sa neurons is presented and shown to be consistent with ontogenetic data and interpretations.

Animals↗

PubMed1979

[Cerebral cortex biopotential and blood oxygenation in glaucomatous patient (author's transl)].

This report presents the data of blood oxygenation investigations (performed according to the method of oxyphemography) of cerebral cortex biopotential determination (EEG) in 32 glaucomatous patients (27 having a bilateral process in developed and advanced stages of the disease. The majority of the patients appeared to have oxygen deficiency (26 subj.) and anomalous EEG (29 subj.). A statistically rather high coefficient of correlation between changes in EEG and OHG deviations was determined. The results enabled the authors to conclude that low cerebral cortex activity in glaucoma patients might be the result of blood oxygen deficiency.

Adult↗

PubMed1979

Effect of polyamines on cell-free protein synthesizing systems from rat cerebral cortex, cerebellum and liver.

The naturally occurring polyamines spermine and spermidine stimulate in low concentrations the amino acid-incorporating activity of cell-free systems from cerebral cortex, cerebellum and liver from the rat to a significant degree, but in high concentrations they have strong inhibiting effects on these systems. Maximal polyamine stimulation is observed with suboptimal Mg2+ concentrations in the reaction mixtures; increasing Mg2+ to 15 mM results in an inhibition of cell-free protein synthesis by polyamines even at low concentrations. The stimulating effect of spermine and spermidine involves a facilitated binding of template RNA and aminoacylated tRNA to the ribosomes. In contrast, the aminoacylation of tRNA is not influenced. These data indicate that spermine as well as spermidine have regulatory functions during the translational process in cerebral cortex, cerebellum and liver. They seem to interact with ribosomal functions and to cause a shift in the optimal Mg2+ concentration required for the synthesis of brain and liver proteins.

Amino Acids↗

PubMed1979

[Effect of tissue hypoxia on the structure of cerebral cortex neurons (light--electron microscopic study)].

An examination of the cerebral cortex of rats exposed to sublethal doses of potassium cyanide has revealed changes in the neurons characteristic of the developing histotoxic hypoxia. The neurons of the large hemisphere cortex showed a pronounced hydropic reaction in their cytoplasm with prevalent involvement of the mitochondria. This form of the pathology was observed irrespectively of the duration of the animals live after the poisoning. In rats which survived for a longer time there appeared hyperchromic neurons the number of which gradually increased. This fact was interpreted as addition of the hypoxic state of the neurons to the cyanide intoxication: this was confirmed by development of signs characteristic of hypoxic hypoxia at this stage. The degree of the changes in the mitochondria was found to correlate with the depth of the impairment of vitally important neuron structures. The data obtained show that the primary morphological changes in the neurons develop in their cytomembranes. The degree of the impairment of the mitochondrial membranes can serve as a criterion of reversibility or irreversibility of the pathological process in the nerve cells.

Animals↗

PubMed1976

The incorporation of radioactive fatty acids and of radioactive derivatives of glucose into the phospholipids of subsynaptosomal fractions of cerebral cortex.

1. Crude synaptosomal fractions (P2) from guinea-pig cerebral cortex were incubated in a Krebs-glucose medium containing labelled fatty acids and [3H]glucose. After the shortest incubation period (7.5 min) a high percentage (50-80%) of the total radioactive fatty acids was found in the P2 fractions. 2. After the incubation, the synaptosomal fractions were submitted to hypo-osmotic disruption and subsynaptosomal fractionation was carried out by using discontinuous-sucrose-gradient centrifugation. The specific radioactivities of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol were determined in fractions D (synaptic vesicles), E (microsomal preparation) and H (disrupted synaptosomes), as were the specific activities of a number of marker enzymes and the distribution of acetylcholine. 3. By using [14C]oleate, [14C]arachidonate, [3H]palmitate and [3H]glucose, the order to specific radioactivities in fraction D was found to be: phosphatidylinositol greater than phosphatidylcholine greater than phosphatidylserine greater than phosphatidylethanolamine. 4. The specific radioactivities of phosphatidylcholine and phosphatidylethanolamine were always higher in fraction D than in fraction E. As fraction E had higher specific activities of several membrane marker enzymes, the enhanced labelling found in fraction D was considered to be localized in the synaptic vesicles. In this fraction, phosphatidylinositol made particularly large contributions to the total phospholipid labelling derived from [14C]arachidonate and [3H]glucose. 5. The similar labelling ratios of fatty acid/glucose in the phospholipids of fractions D and E, and the high specific radioactivities in the total phospholipid of the soluble fraction O, suggested intrasynaptosomal phospholipid transport.

Acetylcholine↗