Jacques Gayet

Ultrastructural study on transcapillary exchanges in the developing telencephalon of the chicken.

Summary Horseradish peroxidase was intravenously injected in the chick during embryonic development and post-hatching growth, in order to study, in the telencephalon, the transcapillary exchanges of the enzyme molecule by electron microscopy. In the embryo, until the 10th day of incubation, the reaction product, making visible the presence of the molecule of peroxidase, was distributed in the intercellular spaces in the neurophil. From this embryonic developmental stage and during the post-hatching growth, the reaction product filled the lumen of the capillaries and some micropinocytotic vesicles with the endothelium near the lumen. The ultrastructural study of the differentiation of the intercellular junctions between the apposed plasma membranes of the endothelial cells, from the 8th day of embryonic development, has shown that the tight junctional complexes become progressively more numerous. It therefore appears that the impediment to the passage of the molecule of peroxidase across the capillary wall, which in the telencephalon is effective from the 10th day of embryonic development, is due to the presence of the tight intercellular junctions between plasma membranes of the endothelium.

Stimulation of Fructose-1,6-Biphosphatase Activity and Synthesis in the Cerebral Cortex of Rats Submitted to the Convulsant Methionine Sulfoximine

Abstract: Purification of rat cerebral cortex fructose‐ l,6‐biphosphatase (FBPase) was performed by substrate elution from phosphocellulose, followed by Sephadex G‐200 column filtration. The purified enzyme exhibited an optimum at pH 7.5, and its catalytic properties were very similar to those of the purified whole‐brain enzyme previously prepared by Majumder and Eisenberg in 1977. The isolated preparation was electrophoretically homogeneous. The molecular weight of the enzyme subunit was 40,000; the hydrophobic amino acids predominated with 592 residues, and tryptophan was not detected. Expressed as μmol fructose‐1,6‐biphosphate hydrolysed per g brain tissue wet weight per min, FBPase activity increased twofold 24 h after an intrapentoned injection of 100 mg per kg body weight of the convulsant methionine sulfoximine (MSO); the increase of the rate of incorporation of [1‐14C]valine into brain FBPase was 2.8‐fold under the same experimental conditions. A rabbit specific antiserum against rat cerebral cortex FBPase was prepared, and immunotitration studies confirmed both an increase in the number of molecules and the activation of brain FBPase, 24 h after administration of MSO. The increase of the number of brain FBPase molecules, induced by MSO, was due to an increase in synthesis of the enzyme, as shown by a double‐label valine incorporation study.

Kinetics of utilization in vivo of glucose carbon in the chick cerebral hemispheres during postnatal growth

Abstract 1. 1. The incorporation of glucose carbon in vivo into glucose, amino acids and carboxylic acids in the cerebral hemispheres was studied during post-hatching growth of the chick after subcutaneous injection of [U-14C]glucose, the animals being decapitated at different times after the injection. 2. 2. The highest rate of incorporation of glucose carbon into the tissue free amino acids occurs between 10 hr and 4 days after hatching. 3. 3. At hatching time, glutamic acid as well as neutral amino acids account for 40% of the whole radioactivity in the amino acid fraction. On the 30th day of postnatal growth, the % of radioactivity in glutamic acid (56%) is higher than that in the neutral amino acids (23%), which is a characteristic of the metabolism in cerebral hemispheres of adult chicks. 4. 4. Studies of the evolution of cerebral glucose concentration during the postnatal growth show an increasing rate between 10 and 48 hr after hatching. 5. 5. On the 2nd, 4th and 30th days of postnatal growth aspartate and glutamate have approximately the same specific radioactivity. On the other hand, at 10 hr after hatching, the specific radioactivity of cerebral aspartate is always higher than that of glutamate. 6. 6. At 10 hr after hatching, the specific radioactivity of cerebral α-alanine is always considerably higher than that of other amino acids. On the 30th day of postnatal growth, and at 10 min after the injection of [U-14C]glucose, the specific radioactivity of α-alanine is higher than that of other amino acids; afterwards, from 10 to 60 min after the injection of labelled glucose, the specific radioactivity of α-alanine decreases and its value tends towards zero.

THE PENETRATION OF THE MEMBRANE OF BRAIN MITOCHONDRIA BY ANIONS

The permeability of the membrane of rat brain non‐synaptosomal mitochondria, towards inorganic and substrate anions, was assessed by measuring the rate of swelling that occurred when mitochondria were suspended in an iso‐osmotic solution of a permeant anion, in the presence of a permeant cation such as NH+4 or K+ in the presence or absence of valinomycin. In NH+4‐phosphate swelling was higher than it was in KCI or K+‐phosphate, which showed the prevalence of the mechanism of phosphate transport previously demonstrated in liver mitochondria. The entry of succinate and L‐malate seemed to require the presence in the inner mitochondrial membrane of specific carriers. as previously postulated for liver mitochondria, but the rate of swelling of brain mitochondria was lower than that of liver organelles. In K+‐succinate, in the presence of antimycin, added ATP induced swelling and this was attributable to the simultaneous permeation both of the anion and the cation. Fumarate did not penetrate into brain mitochondria. Practically no swelling was recorded in NH+4 or K+‐citrate, which indicated that this anion penetrated poorly into the isolated brain mitochondria even in the presence of malate. Swelling occurred in NH+4‐L‐glutamate in the presence of rotenone, and the entry of this anion seemed to follow a gradient of concentration although the presence of a specific translocator in the inner mitochondrial membrane might be concerned. The entry of glutamate was independent of that of phosphate and N‐ethylmaleimide appeared to be a specific inhibitor of this entry. Swelling in K+‐L‐glutamate, in the presence of rotenone, was enhanced by the addition of valinomycin or ATP but in the latter case when osmotic equilibrium was reached swelling was not reversed by oligomycin. In conclusion, the lesser extent of swelling of isolated brain mitochondria compared with liver mitochondria could be attributed to the heterogeneity of the populations of these organelles, each population possessing its own characteristics of membrane permeability. Observations of electron micrographs of brain mitochondria incubated in iso‐osmotic substrate anions confirmed the heterogeneous rate of swelling of these particles.

Diffusion of horseradish peroxidase perfused through the lateral ventricle of the chick telencephalon

SummaryHorseradish peroxidase, perfused into the lateral ventricle of chick brain, freely and slowly diffuses through the cerebral extracellular spaces. The layer of astrocytic end-feet surrounding blood capillaries does not constitute a barrier to the tracer which permeates the basal lamina, diffuses between the pericytic cells and finally accumulates in the intercellular space beneath the tight junctions between contiguous endothelial cells. No evidence was found for transport by micropinocytotic vesicles from the cerebral parenchyma to the capillary lumen.

Glucose and amino acid metabolism in chick telencephalon slices: Changes with incubation conditions and animals' development

Glucose and amino acid metabolism in 1- and 30-day-old chick telencephalon slices was studied in two incubation media in the presence or in the absence of a continuous oxygenation. Medium 1 has a composition and a tonicity similar to cerebrospinal fluid, medium 2 is hypertonic and does not contain any K+ ions. The incorporation of glucose carbon into amino acids and the distribution of radioactivity between the different amino acids are close to the ones observed in the chick brain in vivo only when the slices are incubated in medium 1, with oxygen at 30 days and without oxygen for the 1-day-old chick. It also appears that if oxygenation is necessary for incubation of mature brain tissue in vitro, the absence of the medium oxygenation is more suitable for the study of glucose metabolism in 1-day-old chick brain slices.

Intranigral methionine sulfoximine induces turning behaviour in the rat

Unilateral infusion of methionine sulfoximine (MSO) into the substantia nigra of the rat induced contralateral rotations appearing progressively after a delay of about 100 min. The rotations were maximally antagonized by picrotoxin and bicuculline intraperitoneally injected at approximately 50-80 min after the intranigral infusion of MSO. Contraversive turning behaviour was absent after intranigral infusion of MSO in unilaterally 6-hydroxydopamine-lesioned rats.

Utilization of acetate by chick brain during postnatal maturation

1. The study of the compartmentation of glutamate metabolism has been performed in the chick brain in vivo and in vitro in the presence of [U-14C]acetate between day 1 and day 30 of postnatal maturation. 2. The compartmentation of glutamate metabolism in vivo appears between day 1 and day 4 after hatching in the cerebral hemispheres and optic lobes. It is however more precocious in the optic lobes. In the cerebellum, it appears later, at about day 4 after hatching. The compartmentation of glutamate metabolism appears at the same time as the rapid incorporation of glucose into amino acids takes place in the cerebral hemispheres and the optic lobes. 3. In the chick telencephalon in vitro, the compartmentation of glutamate metabolism is visible from day 1 after hatching onwards. This difference is undoubtedly linked to the absence of an interference of glucose metabolism with acetate metabolism in vitro, and to the presence of a third compartment in the cerebral slices.

Ultrastructural and biochemical studies of the swelling of developing chick telencephalic slices

SummaryAn ultrastructural and biochemical study of the importance and localization of tissue swelling was performed on telencephalic slices of 1- and 30-day-old chicks incubated in an oxygenated or a non-oxygenated physiological medium. The swelling of slices is greater for 30-day-old chick material than for that from 1-day-old chicks. It also reaches higher values in the non-oxygenated than in the oxygenated medium. When the 30-day-old chick telencephalic slices are incubated in an oxygenated medium, swelling mainly affects astrocytes, and especially the astrocytic endfeet. When they are incubated in a non-oxygenated medium, the astrocytes and astrocytic endfeet are very swollen and in addition the swelling also affects the neurons and their organelles. Extracellular space is increased. When 1-day-old chick telencephalic slices are incubated in a non-oxygenated medium, the tissue structures are well preserved. Swelling predominantly affects astrocytes and astrocytic endfeet. Neurons are not affected and the extracellular space is reduced. However, when they are incubated in an oxygenated medium, tissue structures are greatly affected showing a high degree of disorganization. Extracellular space is greatly increased. This study thus indicates that the best incubation conditions are an oxygenated medium for 30-day-old chick telencephalic slices which are characterized by an aerobic metabolism, and a non-oxygenated medium for 1-day-old chick telencephalic slices which have a predominantly anaerobic metabolism.