Ultrastructural characterization of rat neurons in primary culture.

Few studies have addressed the ultrastructure and morphology of neurons in primary pure culture. We therefore use immunohistochemistry and electron microscopy to investigate the ultrastructure of cultured neurons during extended incubation in vitro. Rat cerebral cortex neurons were cultured in Neurobasal™ medium. Adherent cells developed as networks of single neurons or clusters depending on the plating density. Almost all surviving cells were neurons as demonstrated by neurofilament immunolabeling. The number of cultured neurons increased substantially to 14-21 days in vitro (DIV) and then plateaued and subsequently declined. From DIV 1-10 neurons extended large neurites, followed by the development of fine and dense neurites, and neurones survived until DIV 30-50. Notably, numerous mitochondria were observed along fibrous elements within neurites, suggestive of active intracellular trafficking. Electron microscopy also revealed that multiple types of synapses were formed between neurons. These ultrastructural results confirm previous reports of electrophysiological activity in cultured neurons. However many neurons contained distorted mitochondria and abnormal organelles including multilamellar vesicles and multivesicular myeloid bodies. The proportion of neurons containing abnormal organelles increased significantly in culture medium supplemented with antibiotics. On long-term culture neuronal death and apoptotic nuclei were observed. Despite the presence of abnormal organelles, the ultrastructure of cultured neurons was very similar to that of in vivo neurons; in vitro culture therefore provides a useful tool for studies on neuronal development, aging, and neurotransmission.

Serotonergic neurotransmission plays a major role in the action of the glycogenic convulsant methionine sulfoximine.

Abnormalities of carbohydrate metabolism and monoamine neurotransmitters have been widely implicated in the pathoetiology of human epilepsy, and glucose hypometabolism and/or tryptophan utilization can be used to localize epileptic foci in the human brain. To investigate the neurochemical changes that underlie seizure susceptibility we studied four strains of mice that respond differently to the convulsant methionine sulfoximine (MSO). Seizures in CBA/J strain were induced by MSO at a dosage half that necessary to provoke seizures in C57BL/6J, BALB/c, or Swiss mice. We report that brain glycogen content in response to MSO administration was markedly increased in all four strains of mice. Of the monoamine neurotransmitters studied, the most prominent change was in brain serotonin (5-hydroxytryptamine, 5-HT) levels that showed a significant reduction following MSO administration. MSO also lowered the concentration of the 5-HT precursor tryptophan. Notably, inhibition of the fall in 5-HT levels by coadministration of 5-hydroxytryptophan delayed the onset of MSO-induced seizures. These results indicate that increased glycogen content and decreased brain levels of 5-HT and tryptophan are hallmarks of MSO action in mice, and suggest that defective serotonergic neurotransmission could trigger glycogen increase and seizure genesis.

Pharmacological, neurochemical, and behavioral profile of JB-788, a new 5-HT1A agonist.

A novel pyridine derivative, 8-{4-[(6-methoxy-2,3-dihydro-[1,4]dioxino[2,3-b]pyridine-3-ylmethyl)-amino]-butyl}-8-aza-spiro[4.5]decane-7,9-dione hydrochloride, termed JB-788, was designed to selectively target 5-HT(1A) receptors. In the present study, the pharmacological profile of JB-788 was characterized in vitro using radioligands binding tests and in vivo using neurochemical and behavioural experiments. JB-788 bound tightly to human 5-HT(1A) receptor expressed in human embryonic kidney 293 (HEK-293) cells with a K(i) value of 0.8 nM. Its binding affinity is in the same range as that observed for the (+/-)8-OH-DPAT, a reference 5HT(1A) agonist compound. Notably, JB-788 only bound weakly to 5-HT(1B) or 5-HT(2A) receptors and moreover the drug displayed only weak or indetectable binding to muscarinic, alpha(2), beta(1) and beta(2) adrenergic receptors, or dopaminergic D(1) receptors. JB-788 was found to display substantial binding affinity for dopaminergic D(2) receptors and, to a lesser extend to alpha(1) adrenoreceptors. JB-788 dose-dependently decreased forskolin-induced cAMP accumulation in HEK cells expressing human 5-HT(1A), thus acting as a potent 5-HT(1A) receptor agonist (E(max.) 75%, EC(50) 3.5 nM). JB-788 did not exhibit any D(2) receptor agonism but progressively inhibited the effects of quinpirole, a D(2) receptor agonist, in the cAMP accumulation test with a K(i) value of 250 nM. JB-788 induced a weak change in cAMP levels in mouse brain but, like some antipsychotics, transiently increased glycogen contents in various brain regions. Behavioral effects were investigated in mice using the elevated plus-maze. JB-788 was found to increase the time duration spent by animals in anxiogenic situations. Locomotor hyperactivity induced by methamphetamine in mouse, a model of antipsychotic activity, was dose-dependently inhibited by JB-788. Altogether, these results suggest that JB-788 displays pharmacological properties, which could be of interest in the area of anxiolytic and antipsychotic drugs.

Abnormal organelles in cultured astrocytes are largely enhanced by streptomycin and intensively by gentamicin.

The effects of two aminoglycoside antibiotics on cultured astrocyte organelles were investigated in rat, sheep, and human cultured astrocytes using transmission electron microscopy. Marked changes in mitochondrial shapes were observed in cultured or subcultured astrocytes obtained from three species, including humans. As well, new types of organelles were observed: (i) numerous concentric membranes forming vesicles, which were termed multilamellar vesicles; and (ii) many vesicles gathering into membranous structures, which were termed multivesicular myeloid bodies. The number of abnormalities increased proportionally with increasing concentrations of the two aminoglycosides (streptomycin and gentamicin). The incorporation of peroxidase or albumin-gold complex in the abnormal vesicles showed that the endolysosomal system was involved in the formation of these vesicles. Our results show that: abnormal organelles are present in cultured astrocytes; these abnormalities are enhanced by streptomycin and gentamicin; and gentamicin induces more abnormalities than streptomycin. The binding of aminoglycosides to membrane phospholipids may explain the formation of the observed abnormalities in rat, sheep, and human cultured astrocytes.

Primary culture of neural precursors from the ovine central nervous system (CNS).

The present study demonstrates that bipotential neural precursors isolated from an early developmental stage of the sheep embryo nervous system can be maintained in vitro in an undifferentiated state for a long period. These precursors multiplied under the action of epidermal growth factor and basic fibroblast growth factor and formed free-floating aggregates of nestin-immunoreactive cells, called neurospheres. These precursors can undergo predominantly neural or glial differentiation according to the culture conditions. Medium supplemented with foetal calf serum mainly favoured cell differentiation predominantly into astrocytes, whereas the defined SATO medium favoured neuronal differentiation. Using various immunomarkers of neurones and astroglial cells, we described the course of differentiation of neuronal and astroglial cells in different culture conditions. The ability to grow neural precursors from common laboratory animals has been useful for studying the cellular and molecular mechanisms underlying the development of the central nervous system. Furthermore, neural progenitors are already being used for in vivo cell therapy in various neurodegenerative disorders. The ovine species is a well-known model for prion diseases, since scrapie is endemic in most countries and has been studied for a long time. In this respect, the availability of ovine neural precursors will add a new perspective to the study of the pathogenicity of prion diseases.

The xenobiotic methionine sulfoximine modulates carbohydrate anabolism and related genes expression in rodent brain.

Methionine sulfoximine is a xenobiotic amino acid derived from methionine. One of its major properties is to display a glycogenic activity in the brain. After studying this property, we investigate here a possible action of this xenobiotic on the expression of genes related to carbohydrate anabolism in the brain. Glycogen was studied by the means of electron microscopy. Astrocytes were cultured and the influence of methionine sulfoximine on carbohydrate anabolism in these cells was investigated. In vivo, methionine sulfoximine induced a large increase in glycogen accumulation. It also enhanced the glycogen accumulation in cultured astrocytes principally, when the medium was enriched in glucose. The gluconeogenic enzyme fructose-1,6-bisphosphatase may account for glycogen accumulation. Plasmids were built using antisens cDNA to permanently block the expression of fructose-1,6-bisphosphatase. An eukaryotic vector was used and the expression of fructose-1,6-bisphosphatase gene was under the control of the promoter of the glial fibrillary acidic protein. In this case, the glycogen content in cultured astrocytes largely decreased. This work shows that methionine sulfoximine enhances energy carbohydrate synthesis in the brain. Since this xenobiotic also enhances the expression of some genes related to one of the key step of glucose synthesis, it is possible that genes may be one target of methionine sulfoximine. Next investigations will study the actual effect of methionine sulfoximine in the cells.

Correlation between brain glycogen and convulsive state in mice submitted to methionine sulfoximine.

It is now well established that in epileptic patients, hypometabolic foci appear during interictal periods. The meaning and the mechanism of such an hypometabolism are as yet unclear. The aim of the present investigation was to look for a putative relationship between glucose metabolism in the brain and the genesis of seizures in mice using administration of the convulsant, methionine sulfoximine. Besides its epileptic action, methionine sulfoximine is a powerful glycogenic agent. We analyzed the epileptogenic and glycogenic effects of methionine sulfoximine in two inbred mouse strains with different susceptibility towards the convulsant. CBA/J mice displayed high response to methionine sulfoximine. The tonic convulsions appeared 5-6 h after MSO administration, without brain glycogen content variations during the preconvulsive period. These mice died of status epilepticus during the first seizure(s). Conversely, C57BL/6J mice displayed low response to MSO. The tonic and clonic seizures appeared 8 to 14 h after MSO administration with only 2% mortality. The seizures were preceded by an increase in brain glycogen content during the preconvulsive period. Moreover, during seizures, C57BL/6J mice were able to mobilize this accumulated brain glycogen, that returned to high value after seizures. The epileptic and glycogenic responses of the parental strains were also observed in mice of the F2 generation. The F2 mice that convulsed early (16%) did not utilize their small increase in brain glycogen content, and resembled CBA/J mice; while the F2 mice that seized tardily (24%) increased their brain glycogen content before convulsion, utilized it during convulsions, and resembled C57BL/6J mice. Sixty percent of the F2 mice presented an intermediate pattern in epileptogenic responses to the convulsant. These data suggest a possible genetic link between the two MSO effects, epileptiform seizures and increase in brain glycogen content. The increase in brain glycogen content and the capability of its mobilization during seizures could delay the seizure's onset and could be considered a "resistance factor" against the seizures.

Early, middle, and late stages of neural cells from ovine embryo in primary cultures.

The utilization of neural cells in culture has importantly increased the knowledge of the nervous system biology. In most studies, the investigations are performed on biological materials coming from common laboratory animals and the extrapolation of the results to other animals is not easy. For some studies, such as developmental biology of the nervous system, prion disease investigations, or agronomical production, the utilization of ovine neural cell cultures presents many advantages. Unfortunately, there are few data on the conditions of culture of such cells. In the present work, we investigated simple ways to obtain neurons and astrocytes from sheep brain. Viable neuronal cell cultures were obtained from 40 to 50 day old fetuses. Their morphologies were quite similar to that of neurons from rodent or chick brain and they were labeled by antineurofilament antibodies. Stages older than 50 days of pregnancy were unable to give viable culture of neurons. The stages of 40 day old fetus to newborn lamb were able to give viable astrocyte cultures. The common protoplasmic astrocytes were obtained and they were labeled by antiglial fibrillary acidic protein antibodies. The astrocytes contained glycogen, thus looking like the common astrocytes from rodents. Neuronal or astroglial cultures can be derived from 26 day old embryos, but the cultures contained contaminating cells. Among the latter cells, there were undifferentiated cells which were flat and epitheloid and which were grouped as islets. These cells could be maintained in culture for a time duration over 7 months, even after two passages. They differentiated principally in astrocytes with a radial configuration. This work shows how some neural cells can be simply and easily cultured from sheep brain. For the first time, neurons were cultured from the sheep embryonic brain. Moreover, stem cells were cultured for more than 7 months and, finally, glycogen accumulation in sheep astrocytes was shown to be the same as that in rodent astrocytes. The oligodendrocyte culture was already documented. Thus, sheep can easily be used as well as other models for neural cell studies.

Direct cloning of astrocytes from primary culture without previous immortalization.

In primary cultures, much evidence shows the existence of different subtypes of astrocytes that are not all identified. One methodology for studying these subtypes can be their cloning. The present investigation shows a method for a direct cloning of astrocytes without previous immortalization. Astrocytes from the cerebral cortex of newborn rats were cultured, purified by shaking, and harvested by trypsinization. One single astrocyte was plated in a small volume of a homemade cloning medium. After getting a colony, successive platings were made using larger and larger vessels, up to 60-mm-diameter petri dishes. Then, subcultures were made. The yield of the cloning was similar to that of common eukaryotic cell clonings. All along the cloning procedure, the cells were positively immunostained with anti-glial fibrillary acidic protein antibodies. Cloned cells from some batches were spindle-shaped, looking like fibroblasts. Nevertheless, they were immunostained with anti-glial fibrillary acidic protein antibodies, unlike true fibroblasts. These spindle-shaped astrocytes were compared to cells from an astrocytoma cell line that had the same shape. The growth pattern of the astrocytoma cells was different from that of the astrocytes cloned from the primary cultures. All the types of studied cells contained glycogen. On the basis of the criteria of morphology, of glial fibrillary acidic protein immunolabeling, and of glycogen synthesis, the cloned cells kept the characteristics of astrocytes. This study shows that it is perfectly possible to get clones of astrocytes from one astrocyte without previous immortalization, giving thus a convenient material for the study of astrocyte biology.

Various fructose-1,6-bisphosphatase mRNAs in mouse brain, liver, kidney and heart.

The mouse fructose-1,6-bisphosphatase (FBPase) cDNA was previously cloned from testicular teratocarcinoma cultured cells (F9 cells). Using this published nucleotide sequence four primer sets were defined and used to amplify FBPase transcript from cerebral cortex, heart, kidney, liver and testis of male C57B1/6 mice. Only one primer set was efficient in all total RNA prepared from the various tissues. The restriction maps of these RNA amplification products suggested the existence of three different FBPase transcripts; this was confirmed by the nucleotide sequences of the FBPase transcripts and by the deduced amino acid sequences. These data are consistent with the existence of three different FBPase genes. This may be relevant in neurological disease in which abnormalities of brain glucose metabolism are involved.

Isolation of carbohydrate metabolic clones from cultured astrocytes.

Astrocytes are the principal sites of glycogen synthesis in the nervous tissue. Growing evidence shows that there are many types of astrocytes. The aim of the present investigation was to isolate different types of astrocytes that display different carbohydrate anabolism. Astrocytes from newborn rat brain were directly cloned from primary cultures without a previous transformation. Many clones were obtained, and they were termed CP clones. Another series of clones, termed SV clones, were obtained after the transfection of the primary cultures by the SV40 T antigen. The effectiveness of the transfection was verified by the rate of DNA synthesis using flow cytometry and by the presence of plasmid DNA in the genomic DNA of the astrocytes using the Southern blot method. After the transfection, the growth velocity increased greatly. The size and shape of the astrocytes were the same for each cell in a given clone, regardless of the cloning method utilized. However, these sizes and shapes could be different from one clone to another in CP clones, whereas all the astrocytes of all the SV clones looked like each other. All the clones obtained stained positively with anti-glial fibrillary acidic protein antibodies. Glycogen stained in the clones using concanavalin A-horseradish peroxidase. The glycogen content was also measured using biochemical analysis. Concordant results obtained using two methods showed that some clones contained an important quantity of glycogen while other clones contained a small amount, in the CP series as well as in the SV series. This property was the same for the intracellular glucose concentrations. The activity of the gluconeogenic enzyme fructose-1, 6-bisphosphatase was measured in each clone using spectrophotometry. This activity was also significantly different from one clone to another. The clones containing large amounts of glycogen had important fructose-1,6-bisphosphatase activity. The present results show that it is possible to clone astrocytes either directly from primary cultures without immortalization or after their transformation. When analyzing these clones, it appears that carbohydrate anabolism can be significantly different from one astrocyte to another. This difference may also exist in vivo.

Methionine sulfoximine increases acetylcholine level in the rat brain: no relation with epileptogenesis.

Methionine sulfoximine induces epileptiform convulsions in rats. A possible involvement of acetylcholine in the onset of convulsions was investigated. A subconvulsive dose of methionine sulfoximine increased the brain acetylcholine concentration. After administration of a convulsive dose, atropine neither prevented the onset of the seizures nor prevented the increase in acetylcholine concentration. Physostigmine enhanced the increase in acetylcholine level but did not modify the time course nor the intensity of the convulsions. L-DOPA suppressed the seizures without inhibiting the increase in acetylcholine level. The choline content decreased after the convulsant dose. The increase in acetylcholine content is therefore not the unique cause of the seizures, which could result from the reduction of striatal inhibition due to a decrease in dopamine level induced by methionine sulfoximine.

Regulation of fructose-1,6-bisphosphatase activity in primary cultured astrocytes.

In the gluconeogenic pathway, fructose-1,6-bisphosphatase (EC 3. 1. 3. 11) is the last key-enzyme before the synthesis of glucose-6-phosphate. The extreme diversity of cells present in the whole brain does not facilitate in vivo study of this enzyme and makes it difficult to understand the regulatory mechanisms of the related carbohydrate metabolism. It is for instance difficult to grasp the actual effect of ions like potassium, magnesium and manganese on the metabolic process just as it is difficult to grasp the effect of different pH values and the influence of glycogenic compounds such as methionine sulfoximine. The present investigation attempts to study the expression and regulation of fructose-1,6-bisphosphatase in cultured astrocytes. Cerebral cortex of new-born rats was dissociated into single cells that were then plated. The cultured cells were flat and roughly polygonal and were positively immunostained by anti-glial fibrillary acidic protein antibodies. Cultured astrocytes are able to display the activity of fructose-1,6-bisphosphatase. This activity was much higher than that in brain tissue in vivo. Fructose-1,6-bisphosphatase in cultured astrocytes did not require magnesium ions for its activity. The initial velocity observed when the activity was measured in standard conditions was largely increased when the enzyme was incubated with Mn2+. This increase was however followed by a decrease in absorbance resulting in the induction, by the manganese ions, of a singular kinetics in the enzyme activity. Potassium ions also stimulated fructose-1,6-bisphosphatase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Some aspects of carbohydrate metabolism in the brain.

A convenient physiology of the nervous system closely depends on the availability of glucose, the lack of which quickly results in syncope and death. Carbohydrate metabolism in the brain was long thought of as being specific and different from liver carbohydrate metabolism. The present report tries to summarize current data and advances in our knowledge about carbohydrate metabolism. Glucose is brought to the brain by blood flowing through a special network of arteries and is quickly catabolized by the glycolytic and tricarboxylic acid cycle pathways to synthesize energy. It is also used in the synthesis of numerous amino acids, nucleotides and NADPH. Glucose can be polymerized into glycogen in the brain. The nerve tissue is capable of synthesizing glucose-6-phosphate in the gluconeogenic pathway since the fructose-1,6-bisphosphatase, the key enzyme believed to be absent, is actually active and has been purified up to electrophoretic homogeneity. Moreover, the possibility of free glucose synthesis by astrocytes exists. Although the exact role of glycogen in the brain is not totally clear, it is known that the polysaccharide content generally decreases when the functioning of the brain is stimulated and increases in sedative state. This carbohydrate can therefore serve as an indicator for the level of brain activity. Through the administration of methionine sulfoximine, it is possible to increase the amount of glycogen in the brain massively and obtain particles similar to those found in the liver. These in vivo findings have been confirmed by studies based on cultured astrocytes. It has been shown with cultured astrocytes that glutamate increases glycogen synthesis in a pathway which still remains to be elucidated. Brain carbohydrate metabolism is thus in many ways similar to liver carbohydrate metabolism. The astrocyte constitutes the main cell implicated in this metabolism. Improvement in our knowledge about brain carbohydrate metabolism should spread the use of brain glucose metabolism in the diagnosis of certain diseases.

Biochemical and ultrastructural study of glycogen in cultured astrocytes submitted to the convulsant methionine sulfoximine.

The convulsant methionine sulfoximine is a potent glycogenic agent in the central nervous system of rodents in vivo. This investigation was undertaken to look for the basic mechanism underlying this property. Astrocytes were cultivated from newborn rat neopallium and glycogen was studied by both biochemical and ultrastructural methods. When the astrocytes were incubated in a medium containing 5.55 mM glucose, methionine sulfoximine (0.55 mM) induced a significant increase in their glycogen content. Glucose content did not change in astrocytes, but it diminished in the medium in all cases. When the decrease in glucose level in the medium was limited, the same glycogenic effects of methionine sulfoximine were observed, but the glycogen contents were higher. The augmentation of the concentration of the convulsant enhanced its glycogenic effect, but this was not directly dose dependent. When the flat and polygonal astrocytes were transformed into process-bearing astrocytes by dibutyryl cyclic AMP methionine sulfoximine always induced an increase in glycogen content. In this case, the values of glycogen contents were lower. In electron microscopy, no glycogen particles were present in the astrocytes even after methionine sulfoximine treatment, contrary to the case in vivo. These results show that the convulsant does not need the presence of neuronal cells to induce glycogen accumulation and that astrocytes may be the direct cell targets. The apparent discrepancy between the biochemical and ultrastructural data is probably due to the relatively low concentration of glycogen in cultured astrocytes.

Correlation between carbohydrate and catecholamine level impairments in methionine sulfoximine epileptogenic rat brain.

This work shows that the convulsant methionine sulfoximine induces an increase in glucose and glycogen levels and a parallel decrease in norepinephrine and dopamine levels in rat brain. Among the epileptogenic agents, methionine sulfoximine is known to have a glycogenic property in the central nervous system. The aim of this work is to look for the neurochemical mechanism underlying this property. For this, catecholamines, glucose, and glycogen were measured at the same time in different areas of the brain in rats submitted to methionine sulfoximine. The convulsant induced an increase in glucose and glycogen levels as previously described and a decrease in dopamine and norepinephrine levels in all the areas of the rat brain. These changes were roughly dose dependent. When L-dihydroxyphenylalanine and benserazide (a decarboxylase inhibitor) were administered with methionine sulfoximine, the latter failed to induce seizures in rat up to 8 h after dosing. Moreover, the glucose and glycogen amounts did not increase. In all these experiments, there was an obvious evidence of parallelism between seizures, increase in carbohydrate levels, and decrease in catecholamine levels. These results allow to conclude that the glycogenic property of methionine sulfoximine in the central nervous system probably results from its ability to decrease norepinephrine and dopamine levels. Because the effect of the convulsant on the catecholamine levels persisted for long, it is normal that glucose and glycogen levels increased during preconvulsive, convulsive and postconvulsive period. Methionine sulfoximine is probably glycogenic in rat brain because it decreases catecholamine levels for a long time.

Possible involvement of indolamines in the glycogenic effect of the convulsant methionine sulfoximine in rat brain.

The aim of the present investigation was to look for the mechanisms causing disturbances in carbohydrate metabolism during the action of the epileptogenic agent methionine sulfoximine. The levels of glucose, glycogen, and indolamines were measured in seven different regions of rat brain. Methionine sulfoximine induced a decrease in serotonin level which was roughly dose-dependent. There were no obvious changes in tryptophan and 5-hydroxyindoleacetic levels in any area. Methionine sulfoximine induced the known increase in glucose and glycogen levels. The direct precursor of serotonin. 5-hydroxytryptophan, and benserazide (a decarboxylase inhibitor) were then injected into rats in association with methionine sulfoximine. In this case, methionine sulfoximine failed to induce seizures. Moreover, the serotonin level was unchanged and the carbohydrate content did not significantly increase. There was only a rise in 5-hydroxyindoleacetic acid level. This work shows a striking parallelism between serotonin decrease and glycogen increase.

Catecholamine and indoleamine levels in chick cultured neurons and embryonic brain.

Catecholamine and indoleamine levels were determined in cultured neurons from chick embryos and in the "homologous" embryonic cerebral hemispheres in order to study their neurotransmission systems. The seeding of a large number of cells resulted in a pure neuronal culture made of clusters interconnected by processes. Norepinephrine, which was absent from the starting material of the culture, appeared on the 2nd day and then decreased. A small amount of epinephrine was present on the 2nd day and decreased thereafter. Dopamine was not detected. In the cerebral hemispheres of chick embryos, dopamine appeared on the 10th day in ovo and increased steadily up to the 18th day. Epinephrine was also present in the cerebral hemispheres. Its level increased up to the 14th day and then decreased. Indoleamines were measured in the same material. The level of serotonin was markedly higher than that of catecholamines and it increased during cultivation. Tryptophan was already present in the starting material and its amount increased during cultivation. The level of 5-hydroxyindoleacetic acid changed like that of serotonin. In the embryonic cerebral hemispheres, the concentration of serotonin was highest on the 12th day after incubation and then decreased. Tryptophan level decreased steadily all during the embryogenesis. These results were discussed on the ground of differences in the synthesized neurotransmitters.

Glycogen synthesis and immunocytochemical study of fructose-1,6-biphosphatase in methionine sulfoximine epileptogenic rodent brain.

The effects of the convulsant methionine sulfoximine (MSO) on the glucose pathway have been investigated in mouse and rat brain. The key gluconeogenic enzyme fructose-1,6-biphosphatase (FBPase) (EC 3.1.3.11) was immunostained by rat anti-FBPase antibody. The rat cortex slices were very lightly stained, almost unstained in controls. After MSO injection, there was a marked staining only in astrocytes (perikarya, processes, and end feet). The activity of this enzyme also increased. MSO induced an increase of 63% in the stability at heating (47 degrees C) and of 36% in the stability at proteolysis (trypsin, 10 micrograms/ml) of FBPase. The convulsant had no effect on the concentrations of the metabolites related to the FBPase-phosphofructokinase step, i.e., fructose-1,6-biphosphate, glyceraldehyde-3-phosphate, and dihydroxyacetone phosphate, before, during, or after the convulsions. These results show that the cellular site of glucose pathway impairment induced by MSO in rodent brain is presumably the astroglial cells and that one mechanism of glycogenesis could be the reinforcement of the molecules of FBPase, which enhances gluconeogenesis. A hypothetical diagram of glucose metabolism under the effect of MSO has been proposed.

Glycogen content and fructose-1, 6-biphosphatase activity in methionine sulfoximine epileptogenic mouse brain and liver after protein synthesis inhibition.

Mice given intraperitoneal injections of methionine sulfoximine (MSO) (100 mg/kg body weight) showed tonic-clonic seizures 7 to 8 h later. The protein synthesis inhibitors actinomycin D and cycloheximide, when combined with MSO delayed the onset of seizures. Methionine completely abolished the convulsions and metyrapone delayed them for some hours. Twenty-four h after the administration of the convulsant, the activity of the gluconeogenic enzyme, fructose-1, 6-biphosphatase (FBPase), and the glycogen content were determined in different areas of the brain. MSO induced an increase in both FBPase activity and glycogen content. These effects were antagonized by the inhibitors of protein synthesis. Metyrapone partly inhibited MSO-induced increases of FBPase activity and glycogen content whereas methionine completely abolished them. MSO decreased glycogen content in liver but had no effect on blood glucose level 24 h after its administration. These findings suggested that in MSO epileptogenic brain, glycogen accumulation may proceed from an enhanced gluconeogenesis.