Glutatmine synthetase activity in the chick brain during postnatal growth. Effect of MSO

Glutamine synthetase activity was estimated in the chick cerebral hemispheres, optic lobes and cerebellum between the 1st and the 30th day of postnatal growth. Glutamine synthetase activity is higher in the cerebellum than in the cerebral hemispheres and lowest in the optic lobes at 1 day after hatching; at 30 days after hatching, it is the same in the optic lobes and in the cerebellum and lowest in the cerebral hemispheres. The great increase of glutamine synthetase activity between the 1st and the 4th day after hatching corresponds to the appearance of the heterogeneity of the chick brain glutamate metabolism. The glutamine synthetase activity is inhibited by MSO $\text{in vivo}$ at a concentration of 100 mg kg ^?1 at values of 87, 90 and 89 % in cerebral hemispheres, optic lobes and cerebellum of 1, 2 and 4-day-old chicks. The enzyme inhibition is less pronounced $\text{in vitro}$ and reaches values of about 25 and 75 % for 1 and 10 mM MSO concentrations respectively in the three brain areas of the 1 to 4-day-old chick and values slightly lower in the 30-day-old chick brain.     

Utilization in vivo of glucose carbon in the optic lobes and the cerebellum of the chick during postnatal growth.

1. The incorporation of glucose carbon in vivo into amino acids was studied in the chick optic lobes and cerebellum during postnatal growth after subcutaneous injection of [U-14C]glucose. 2. The rapid incorporation of glucose carbon into free amino acids appears between the 1st and the 2nd day of postnatal growth in the optic lobes and between the 1st and the 4th day after hatching in the cerebellum. 3. The period during which the properties of mature brain metabolism are obtained is characterized in both structures during the first 48 hr of postnatal growth by changes in the specific radioactivity of some amino acids such as aspartate and alpha-alanine, and also by transient increases of glucose and glutamine concentrations. 4. The gamma-aminobutyrate content in the optic lobes is very high; the cerebellum on the contrary is characterized by its low gamma-aminobutyrate concentration linked to a very fast metabolism of this amino acid.     

Glutaminase activity in the chick central nervous system during postnatal growth.

1. Glutaminase activity was evaluated in the chick cerebral hemispheres, optic lobes and cerebellum between the 1st and 30th day of postnatal growth. 2. Glutaminase activity is higher in the cerebral hemispheres than in the optic lobes and is lowest in the cerebellum. 3. It seems to be inversely related to the magnitude of the variations of glutamine concentration in the three areas. 4. No direct relation exists between enzyme activity and glutamate concentration in the three tissues.     

Stimulation of brain development in chick embryo by elevated temperature

Summary Experimental chick embryos were incubated at 37.5°C till day 7 and after day 10, and at 40.5°C on days 7–10; their optic lobes and cerebral hemispheres at day 10 and at hatching were compared with controls incubated at 37.5°C only. Cell numbers at day 10 were directly counted by a new method involving formalin fixation and cell disaggregation by gentle sonication. At hatching, body weights, organ weights and organ DNA (cell numbers) were the same in experimentals and in controls, for both optic lobes and cerebral hemispheres, though the protein contents were significantly higher in experimentals. However, at 10 days (end of neuron proliferation) the weights and the cell numbers in experimentals were significantly higher. Two possible explanations have been offered: 1. Elevated neuron population in experimental animals at day 10 is followed by their elevated death rate, or 2. The increment in neuron number is permanent but at hatching it is overshadowed by the population of other cells.An abstract of this work has been presented (Zamenhof, 1975)     

The infraslow potential oscillations in developing chick embryo brain. Correlations with neuronal activity.

The correlations between infraslow potential oscillations [ISPO] and EEG activity were studied in the brain hemispheres and optic lobes of chick embryos from the 15th to 21st day of incubation. 1. The ISPO of the optic lobes remained unchanged during and after peripheral optic stimulation -- not only in 17-day-old embryos (the prefunctional stage), but also in 19- and 21-day-old embryos, in which optic evoked activity in the optic tectum is already well developed. 2. The intracerebral administration of strychnine, GABA and sodium glutamate had no effect on the ISPO of the brain hemispheres in 15-day-old embryos. 3. The effect of these neurotropic drugs in 20-day-old embryos varied. Strychnine evoked concomitant activation of ISPO and the EEG, sodium glutamate simultaneously depressed both activities, while GABA inhibited EEG activity without affecting ISPO patterns. 4. These results supported our conclusion that neuronal activity plays a secondary role in the ISPO generation process in developing brain tissue.     

GLUTAMATE METABOLISM IN OCTOPUS BRAIN IN VIVO;ABSENCE OF A WAELSCH EFFECT

Abstract— The metabolism of [U-¹⁴C]glutamate was followed in vivo in the octopus Eledone cirrhosa following intracranial injection, and compared with that in the mammalian brain.
By contrast with the rat brain, the specific activity of glutamine recovered from Eledone optic and vertical lobes was lower than that of glutamate at short time intervals after injection. Thus the Waelsch effect was not apparent in this species. Again, in contrast with the rat brain, radioactivity could be found in alanine but not in GABA following [U-¹⁴C]glutamate injection. This was compatible with observations made previously in vitro.
The significance of these intraspecies differences in metabolism and compartmentation is discussed.     

The development of superficial infraslow potential oscillations of the brain in chick embryos.

The development of superficial infraslow potential oscillations (ISPO) of brain hemispheres, cerebellum and optic lobes was studied in chick embryos between day 9 and 21 of incubation. The ISPO were firstly registered in brain hemispheres at day 10 of incubation, i.e. 5 days before the onset of spontaneous EEG activity. The ISPO in 10-day-old embryos had an average frequency of 9.9 c/min and an amplitude of 0.14 mV. During further development till hatching the frequency decreased to 7.5 c/min and the amplitude increased to 1.39 mV. Similar ISPO with the same developmental trend were also registered from the surface of the cerebellum and optic lobes. Superficial ISPO were not synchronized either between both hemispheres or between different fields of the same hemisphere.     

Effects of corticosterone on brain cholinergic enzymes in chick embryos

The effects of corticosterone on the cholinergic enzymes, choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) were studied in the chick embryonic brain. Chick embryos received either 0.25, 0.5, or 1.0 g of corticosterone via the air sac daily for three days during either embryonic days 6 through 8 (E6-E8), of cerebral neurogenesis, or days 10 through 12 (E10-E12), a period of cerebellar neurogenesis. Enzyme activities were determined in cerebral hemispheres, optic lobes, cerebellum and remaining brain at 10, 15, and 20 days of incubation. In embryos treated from E6 to E8, ChAT activity was generally higher at day 10 in cerebral hemispheres and optic lobes (cerebellum was not determined) while AChE activity was not affected. At day 20 ChAT activity of treated chick embryos was lower in the cerebral hemispheres and optic lobes, but not in the cerebellum; AChE activity was higher in the cerebral hemispheres, lower in the optic lobes, and not changed in the cerebellum as compared to controls. However, in embryos treated from E10 to E12 both cerebellar ChAT and AChE activities were higher at day 15 in comparison to controls. These data show that the hormonal effects were most prominent only in the brain areas undergoing neurogenesis during the period of hormonal treatment. Since AChE activity is also present in nonneuronal cells, the observed alterations caused by corticosterone may reflect glial cell responses to the hormone. Whether the hormone affects the final number and/or maturation of cholinergic neurons and/or glial cells remain to be investigated.     

Purification and characterization of cytosolic aldolase from carrot storage root.

The time courses of incorporation of 13C from 13C-labelled glucose or acetate into cerebral amino acids (glutamate, glutamine and 4-aminobutyrate) and lactate were monitored by using 13C-n.m.r. spectroscopy. When [1-13C]glucose was used as precursor the C-2 of 4-aminobutyrate was more highly labelled than the analogous C-4 of glutamate, whereas no label was observed in glutamine. A similar pattern was observed with [2-13C]glucose: the C-1 of 4-aminobutyrate was more highly labelled than the analogous C-5 of glutamate. Again, no labelling of glutamine was detected. In contrast, [2-13C]acetate labelled the C-4 of glutamine and the C-2 of 4-aminobutyrate more highly than the C-4 of glutamate; [1-13C]acetate also labelled the C-1 and C-5 positions of glutamine more than the analogous positions of glutamate. These results are consistent with earlier patterns reported from the use of 14C-labelled precursors that led to the concept of compartmentation of neuronal and glial metabolism and now provide the possibility of distinguishing differential effects of metabolic perturbations on the two pools simultaneously. An unexpected observation was that citrate is more highly labelled from acetate than from glucose.     

Developmental Changes in β-Citryl-L-G1utamate Concentration and Its Synthetic and Hydrolytic Activities in Neuronal Cells Cultured from Chick Embryo Optic Lobes

Developmental changes in the concentration of beta-citryl-L-glutamate(beta-CG) have been examined in the cerebrum and optic lobe of the developing chick brain and in primary cultured neuronal cells from the chick embryo optic lobes with gas chromatographic and HPLC methods originated in our studies. A sharp peak was shown by beta-CG, with a maximal concentration at 13 days of incubation in the optic lobe of the developing chick brain but decreasing markedly to adult levels. The developmental change in primary cultured neurons was similar to that in the optic lobe of the developing chick brain. Changes in synthetic and hydrolytic activities of beta-CG were studied during growth of primary cultured neurons. Incorporation of radioactivities from radiolabeled pyruvate and alanine into beta-CG increased significantly on day 3 of culture, reaching a plateau on day 6, whereas that from radioactive glutamine and glutamate increased gradually from day 3 to day 12 of culture. The hydrolyzing enzyme activity of beta-CG during neuron growth was low until day 3 of culture, when it increased significantly until day 12. Similar developmental changes were observed in the developing chick embryo optic lobes.     

CHOLINE ACETYLTRANSFERASE (ChAc) ACTIVITY IN DEVELOPING CHICK OPTIC CENTRES AND THE EFFECTS OF MONOLATERAL REMOVAL OF RETINA AT AN EARLY EMBRYONIC STAGE AND AT HATCHING

The choline acetyltransferase (ChAc) activity was measured in the optic centres of chick embryos after early removal of the optic cup and of young chicks after monolateral extirpation of the right eyeball after hatching. The contralateral optic lobes were thus deprived of their complement of retinal fibres. The following results were obtained: in chick embryos the ChAc was slightly lower in the deafferented lobe between the 10th and the 14th day of incubation; between the 14th and the 17th day a critical fall in activity was observed leading to a significant ChAc loss of 71 per cent. In eye deprived chicks no significant change in total ChAc activity occurred during the first postoperative month; significant changes were found only in the second month. The results reached so far suggest that removal of retinal fibres does not cause short term changes in optic centre ChAc in either the embryo or the chick. ChAc contained in nerve cell bodies seems independent of synapses and its behaviour is interpreted as a reflection of metabolic disturbance of the centre.     

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.     

In vivo 13C NMR studies of compartmentalized cerebral carbohydrate metabolism

Localized 13C nuclear magnetic resonance (NMR) spectroscopy provides a unique window for studying cerebral carbohydrate metabolism through, e.g. the completely non-invasive measurement of cerebral glucose and glycogen metabolism. In addition, label incorporation into amino acid neurotransmitters such as glutamate (Glu), GABA and aspartate can be measured providing information on Krebs cycle flux and oxidative metabolism. Given the compartmentation of key enzymes such as pyruvate carboxylase and glutamine synthetase, the detection of label incorporation into glutamine indicated that neuronal and glial metabolism can be measured in vivo. The purpose of this paper is to provide a critical overview of these recent advances into measuring compartmentation of brain energy metabolism using localized in vivo 13C NMR spectroscopy. The studies reviewed herein showed that anaplerosis is significant in brain, as is oxidative ATP generation in glia and the rate of glial glutamine synthesis attributed to the replenishment of the neuronal Glu pool and that brain glycogen metabolism is slow under resting conditions. This new modality promises to provide a new investigative tool to study aspects of normal and diseased brain hitherto unaccessible, such as the interplay between glutamatergic action, glucose and glycogen metabolism during brain activation, and the derangements thereof in patients with hepatic encephalopathy, neurodegenerative diseases and diabetes.     

Changes in sterol metabolism in the skin of developing chick embryo and alterations in the presence of an anticholesterolemic agent and a chemical carcinogen

Changes in sterol metabolism in the skin of chick embryo during its development were studied with embryonal chick skin and with the cultured skin tissues. Changes in sterol metabolism of the skin of chick embryo began to appear at day 17, as observed by the accumulation of dihydrolanosterol, and the ratio of dihydrolanostrol:cholesterol increased thereafter until hatching. A similar change in sterol metabolism was also observed with the cultured skin tissue of chick embryo, although the stages of development seem to have been delayed by 3 days. The active sterol metabolism of the cultured skin tissue was also confirmed by studies of incorporation of [2-14C]acetate into sterols. 20,25-Diazacholesterol almost completely inhibited the incorporation of [2-14C]acetate into C27 sterols, whereas a chemical carcinogen, 4-hydroxyaminoquinoline 1-oxide, inhibited the incorporation of [2-14C]acetate into lathosterol but not that into cholesterol.     

Glycosyltransferases in Different Brain Regions During Chick Embryo Development

Glycosphingolipids, in particular gangliosides, play a crucial role in neuronal development and are known to change dramatically in total content and distribution in different brain areas during embryogenesis. In the present work we analyzed the activity of enzymes involved in the metabolism of gangliosides, at different periods of functional maturation in different regions of chick embryo brain. Our data demonstrate differences in the enzymatic activities in the examined areas; these differences might be correlated with the functional lateralization occurring in the brain during development. Significative differences were found in glycosphingolipid composition between controlateral cerebral hemispheres and optic lobes; these results together with previous data we found, contribute to reinforce our hypothesis on the occurrence of biochemical lateralization during early brain development.     

Astrocyte metabolism is disturbed in the early development of experimental hydrocephalus

The proper diagnosis of the arrested or the progressive form of hydrocephalus has a critical impact on treatment, but remains difficult. The assessment of early changes in cerebral metabolism might help in the development of adequate non-invasive diagnostic tools. This study examined the alterations in label incorporation in neurotransmitter amino acids and other compounds in kaolin-induced progressive hydrocephalus in rats by means of magnetic resonance spectroscopy (MRS) combined with the administration of [1-13C]glucose and [1,2-13C]acetate. Some 2, 4 and 6 weeks after kaolin injection into the cisterna magna, cerebrum, brainstem and cerebellum were dissected. Interestingly, labelling of most amino acids derived from [1-13C]glucose showed no alterations, whereas labelling from [1,2-13C]acetate was affected. Two weeks after induction of hydrocephalus the taurine concentration was decreased, whereas the concentration of [1,2-13C]lactate was increased in the cerebrum and that of [1,2-13C]GABA in the brainstem. Furthermore, labelling from [1,2-13C]acetate was significantly decreased in [4,5-13C]glutamate, [1,2-13C]glutamate and [1,2-13C]GABA in cerebrum from 4 weeks after hydrocephalus induction. The concentration of N-acetylaspartate, a neuronal marker, was unchanged. However, labelling of the acetyl group from [1-13C]glucose was decreased in cerebellum and brainstem at 6 weeks after the induction of hydrocephalus. As glucose is metabolized predominately by neurones, whereas acetate is exclusively taken up by astrocytes, these results indicate that mostly astrocytic, and only later neuronal, metabolism is disturbed in the kaolin model of hydrocephalus. If verified in patients using in vivo MRS, impaired astrocyte metabolism might serve as an early indication for operative treatment.     

Chondroitin sulfate proteoglycan expression during neuronal development.

Proteoglycans of developing chick brain were distinguished on the basis of reactivity with four well characterized antibody reagents (S103L, to the CS-rich domain; HNK-1, to 6-sulfated glucuronic acid; 1-C-3, to the HABr region and 5-D-4, to KS chains). One chondroitin sulfate proteoglycan reacted exclusively with S103L and 1-C-3 and not with the other two antibodies, hence is designated the S103L reactive brain CSPG. The other proteoglycan reacted exclusively with HNK-1 and 5-D-4 and not with S103L and 1-C-3, hence it is designated the HNK-1 reactive brain CSPG. In addition to these immunological distinctions, the S103L and HNK-1 CSPGs exhibited significant biochemical differences at both the protein and carbohydrate levels. Most interestingly, both CSPGs were found in all regions of the brain, and were expressed in a developmentally regulated pattern. The S103L CSPG was not detectable prior to embryonic day 7, increased to a maximum at day 13-15 and declined by day 20 in most brain regions examined. In contrast, the HNK-1 CSPG was present as early as embryonic day 4 and remained constant through hatching. Neuronal cultures established from embryonic day 6 (E6) cerebral hemispheres represent an in vitro paradigm that mimics in vivo neuronal development and differentiation. In this culture system we found that the expression of the S103L and HNK-1 CSPG followed a pattern similar to that observed in developing brain and further, that neurons are probably the sole source of S103L CSPG in cerebral cortex during neuroembryogenesis.     

Collagen-tailed and globular forms of acetylcholinesterase in the developing chick visual system

We have carried out a comparative study of the developmental profiles of the enzyme acetylcholinesterase, and of its collagen-tailed and globular structural forms, solubilized in the presence of 1 M NaCl, 1% (w/v) sodium cholate and 2 mM EDTA, in the chick retina and optic lobes. The overall acetylcholinesterase activities, both per mg protein and per embryo or chick, are substantially higher in tectum than in retina, from embryonic day 16. The A₁₂ collagen-tailed form of the enzyme is present in similar amounts in the embryonic retina and optic tectum; however, while the A₁₂ activity increases significantly in retina after birth, both by percentage and in absolute terms, the tectal tailed enzyme follows a declining developmental profile, reaching a minimum after 6 months of life. On the other hand, the globular G₄ species shows developmental profiles, both in retina and tectum, rather similar to those obtained for the overall enzyme activity, while the G₂ and G₁ forms are present in comparable concentrations in both tissues. Besides, G₄ is the predominant globular form in the chick optic lobe after hatching, G₂ and G₁ being enriched in the embryonic tectum. In the case of retina, however, all the globular forms contribute more evenly to the total acetylcholinesterase activity, along the developmental period considered.The potential significance of some of the postnatal developmental profiles is discussed in terms of the progressive adjustment of retina and tectum to the requirements of visual function.     

Cortical Bilateral Adaptations in Rats Submitted to Focal Cerebral Ischemia: Emphasis on Glial Metabolism

This study was performed to evaluate the bilateral effects of focal permanent ischemia (FPI) on glial metabolism in the cerebral cortex. Two and 9 days after FPI induction, we analyze [¹⁸F]FDG metabolism by micro-PET, astrocyte morphology and reactivity by immunohistochemistry, cytokines and trophic factors by ELISA, glutamate transporters by RT-PCR, monocarboxylate transporters (MCTs) by western blot, and substrate uptake and oxidation by ex vivo slices model. The FPI was induced surgically by thermocoagulation of the blood in the pial vessels of the motor and sensorimotor cortices in adult (90 days old) male Wistar rats. Neurochemical analyses were performed separately on both ipsilateral and contralateral cortical hemispheres. In both cortical hemispheres, we observed an increase in tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and glutamate transporter 1 (GLT-1) mRNA levels; lactate oxidation; and glutamate uptake and a decrease in brain-derived neurotrophic factor (BDNF) after 2 days of FPI. Nine days after FPI, we observed an increase in TNF-α levels and a decrease in BDNF, GLT-1, and glutamate aspartate transporter (GLAST) mRNA levels in both hemispheres. Additionally, most of the unilateral alterations were found only in the ipsilateral hemisphere and persisted until 9 days post-FPI. They include diminished in vivo glucose uptake and GLAST expression, followed by increased glial fibrillary acidic protein (GFAP) gray values, astrocyte reactivity, and glutamate oxidation. Astrocytes presented signs of long-lasting reactivity, showing a radial morphology. In the intact hemisphere, there was a decrease in MCT2 levels, which did not persist. Our study shows the bilateralism of glial modifications following FPI, highlighting the role of energy metabolism adaptations on brain recovery post-ischemia.