DETERMINATION OF BRAIN-SPECIFIC ANTIGENS IN SHORT TERM CULTIVATED RAT ASTROGLIAL CELLS AND IN RAT SYNAPTOSOMES

—The brain-specific antigens 14·3·2, GFA, A5, F3, D1, D2, D3 and C1 were quantitated in a short-term astroglial cell culture taken as a model of glial cells, and in synaptosomes, synaptosomal membranes and synaptic vesicles as neuronal material. Furthermore, the antigens were quantitated in newborn rat brain, as this served as the starting material for the cell culture. The membrane antigens C1, D1, D2 and D3 were absent from the cultured astroglia, indicating a neuronal origin for these antigens. C1 was enriched 3-fold in synaptosomes and synaptosomal membranes and more than 10-fold in synaptic vesicles indicating that this antigen might be a marker protein for nerve endings. The name Synaptin is introduced for this antigen. Conversely, the data on the antigens D1, D2 and D3 indicated that these antigens were not restricted to the synaptosomes although they were of neuronal origin. Trace amounts of the cathodal part of the heterogeneous cytoplasmic antigen 14·3·2 were present in the cell culture, possibly originating from a few contaminating neurons. The cytoplasmic antigens A5 and F3 were found both in the astroglial culture and in the synaptosomal fraction. F3, however, was found in low concentration in the synaptosomes and 3-fold enriched in newborn rat brain compared to rat brain from 35-day-old rats or to 21-day-old brain cell cultures. It was therefore regarded as a brain specific fetal antigen. The antigen GFA was highly enriched in the astroglial culture compared to whole brain and only trace amounts were found in the synaptosomal fraction supporting the astroglial origin of this antigen.     

Modifications in energy metabolism during the development of chick glial cells and neurons in culture

Developmental changes in lactate dehydrogenase (LDH), enolase, hexokinase (HK), malate dehydrogenase (MDH), and glutamate dehydrogenase (GDH) activities were measured in cultures of pure neurons and glial cells prepared from brains of chick embryos (8 day-old for neurons, 14 day-old for glial cells) as a function of cellular development with time in culture. The modifications observed in culture were compared to those measured in brain extracts during the development of the nervous tissue in the chick embryo and during the post-hatching period. A significant increase of MDH, GDH, LDH, and enolase activities are observed in neurons between 3 and 6 days of culture, whereas simultaneously a decrease of HK values occurs. In the embryonic brain between 11 and 14 days of incubation, which would correspond for the neuronal cultures to day 3 through 6, modifications of MDH, GDH, HK, and enolase levels are similar to those observed in neurons in culture. Only the increase of LDH activity is less pronounced in vivo than in cultivated cells. The evolution of the tested enzymatic activities in the brain of the chick during the period between 7 days before and 10 days after hatching is quite similar to that observed in cultivated glial cells (prepared from 14 day-old embryos) between 6 and 18 days of culture. All tested activities increased in comparable proportions. The modifications of the enzymatic profile indicate that some maturation phenomena affecting energy metabolism of neuronal and glial elements in culture, are quite similar to those occuring in the total nervous tissue. A relationship between the development of the energy metabolism of the brain and differentiation processes affecting neuroblasts and the glial-forming cells is discussed.     

Changes in Diffusion Kurtosis Imaging and Magnetic Resonance Spectroscopy in a Direct Cranial Blast Traumatic Brain Injury (dc-bTBI) Model

Explosive blast-related injuries are one of the hallmark injuries of veterans returning from recent wars, but the effects of a blast overpressure on the brain are poorly understood. In this study, we used in vivo diffusion kurtosis imaging (DKI) and proton magnetic resonance spectroscopy (MRS) to investigate tissue microstructure and metabolic changes in a novel, direct cranial blast traumatic brain injury (dc-bTBI) rat model. Imaging was performed on rats before injury and 1, 7, 14 and 28 days after blast exposure (~517 kPa peak overpressure to the dorsum of the head). No brain parenchyma abnormalities were visible on conventional T2-weighted MRI, but microstructural and metabolic changes were observed with DKI and proton MRS, respectively. Increased mean kurtosis, which peaked at 21 days post injury, was observed in the hippocampus and the internal capsule. Concomitant increases in myo-Inositol (Ins) and Taurine (Tau) were also observed in the hippocampus, while early changes at 1 day in the Glutamine (Gln) were observed in the internal capsule, all indicating glial abnormality in these regions. Neurofunctional testing on a separate but similarly treated group of rats showed early disturbances in vestibulomotor functions (days 1–14), which were associated with imaging changes in the internal capsule. Delayed impairments in spatial memory and in rapid learning, as assessed by Morris Water Maze paradigms (days 14–19), were associated with delayed changes in the hippocampus. Significant microglial activation and neurodegeneration were observed at 28 days in the hippocampus. Overall, our findings indicate delayed neurofunctional and pathological abnormalities following dc-bTBI that are silent on conventional T2-weighted imaging, but are detectable using DKI and proton MRS.     

COMPARTMENTATION AND LABELLING OF AMINO ACIDS IN THE DEVELOPING RAT BRAIN AFTER INJECTION OF [U-14C]RIBOSE

Abstract— [U-¹⁴C]Ribose was given by subcutaneous injection to young rats aged 2–56 days. During the first week after birth ¹⁴C in the brain was found mainly combined in glucose, fructose and sedoheptulose which contained 46–57 per cent of the ¹⁴C in the acid soluble metabolites in the rat brain. In contrast, during the critical period (10–15 days after birth) the ¹⁴C in the free sugars decreased from 24 to 3 per cent, while the ¹⁴C content of amino acids in the brain increased from 11 to 44 per cent of the total perchloric acid-soluble ¹⁴C. The increase in labelling of amino acids during the critical period was attributed to increased glycolysis and increased oxidation of pyruvate. The relative specific radioactivity of y -aminobutyrate and aspartate in the rat brain at 28 days after birth was equal to or greater than the relative specific radioactivity of glutamate. Assuming that the increase in amino acid content following the cessation of cell proliferation in the brain is located mainly in cell processes (cytoplasm of axons, dendrites, glial processes and nerve terminals), tentative values were estimated for the pool sizes of glutamate, glutamine, aspartate and y -amino butyrate.     

Differences in the time course of haloperidol-induced up-regulation of rat striatal and mesolimbic dopamine receptors

Regional differences in the onset and persistence of increased dopamine D2 receptor density in rat brain were studied following daily injections of haloperidol for 3, 7, 14, or 28 days. Striatal [3H]-spiroperidol Bmax values were significantly increased following 3-28 days of haloperidol treatment, as compared to saline controls. Olfactory tubercle Bmax values were significantly increased only after 14 or 28 days of haloperidol treatment. Nucleus accumbens Bmax values were significantly increased only in the 14-day drug treatment group, suggesting that dopamine D2 receptor up-regulation in nucleus accumbens may reverse during ongoing neuroleptic treatment. These findings suggest that important differences in adaptive responses to chronic dopamine blockade may exist between dopaminergic synapses located in various rat brain regions.     

Neuronal and Glial Expression of the Multidrug Resistance Gene Product in an Experimental Epilepsy Model

1. Failure of anticonvulsive drugs to prevent seizures is a common complication of epilepsy treatment known as drug-refractory epilepsy but their causes are not well understood. It is hypothesized that the multidrug resistance P-glycoprotein (Pgp-170), the product of the MDR-1 gene that is normally expressed in several excretory tissues including the blood brain barrier, may be participating in the refractory epilepsy. 2. Using two monoclonal antibodies against Pgp-170, we investigated the expression and cellular distribution of this protein in the rat brain during experimentally induced epilepsy. Repeated seizures were induced in male Wistar rats by daily administration of 3-mercaptopropionic acid (MP) 45 mg/kg i.p. for either 4 days (MP-4) or 7 days (MP-7). Control rats received an equivalent volume of vehicle. One day after the last injection, rats were sacrificed and brains were processed for immunohistochemistry for Pgp-170. As it was previously described, Pgp-170 immunostaining was observed in some brain capillary endothelial cells of animals from control group. 3. Increased Pgp-170 immunoreactivity was detected in MP-treated animals. Besides the Pgp-170 expressed in blood vessels, neuronal, and glial immunostaining was detected in hippocampus, striatum, and cerebral cortex of MP-treated rats. Pgp-170 immunolabeled neurons and glial cells were observed in a nonhomogeneous distribution. MP-4 animals presented a very prominent Pgp-170 immunostaining in the capillary endothelium, surrounding astrocytes and some neighboring neurons while MP-7 group showed increased neuronal labeling. 4. Our results demonstrate a selective increase in Pgp-170 immunoreactivity in the brain capillary endothelial cells, astrocytes, and neurons during repetitive MP-induced seizures. 5. The role for this Pgp-170 overexpression in endothelium and astrocytes as a clearance mechanism in the refractory epilepsy, and the consequences of neuronal Pgp-170 expression remain to be disclosed.     

Regional changes in the concentrations of glutamate,glycine, taurine,and GABA in the vitamin B-6 deficient developing rat brain: Association with neonatal seizures

It is well known that a dietary restriction of vitamin B-6 during gestation and lactation produces spontaneous seizures in neonatal animals. Since pyridoxal phosphate, one of the biologically active forms of vitamin B-6, is the cofactor for GAD the neonatal seizures have been attributed to low levels of brain GABA as a result of cofactor depletion. Although GABA levels are significantly lower in B-6 restricted neonatal rats with spontaneous seizures, seizure activity is not present in B-6 deficient adult rats or 28 day old rats in the present study, despite significantly low levels of brain GABA. These facts suggest that depletion of GABA is not the only biochemical alteration essential for the emergence of seizures. In the present study, the effect of vitamin B-6 undernutrition on the concentrations of the neuroactive amino acids, Glu, Gly, Tau, and GABA was determined in selected regions of the developing rat brain. The results show that the concentrations of Glu, Tau, and GABA were significantly lower and GLY significantly higher in selected brain regions of the B-6 restricted 14 day old rat compared to control tissue. Most of these changes were unique to 14 days of age, the time when spontaneous seizures are observed, and not present at 28 or 56 days of age when seizures are absent. This pattern of amino acid changes in the brain and the magnitude of the changes was consistent with those measured in a variety of chemically-induced animal models of epilepsy and in human epileptic foci. The regional distribution of amino acid changes was associated with brain regions which have been suggested to be responsible for the initiation and propagation of seizure activity. Two unique findings were also made in this study. First, there was a regional brain heterogeneity in the age-associated loss of brain Tau concentrations with the pons/medulla and substantia nigra appearing to be highly vulnerable and the hippocampus quite resistant to the loss of Tau. A second finding was the normalization of the neonatal GABA deficit in most brain regions by 56 days of age. The normalization of brain GABA was present in the face of continued dietary vitamin B-6 restriction. In summary, this study shows that the neuroactive amino acids Glu, Gly, Tau, and GABA are markedly altered in the seizure-prone vitamin B-6 restricted neonatal rat brain. The alterations in the brain concentration of Glu, Gly, and Tau may play an equally important role as GABA in the underlying mechanism of seizures associated with this condition.Abbreviations GAD Glutamic acid decarboxylase - GABA gamma-aminobutyric acid - Glu glutamate - Gly glycine - Tau taurine - CNS central nervous system - CTX cortex - HIPP hippocampus - C/P caudate/putamen - SN substantia nigra - Cb cerebellum - P/M pons/medulla     

Developmental profile of three enolase isozymes in rat brain determination from one-cell embryo to adult brain

Levels of three enolase isozymes (αα, αγ and γγ) were determined in rat tissues from one-cell embryo to adult brain with a sensitive enzyme immunoassay system. Each embryo of the early stage (gestational age, 0–3 days) contained about 5 × 10^?17 mol of αα enolase. The nervous system-specific αγ and γγ enolases would be detected in the embryos of 6–8 days, which contain no histologically recognizable neurones. The 8-day embryos contained 4.3 × 10^?17 and 3.4 × 10^?16 mol of αγ and γγ enolases. Amounts of all the three enolases were increased with growth of the embryo. The nervous system-specific enolases (αγ and γγ) in the brain kept increasing until 1–2 months of postnatal age, whereas the αα enolase level in the brain was relatively constant after the 15-day embryo through the adult rat.     

Udp-galactose: glycoprotein galactosyltransferase activity in a clonal line of rat brain.

1. UDPgalactose:glycoprotein galactosyltransferase (EC 2.4.1.-) activity was demonstrated in homogenates from whole rat brain, isolated neuromal perikarya, enriched glial cell fractions, and cultured rat glial tumor cells (clone C6). 2. Galactosyltransferase activity was enriched 3-9-fold in neuronal perikarya and 1.4--1.8-fold in the glial cell fraction over the activity in whole brains from 19- and 40-day-old rats. The activity of galactosyltransferase in neuronal perikarya decreased with age. Extensive contamination of the glial cell fraction with membranous fragments appeared to obscure the precise specific activity of this fraction. 3. The specific activity of the enzyme in glial tumor cells was 4--8-fold higher than in brain tissue when the enzyme was assayed under identical conditions using endogenous and different exogenous acceptors. 4. Galactosyltransferase activities from adult brain and glial tumor cells had similar properties. They both required Mn-2 plus and Triton, and exhibited pH optima between 5 and 7. The apparent Km of the enzyme for UDPgalactose was 1.3-10-minus 4 M for brain tissue and 2.2-10-minus 4 M for glial tumor cells. 5. The high galactosyltransferase activity in glial tumor cells and in neuronal perikarya of younger rats is compatible with the possibility of a role of this enzyme in developing brain.     

Increased Concentrations of 3-Hydroxykynurenine in Vitamin B6 Deficient Neonatal Rat Brain

Increased concentrations of the endogenous tryptophan metabolite 3-hydroxykynurenine (3-HK) were measured in the brains of vitamin B6 deficient neonatal rats. Mean concentrations of 3-HK in B6 deficient cerebellum, corpus striatum, frontal cortex, and pons/medulla ranged from 9.7 to 18.6 and 102 to 142 nmol/g of wet tissue at 14 and 18 days of age, respectively. 3-HK was not significantly increased in control neonatal or adult rat brain, vitamin B6 deficient rat brain at 7 days of age, or in brains from adult rats deprived of vitamin B6 for 58 days. The administration of daily intraperitoneal injections of vitamin B6 from the 14th to the 18th day of age decreased the concentration of 3-HK to control levels. 3-HK has been shown by other investigators to produce seizures when injected into the cerebral ventricles of adult rodents. Thus, our studies show the accumulation in brain of a putative endogenous convulsant as the result of a nutritional deficiency.     

Determination and characterization of neuron specific protein (NSP) associated enolase activity.

Fractionation of the 40–80% (NH₄)₂SO₄ fraction of a soluble rat brain extract on DEAE cellulose resolves three species of enolase activity, two of which react with antiserum to neuron specific protein from rat (NSP-R) and one which does not react. Direct assay of pure neuron specific protein from rat, cat and human brain (NSP-R, NSP-C, NSP-H) as well as bovine brain 14-3-2, using 2 different assay systems demonstrate that all these preparations display enolase activity of between 40 and 70 units/mg. This activity is Mg⁺⁺ dependent and inhibited by fluorophosphate in all cases. Kinetic parameters such as Km for Mg⁺⁺, 2 PGA, and pH optimum were determined for the 2 different NSP preparations and also for bovine brain 14-3-2 protein.     

Comparative study of appearance of water-soluble antigens and brain esterase fractions in ontogenesis of two strains of rats

By means of immunoelectrophoresis of rat brain, 12 water-soluble antigens were detected, five of which were found to be specific to the brain. Histochemical reactions have identified two antigens that are not specific to the brain, lactate dehydrogenase and esterase. By means of enzymoelectrophoresis, 14 esterase fractions were determined. An immunoautoradiographic study of the synthesis of some antigens specific to the brain was carried out. It was found that rats responding to sound by epileptic seizures develop more slowly than normal rats. In particular, in rats resistant to auditory stimulus the antigenic spectrum typical of the brain of the adult rat (12 antigens) is formed by day 14 of postnatal life, while in rats of the sensitive strain this pattern takes 17 days to completely form. The last brain-specific antigen for rats susceptible to audiogenic epilepsy appears on day 17, while this antigen is present in normal rats on day 14. Fast-moving esterase fractions are detected earlier in ontogenesis in rats resistant to sound than in sensitive rats.     

THE NATURE OF THE TWO PROTEINS OF BRAIN SPECIFIC ANTIGEN 14-3-2

The three isoenzymes of rat brain enolase (2-phospho d -glycerate hydrolase EC 4.2.1.11.) χχ, χγ and γγ were separated by ion-exchange chromatography and were tested for reaction with an antiserum against brain specific antigen 14-3-2. This monospecific antiserum affects the enolase activity of only the χγ and γγ isoenzymes. Immunoelectrophoretic experiments show that the two proteins which react as 14-3-2 both contain γ enolase subunits, and one of these also contains χ enolase subunits. It is concluded that the 14-3-2 antigen and the γ enolase subunit are identical, and that the two proteins which react immunologically as 14-3-2 are the χγ and γγ enolase isoenzymes.     

Quantitative autoradiography of the development of mu opiate binding sites in rat brain

The regional developmental appearance of mu binding sites in rat brain was examined by quantitative autoradiography of 3H-dihydromorphine binding in rats 2, 14, 21, and 28 days old. Labeling with 3H-dihydromorphine was heterogeneous in adult rat brains, as previously reported by other laboratories. Levels of 3H-dihydromorphine binding ranged from approximately 250 nCi/g tissue in the interpeduncular nucleus and 100 nCi/g tissue in the habenula to 40 nCi/g tissue in the hypothalamus and periaqueductal gray. Some areas, particularly white matter regions, had no detectable specific binding. The density of 3H-dihydromorphine binding increased in all regions between 2 and 28 days of age. The increases in 3H-dihydromorphine binding in various regions of rat brain developed at different rates. Maximal densities were seen by 14 days of age in most regions examined, including the caudate, hippocampus, amygdala, and hypothalamus. Binding in the medial thalamus and quadrigeminal plate, however, did not reach maximal levels until 21 days. Although quantitative autoradiography offers major advantages in the examination of the regional distribution of opiate binding sites, variability both between sections from the same brain and between sections from different brains demonstrate some of the difficulties associated with this type of experimental approach.     

Postnatal changes of cathepsin D activity in rat liver and brain

Total and specific activity of cathepsin D (EC. 3.4.23.5) were measured in rat liver and brain from 1 to 98 days of age. The activity of cathepsin D in the liver of adult and newborn rats was the same while in the rat brain it was higher in adult than in newborn rats. In the liver maximum specific activity of cathepsin D occurred on the 10th postnatal day and minimum on the fourth day of age. In the brain maximum specific activity of the enzyme occurred on the 14th postnatal day. Total activity of cathepsin D increased after birth in rat liver and brain. These results are discussed in relation to the functional role of cathepsin D in the rat liver and the brain.     

Nutrition and fetal brain maturation : I. Responses in vitro and in vivo

The glycolytic enzyme enolase increases during the perinatal period of brain development and was utilized as a marker for examining the effect of culture environment on differentiation of cells from 20-day fetal rat brain. Enolase activity in cell cultures increased from 0.91 +/- 0.03 (Day 0) to 2.11 +/- 0.10 mumol/min/mg protein (Day 6). Comparable levels were not reached in vivo until neonatal pups were 15 days old. The in vitro increase was inhibited by both cycloheximide and actinomycin D. Enolase activity in the cells responded to alterations in both incubation media and homologous serum. After 6 days in culture, cells incubated in rat serum (10%) added to MEM or RPMI produced twice as much enolase activity as cells incubated similarly in Ham's medium, i.e., 1.96 +/- 0.09 and 1.85 +/- 0.21 vs 1.02 +/- 0.09, P less than 0.001. Results of a comparable magnitude were obtained when fetal calf serum replaced adult rat serum, but enolase production was somewhat lower when newborn calf serum replaced adult rat or fetal calf serum. When cells were incubated for 6 days with graded concentrations of adult rat serum (2.5-15%), enolase activity increased progressively. The pattern of enolase response suggests that the fetal rat brain cell model described herein will provide a sensitive probe with which to gain insight into nutrition and fetal brain development.     

Changes of Enzymes Involved in Prostaglandin Metabolism and Prostaglandin Binding Proteins in Rat Brain During Development and Aging

In the developing rat brain, the enzymatic formation of prostaglandin D2 from prostaglandin H2 increased 60-fold from day 12 of gestation to birth. The activity still rose gradually to the highest level (90 nmol/min/g wet tissue) at day 7 after birth. The activities of prostaglandin E2 and F2 alpha synthetases in rat brain were highest at gestational age 19 days (30 nmol/min/g wet tissue), respectively. The specific activity of NADP-dependent 15-hydroxy-prostaglandin D2 dehydrogenase in rat brain was highest at the earliest gestational age we examined (day 12 of gestation). The specific bindings of prostaglandin D2 and E2 to the crude mitochondrial fraction of rat brain were observed from day 16 of gestation and increased to day 7 after birth. Although the activities of the enzymes responsible for prostaglandin metabolism were unchanged postmaturationally, the maximal concentrations of the binding sites on the synaptic membrane for both prostaglandins D2 and E2 decreased with constant affinity to less than one-sixth with age from 1 week to 24 months after birth. These results indicate that prostaglandins may play important roles during maturation and aging in rat brain.