The Effects of Chronic Manganese Chloride Treatment Expressed as Age-Dependent, Transient Changes in Rat Brain Synaptosomal Uptake of Amines

Abstract: As a result of chronic manganese treatment of rats from conception onwards, a decrease was observed in the uptake of dopamine, but not of noradrenaline or serotonin, by synaptosomes isolated from hypothalamus, striatum, and midbrain and in choline uptake by hypothalamic synaptosomes obtained from 70–90-day-old animals. In 100–120-day-old manganese-treated rats the only difference observed was increased choline uptake by striatal synaptosomes. All comparisons were with age-matched controls. These results, which are consistent with views of a dopaminergic and cholinergic involvement in manganese encephalopathy, point out that changes in these systems are observable only at specific times during manganese intoxication.     

Brain Regional Distribution of Glutamic Acid Decarboxylase, Choline Acetyltransferase, and Acetylcholinesterase in the Rat: Effects of Chronic Manganese Chloride Administration after Two Years

Abstract: Rats were treated chronically with manganese chloride from conception onward for a period of over 2 years in order to study the effects of manganese and aging on the activities of glutamic acid decarboxylase (GAD), choline acetyltransferase (ChAT), and acetylcholinesterase (AChE) in hypothalamus, cerebellum, pons and medulla, striatum, midbrain, and cerebral cortex (which included the hippocampus). Manganese-treated 2-month-old and 24- to 28-month-old rats and age-matched controls were studied. In control rats during aging the activities of GAD decreased in hypothalamus (19%), pons and medulla (28%), and midbrain (22%) whereas the activities of AChE decreased in all regions (20–48%), particularly in the striatum (44–48%). Changes in ChAT activities in aging were observed only in one region—a decrease (23%) in the striatum. Life-long treatment with manganese appeared to abolish partially the decreases in aging in AChE activities in hypothalamus, cerebellum and striatum, and striatal ChAT activity. Manganese treatment also seemed to abolish the age-related decreases in GAD activities, since GAD activities in various brain regions of manganese-treated senescent rats were not significantly different from those of control young rats. These results are discussed in relation to other metabolic changes associated with aging and manganese toxicity.     

Acetylcholinesterase Activity in Regions of the Rat Brain Following a Convulsion

The effects of electroconvulsive shock on the levels of acetylcholinesterase in several brain regions of the rat were studied. Hippocampus, mesencephalon, cortex, and striatum exhibited rapid changes in acetylcholinesterase activity during the first few minutes following the convulsion, whereas brainstem and basal forebrain levels remained unchanged. In both hippocampus and midbrain there was a sustained decrease in activity: the total acetylcholinesterase activity was decreased by up to 40% within 2 min of the convulsion and did not return to control values for another 3 h. Thirty minutes after a flurothyl-induced convulsion there was a similar fall in acetylcholinesterase activity in both these regions, whereas a subconvulsive electric shock produced no change. It is concluded that a convulsion produces significant short-term decreases in acetylcholinesterase activity in areas of the rat brain that are involved in the generation and propagation of seizures, and the question is raised of whether this is related to the increase in seizure threshold that follows a convulsion.     

Soluble 5''-Nucleotidase Activities in Rat Brain

5'-Nucleotidase activity was assayed in 105,000-g supernatants from rat brain by following conversion of [3H]AMP into adenosine. The effect of ATP on this process was complex and suggested the presence of at least two soluble 5'-nucleotidase activities: one inhibited by ATP and another activated by ATP. The relative proportions of these activities differed considerably among brain regions. Activity changes induced by hypothyroidism also suggested that these activities may be regulated independently. These findings may have consequences for the regional regulation of adenosine formation in the brain.     

The Regional Distribution of Monoamine Oxidase Activities Towards Different Substrates: Effects in Rat Brain of Chronic Administration of Manganese Chloride and of Ageing

Abstract: The effects of chronic manganese chloride administration (1 mg MnCl₂ 4H₂O/ml of drinking water) and ageing on the regional distribution of monoamine oxidase (MAO, EC 1.4.3.4) were studied in 2-month- and 24–28-month-old rats. In both the control and Mn-treated rats, the serotonin oxidation (type A) rates decreased in hypothalamus, pons and medulla, striatum, midbrain and cerebral cortex, but not in cerebellum, in ageing. On the other hand the benzylamine oxidation (type B) rates in hypothalamus, striatum and cerebral cortex increased in ageing. In all regions except the cerebellum, there was a uniform decrease in the A/B ratio. This decrease was verified by differential inhibition studies using clorgyline and l -deprenyl, specific type A and type B inhibitors respectively. The dopamine-oxidising rates decreased in all regions, except the cerebral cortex and the cerebellum, in ageing control rats. This age-related decrease was not seen in the striatum and midbrain of manganese-treated rats. In these rats the other effect was an age-related increase in the rate of oxidation of all the amines in the cerebellum, not observed in control rats. These selective effects of manganese are only seen when comparing age-related changes in both groups of animals, since comparison of manganese-treated rats with age-matched controls showed a significant difference only in the rate of serotonin oxidation in the cerebellum of 2-month-old rats. The relationship of these observations to the effects of ageing and manganese encephalopathy on specific amine systems is discussed.     

The Posttranslational Arginylation of Proteins in Different Regions of the Rat Brain

The posttranslational incorporation of arginine into proteins catalyzed by arginyl-tRNA protein transferase was determined in vitro in different rat brain regions. The incorporation was found in all the regions studied, although with different specific activities (pmol [14C]arginine incorporated/mg protein). Of the regions studied, hippocampus had the highest specific activity followed by striatum, medulla oblongata, cerebellum, and cerebral cortex. Electrophoretic analysis of the [14C]arginyl proteins from the different regions followed by autoradiography and scanner densitometry showed at least 13 polypeptide bands that were labeled with [14C]arginine. The radioactive bands were qualitatively coincident with protein bands revealed by Coomassie Blue. There were peaks that showed different proportions of labeling in comparison with peaks of similar molecular mass from total brain. Most notable because of their high proportions were those of molecular mass 125 kDa in hippocampus, striatum, and cerebral cortex; 112 and 98 kDa in striatum and cerebellum; and 33 kDa in hippocampus and striatum. In lower proportions than in total brain were the peaks of 33 kDa in medulla oblongata and cerebral cortex and of 125 kDa in medulla oblongata.     

Ontogeny of Brain Neurotensin in the Rat: A Radioimmunoassay Study

Abstract: Concentrations of neurotensin-like immunoreactivity in several discrete brain areas were measured by radioimmunoassay in rats of different ages (2, 5, 10,20, 30, and 60 days). Neurotensin-like immunoreactivityincreased steadily from 2 to 30 days of age in the hypothalamus and then plateaued, while the preoptic area andamygdala concentrations peaked at 30 days of age. Brain-stem neurotensin-like immunoreactivity concentrationsincreased from 2 to 10 days of age and decreased from20 to 60 days of age. Several other regions showed nosignificant age-associated changes in neurotensin-like im-munoreactivity.     

Effect of Chronic Manganese Treatment on Adenosine Tissue Levels and Adenosine A2a Receptor Binding in Diverse Regions of Mouse Brain

In the present study the effects of chronic manganese (Mn) treatment on adenosine A_2a receptor binding in mouse brain have been assessed. Male albino mice were divided in two groups: In the Mn-treated group, the animals were injected intraperitoneally (i.p.) with MnCl₂ (5 mg/kg/day) five days per week during 9 weeks; in the control group, they were injected likewise with a saline solution. A significant decrease of the Kd without alteration of Bmax in the cerebellum and, an increase of the Kd and Bmax in hippocampus of mice treated with Mn were found. Also, an increase of Kd in frontal cortex was observed. The binding parameters in caudate nucleus, olfactory bulb and hypothalamus were not altered by Mn. A significant decrease in the adenosine concentration in caudate nucleus, olfactory bulb and hypothalamus, without significant changes in hippocampus, frontal cortex and cerebellum was also detected. These findings suggest that chronic administration of Mn could affect adenosine receptor function and turnover, depending on the brain region analyzed.     

Correlation of Hexokinase Content and Basal Energy Metabolism in Discrete Regions of Rat Brain

Brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) levels in seven regions of rat brain were estimated by photometric measurement of immunofluorescence in cryostat-cut sections. When compared with basal rates of glucose metabolism in these regions, estimated by the 6-[14C]glucose method, a significant correlation was observed. Thus, hexokinase content reflects metabolic energy demands.     

Polymorphism of Acetylcholinesterase in Discrete Regions of the Developing Human Fetal Brain

The molecular forms and membrane association of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) and pseudocholinesterase (acylcholine acylhydrolase, EC 3.1.1.8) were determined in the presence of protease inhibitors in dissected regions of developing human fetal brain, as compared with parallel areas from mature brain. All areas contained substantial cholinesterase activities, of which acetylcholinesterase accounted for almost all the activity. Two major forms of acetylcholinesterase activity, sedimenting at 10-11S and 4-5S, respectively, were detected on sucrose gradients and possessed similar catalytic properties, as judged by their individual Km values toward [3H]acetylcholine (ca. 4 X 10(-4) M). The ratio between these forms varied by up to four- to fivefold, both between different areas and within particular areas at various developmental stages, but reached similar values (about 5:2) in all areas of mature brain. Acetylcholinesterase activity was ca. 35-50% low-salt-soluble and 45-65% detergent-soluble in various developmental stages and brain areas, with an increase during development of the detergent-soluble fraction of the light form. In contrast, pseudocholinesterase activity was mostly low-salt-soluble and sedimented as one component of 10-11S in all areas and developmental stages. Our findings suggest noncoordinate regulation of brain acetylcholinesterase and pseudocholinesterase, and indicate that the expression of acetylcholinesterase forms within embryonic brain areas depends both on cell type composition and on development.     

The Effect of Visual Deprivation on β-Adrenergic Receptors in the Visual Centres of the Rat Brain

The levels of binding of [3H]dihydroalprenolol to beta-adrenergic receptors in the visual centres and frontal cortex from brains of control, dark-reared and monocularly deprived rats were compared. Receptor binding is changed in monocularly deprived rats in the lateral geniculate nuclei and superior colliculi of both sides. Scatchard analyses indicated that the changes in the [3H]dihydroalprenolol binding in the lateral geniculate nuclei were due to alterations in both receptor affinity and receptor number. No effect of dark-rearing could be detected.     

Chronic Manganese Treatment of Rats Alters Synaptosomal Uptake of Dopamine and the Behavioural Response to Amphetamine Administration

Abstract: Chronic manganese treatment from conception onward resulted in increased striatal synaptosomal uptake of dopamine, but not of a variety of other neurotransmitters/precursors in 80-day-old rats. The open-field behaviour of these manganese-treated 80-day-old rats was no different from that of untreated age-matched rats. However, amphetamine administration (1 mg/kg body weight) increased activity to a significantly lower extent in manganese-treated rats. These observations indicate that chronic manganese treatment results in marked alterations of activities associated with the dopaminergic system.     

Ontogeny and Subcellular Distribution of Rat Brain Tele-Methylhistamine

Abstract: The whole brain content and subcellular distribution of histamine and its metabolite, tele-methylhistamine, were studied during postnatal development of the rat. Brain methylhistamine levels were similar to or greater than histamine levels, indicating that histamine methylation is a major metabolic pathway in neonatal brain, as it is in adults. When calculated per brain, histamine, methylhistamine, and histamine methyltransferase were all maximal 10 days after birth. In neonates, brain histamine was found almost entirely in nuclear fractions, whereas methylhistamine was found almost exclusively in supernatant fractions. By day 20, however, a greater proportion of both amines was localized in subcellular fractions containing synaptosomes, a finding consistent with histamine's suggested transmitter role. The ontogenic pattern of brain methylhistamine questions the mast cell origin of neonatal histamine, but may be consistent with a role for histamine in brain development.     

Ontogenesis of Adenosine Deaminase Activity in Rat Brain

The activity of adenosine deaminase (ADA) was determined in whole brain of rats at the embryonic age of 15 days through to adulthood and in nine brain regions in rats 1 day old through to adulthood. In 1-day-old rats, the highest activity was seen in olfactory bulbs (550 +/- 15 nmol/mg protein/30 min) and this was 4.5-fold higher than that in the pons, which was the lowest. In adult animals, olfactory bulb still contained the greatest activity, which was about eightfold higher than hippocampus, which had the lowest. Except for hypothalamus, where ADA activity increased nearly twofold in rats between the ages of 1 and 50 days, significant decreases of as much as fivefold were found in whole brain, superior colliculus, cortex, hippocampus, cerebellum, olfactory bulbs, and olfactory nucleus. In contrast, ADA activity in pons and subcortex remained relatively constant throughout the developmental period. The Km values for ADA in whole brain at 18 days gestation (48 +/- 5 microM) were not significantly different from that observed in adult rats (38 +/- 7 microM), whereas the Vmax values decreased significantly from 339 +/- 9 to 108 +/- 8 nmol/mg protein/30 min. Taken together, the developmental patterns observed in the various brain regions appear not to correspond to any one particular process such as periods of rapid cell proliferation, cell death, synaptogenesis, or myelination. Nor do they correspond to known developmental profiles of transmitters, their receptors, or their metabolic enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterogeneity of Rat Brain Acetylcholinesterase: A Study by Gel Filtration and Gradient Centrifugation

Abstract: According to their solubilization properties, two classes of acetyl-cholinesterases (AChE) can be detected in the adult rat brain: a "soluble" species (easily solubilized without detergent), and a membrane-bound species (solubilized only in the presence of detergent). The latter was found to be homogeneous by gel filtration (Stokes radius 8.05 ± 0.35 nm) and sucrose gradient centrifugation (9.75 ± 0.2 S) in the presence of Triton X-100. The "soluble" AChE gives three stable species in the presence of the same detergent with Stokes radii and sedimentation constants of 10.9 ± 0.5 nm and 16 ± 2 S; 6.75 ± 0.30 nm and 10.7 ± 0.4 S; 5.37 ± 0.35 nm and 4.37 ± 0.1 S. Co-chromatography and co-sedimentation or the reduction and alkylation of disulfide bridges show that all the soluble species are different from the membrane-bound AChE. The possibility that soluble and membrane-bound AChE are completely different molecules is discussed.     

Ontogeny and Cellular Localization of the Pyruvate Recycling System in Rat Brain

Abstract: The ontogeny of the cerebral pyruvate recycling pathway and the cellular localization of associated enzymes, malic enzyme (ME) and phosphoenolpyruvate carboxykinase (PEPCK), have been investigated using a combination of ¹³C NMR spectroscopy, enzymatic analysis, and molecular biology approaches. Activity of the pathway, using [1,2-¹³C₂]acetate as a substrate, was detected by ¹³C NMR in brain extracts 3 weeks after birth, increasing progressively up to the third month of age. In whole-brain homogenates, ME activity increased to adult levels with the same time course as the recycling pathway. PEPCK activity was low during the first 2 weeks of life and decreased further toward adulthood. ME and PEPCK activity were found in primary cultures of astrocytes and in synaptosomal fractions of adult brain. Primary cultures of cortical neurons showed PEPCK activity but no detectable ME activity. The cytosolic ME gene was expressed in primary cultures of neurons and in astrocytes as well as in the neonatal and adult brain. The PEPCK gene was expressed both in primary cultures of cortical neurons and in astrocytes, but the level of its expression in the neonatal and adult brain was undetectable.     

The Activities of Some Energy-Metabolising Enzymes in Nonsynaptic (Free) and Synaptic Mitochondria Derived from Selected Brain Regions

Abstract: The enzyme complement of two different mitochondrial preparations from adult rat brain has been studied. One population of mitochondria (synaptic) is prepared by the lysis of synaptosomes, the other (nonsynaptic or free) by separation from homogenates. These populations have been prepared from distinct regions of the brain: cortex, striatum, and pons and medulla oblongata. The following enzymes have been measured: pyruvate dehydrogenase (EC 1.2.4.1), citrate synthase (EC 4.1.3.7), NAD-linked isocitrate dehydrogenase (EC 1.1.1.41), NADP-linked isocitrate dehydrogenase (EC 1.1.1.42), fumarase (EC 4.2.1.2), NAD-linked malate dehydrogenase (EC 1.1.1.37), D-3-hydroxybutyrate dehydrogenase (EC 1.1.1.30), and mitochondrially bound hexokinase (EC 2.7.1.1) and creatine kinase (EC 2.7.3.2). The nonsynaptic (free) mitochondria show higher enzyme specific activities in the regions studied than the corresponding values recorded for the synaptic mitochondria. The significance of these observations is discussed in the light of the different metabolic activities of the two populations of mitochondria and the compartmentation of the metabolic activities of the brain.     

Ontogeny of the GTP-Binding Protein Go in Rat Brain and Heart

We determined the ontogeny of the GTP-binding protein Go in rat brain and heart by employing highly sensitive enzyme immunoassay methods. In the brain, the alpha subunit of Go (Go alpha) gradually increased and reached adult levels approximately 20 and 30 days after birth in cerebral cortex and cerebellum, respectively. Concentrations of beta subunits, which were also quantified by the immunoassay, were almost equal to those of Go alpha in the brain of rats younger than 10 days, but were higher than those of Go alpha after 10 days. These results suggest that late development of GTP-binding proteins other than Go. Go alpha was immunohistochemically positive in neuropils and negative in cell bodies at any age tested. In the heart, the concentrations of Go alpha increased up to several times of the adult level just after birth, and then gradually decreased after the 20th postnatal day. The level of Go alpha in the liver, however, was very low and constant throughout ontogenic development. An immunohistochemical study indicated that Go alpha was positive in the cardiac muscle of young rat, but negative in that of adult rat. These results indicate that Go alpha exists in cells other than those of nervous tissues and neuroendocrine cells in some periods of ontogenic development.     

A Comparison of the Distribution of Neurotransmitters in Brain Regions of the Rat and Guinea-Pig Using a Chemiluminescent Method and HPLC with Electrochemical Detection

Six brain areas of rats and guinea-pigs, killed by microwave irradiation, were used for the concomitant measurement of the levels and regional distribution of cholinergic, biogenic amine, and amino acid neurotransmitters and metabolites. Acetylcholine (ACh) and choline (Ch) were quantified by chemiluminescence; noradrenaline (NA), dopamine (DA), 5-hydroxytryptamine (5-HT), and their metabolites by HPLC with electrochemical detection (HPLC-EC); and six putative amino acid neurotransmitters by HPLC-EC following derivatisation. The levels and regional distribution of these transmitters and their metabolites in the rat were similar to those reported in previous studies, except that biogenic amine transmitter levels were higher and metabolite concentrations were lower. The guinea-pig showed a similar regional distribution, but the absolute levels of ACh were lower in striatum and higher in hippocampus, midbrain-hypothalamus, and medulla-pons. In all areas, the levels of Ch were higher and those of NA, 5-HT, and taurine were lower than in the rat. The most marked differences between the rat and guinea-pig were in the relative proportion of DA metabolites and 5-HT turnover, as estimated by metabolite/transmitter ratios. This study can be used as a basis for a comprehensive understanding of the central effects of drugs on the major neurotransmitter systems.     

Divergent Regulation of Acetylcholinesterase and Butyrylcholinesterase in Tissues of the Rat

Abstract: Investigating the possibility that acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) are regulated in a coordinated manner, we have examined the natural variation in activity of these two enzymes in several tissues of adult male Sprague-Dawley, Fischer-344, and Wistar-Furth rats. Both enzymes varied greatly in mean activity among brain, diaphragm, atria, serum, superior cervical ganglia, and liver. In Sprague-Dawley rats there were also large individual variations with up to a fivefold range of AChE activities and up to a 100-fold range of BuChE activities in a given tissue. Individual variations in cholinesterase activities appeared to be smaller in the inbred Fischer-344 or Wistar-Furth rats. Experiments with internal standards of partially purified AChE and BuChE indicated that the individual variations probably reflected differences in the intrinsic content or specific activity of the tissue enzymes. Comparison of the AChE activities in different tissues of a given group of rats failed to reveal statistically significant correlations in any strain (i.e., the relative activity of any one tissue was no guide to the relative activity of any other tissue in the same rat). This result indicates that the regulation of AChE is tissue-specific. By contrast, BuChE activity showed highly significant correlations among the majority of the tissues examined in the Sprague-Dawley rats, implying that widely dispersed factors can affect the regulation of this enzyme. Body-wide regulation is not necessarily the rule, however, since only a single tissue pair in the inbred Fischer rats and none of the pairs in the Wistar-Furth rats showed significant correlations of BuChE activity. In general, AChE and BuChE activities were not correlated with each other to a statistically significant degree. We conclude that the control of these enzymes normally involves different mechanisms and is strongly affected by the genetic background of the sample population.