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.     

The Ontogeny of Acetylcholinesterase Activities in Rat Brain Regions and the Effect of Chronic Treatment with Manganese Chloride

Abstract: Acetylcholinesterase activities were determined in the rat cerebral cortex, striatum, midbrain, pons and medulla, hypothalamus, and cerebellum at 5, 12, 20, 30, and 60 days after birth. The ontogeny of the enzyme differed in the various regions, occurring earlier in the more caudal regions, except in the cerebellum where there was no increase. Chronic manganese treatment from conception did not influence the developmental profile of this cholinergic marker.     

Substrate Selectivity of Type A and Type B Monoamine Oxidase in Rat Brain

Abstract: Use of the irreversible inhibitors clorgyline and deprenyl showed that rat brain mitochondria contain type A and type B monoamine oxidase (MAO). Tyramine is a substrate for both types of MAO, whereas serotonin is a preferential substrate for type A MAO. In contrast to MAO in other tissues, type A MAO in brain tissue oxidizes β-phenylethylamine (PEA) at high concentrations (0.5 and 1.0 mM). The proportions of type A and type B MAO activities in the mitochondria estimated from the double-sigmoidal inhibition curves of tyramine oxidation were about 70:30 irrespective of the concentration of tyramine. With PEA as substrate, the ratios of type A to type B activities were found to increase from low values at low concentrations to about 1 at 0.5-1.0 mM-PEA, and even higher at further increased concentrations of PEA. At very low (0.01 mM) and high (10.0 mM) concentrations of PEA, single-sigmoidal curves were obtained; with the high PEA concentration the activity was highly sensitive to clorgyline, whereas with the low concentration it was highly sensitive to deprenyl. In deprenyl-pretreated mitochondrial preparations, all the remaining activity towards 0.5-1.0 mM-PEA was shown to be highly sensitive to clorgyline, demonstrating that this activity was indeed due to oxidation by type A MAO. The opposite result was obtained with deprenyl as inhibitor of clorgyline-pretreated preparations, demonstrating that PEA at this concentration was also oxidized by type B MAO in rat brain mitochondria. The K₃ values of type A and type B MAO for PEA were significantly different. On Lineweaver-Burk analysis, plots with PEA as substrate for type A MAO in a deprenyl-treated preparation were linear over a wide concentration range, whereas those for type B MAO in a clorgyline-treated preparation were not linear, but showed substrate inhibition at higher concentrations of the substrate. It is concluded from the present findings that the effect of the substrate concentration must be considered in studies on the characteristics of multiple forms of MAO in various organs and species.     

Gonadal Influences on the Sexual Differentiation of Monoamine Oxidase Type A and B Activities in the Rat Brain

Abstract: The sex-dependent differentiation of monoamine oxidase (MAO) in the hypothalamus of 60-day-old, Charles River rats was found to involve only type A (MAO-A), and not type B (MAO-B) enzyme. In vivo inhibition of type A by clorgyline, and type B by (−)deprenyl, however, tended to decrease the specific activity of both types of MAO to a smaller extent in the female than in the male hypothalamus. When masculinization was prevented by neonatal administration of estradiol (E) to males, hypothalamic MAO-A and MAO-B activities increased in both control and MAO-inhibited rats. Androgenization of females, however, had little effect on the MAO activity. Whereas the effects of neonatal estrogenization were attributable neither to a direct influence of E nor to a sexual difference in the peripheral clearance of the MAO-inhibitor used, single, high doses of steroids to adult, but not to newborn rats, did acutely affect the kinetics of MAO-A. The activity of MAO-A was also decreased by high concentrations of E or TS in vitro. The imprinting for patterns of hypothalamic MAO-A and MAO-B in the two sexes results, probably, from genetic predetermination. Neonatal changes in the homeostasis of gonadal hormones may result in type-MAO nonspecific effects in adulthood, whereas the short-term effects of high concentrations of steroids may be selective for the A form.     

Selective Effects of Neonatal Hypothyroidism on Monoamine Oxidase Activities in the Rat Brain

Hypothyroidism of mild intensity was obtained with prenatal and neonatal submission of Long-Evans rats to an iodide-rich diet. Chronic daily administration of methimazole to iodide-supplemented Long-Evans pups or to iodine-deprived Charles-River rats through the first 29–30 days of age provoked severe hypothyroidism. Monoamine oxidase type A (MAO-A) and not type B (MAO-B) activity was consistently, although slightly (by approximately 20%), increased in the hypothyroid brain. Triiodothyronine (T₃)-induced hyperthyroidism did not affect MAO activity. Replacement therapy with T₃ did not normalize MAO-A activity in hypothyroidism. Methimazole displayed a competitive and reversible in vitro inhibition of MAO-A but not MAO-B activity. Although this effect was obtained at concentrations far higher than those estimated to reach the brain after a single injection of the goiterogen, the occurrence of accumulation processes in the metabolism-deficient hypothyroid neonate rs cannot be excluded. Thus, MAO-A activity might be either directly depressed during the goiterogenic treatment, or increased as the result of some kind of rebound effect after interruption of methimazole administration.     

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.     

Interaction of N-(2-Nitro-4-Azidophenyl)-Serotonin with Two Types of Monoamine Oxidase in Rat Brain

The effects of N-(2-nitro-4-azidophenyl) serotonin (NAP-5-HT) on types A and B monoamine oxidase (MAO) in rat brain cortex were studied. In the dark this compound acted as a competitive inhibitor for both types A and B MAO (Ki values of 0.19 microM and 0.21 microM for types A and B MAO, respectively). Upon photolysis, NAP-5-HT became an irreversible inhibitor for only type B MAO. A 50% inhibition was obtained by irradiation of the enzyme in the presence of 35 nM NAP-5-HT. Furthermore the inhibition of type B MAO could be protected by including its substrate phenylethylamine during the irradiation. Under the same photolytic conditions photodependent inhibition of type A MAO by NAP-5-HT was not clearly observed. These results provide further evidence that there is a fundamental difference in the active site of the two types of MAO in brain. NAP-5-HT may be a useful photoaffinity probe for characterizing the active site of type B MAO.     

Differential Effects of Angiotensin II and Eledoisin on Monoamine Oxidase A and B Activities in Rat Brain

Intracerebroventricular injections of angiotensin II caused 108, 62, and 54% increases in monoamine oxidase A activities in rat hippocampus, hypothalamus, and striatum, respectively. These activatory effects were abolished by simultaneous injections of eledoisin. No significant changes of monoamine oxidase B activities were found under the same experimental conditions. Neither angiotensin II nor elodoisin changed substrate/inhibitor affinities of both isoenzymes. These data indicate that angiotensin II and tachykinin transmitter systems may exert opposite, long-term regulatory effects on monoaminergic neurons in rat brain.     

Regional Distribution of Calmodulin Activity in Rat Brain

Calmodulin activity in 68 discrete areas of rat brain, obtained by micropunch technique, was assessed by its capacity to activate a calmodulin-sensitive form of phosphodiesterase. In general, the activity of calmodulin was higher in the telencephalon, limbic system, and hypothalamus than in the mesencephalon, pons, cerebellum, and medulla. However, there were substantial differences in calmodulin activity in discrete nuclei of each region. The regional distribution of calmodulin activity in rat brain does not appear to correlate with that of any of the known putative neurotransmitters or peptides.     

Regional Distribution of Catalase in the Adult Rat Brain

Catalase activity was measured in 11 areas of perfused adult rat brain. The hypothalamus and substantia nigra contained the highest activities. The corpus callosum. a white-matter structure, contained intermediate activity. The caudate-putamen and frontal cortex contained the lowest activities. Regional catalase bears some relationship to the reported distribution of microperoxisomes, but considerable activity is present in areas with few microperoxisomes. Catalase may function as one of the systems detoxifying H₂O₂ formed in CNS amine metabolism.     

Properties and Regional Distribution of Pyruvate Dehydrogenase Kinase in Rat Brain

A method is described to measure directly in rat brain the activity of pyruvate dehydrogenase kinase (PDHa kinase; EC 2.7.1.99), which catalyzes the inactivation of pyruvate dehydrogenase complex (PDHC, EC 1.2.4.1, EC 2.3.1.12, and EC 1.6.4.3). The activity showed the expected dependence on added ATP and divalent cation, and the expected inhibition by dichloroacetate, pyruvate, and thiamin pyrophosphate. These results, and the properties of pyruvate dehydrogenase phosphate phosphatase (EC 3.1.3.43), indicate that the mechanisms of control of phosphorylation of PDHC seem qualitatively similar in brain to those in other tissues. Regionally, PDHa kinase is more active in cerebral cortex and hippocampus, and less active in hypothalamus, pons and medulla, and olfactory bulbs. Indeed, the PDHa kinase activity in olfactory bulbs is uniquely low, and is more sensitive to inhibition by pyruvate and dichloroacetate than that in the cerebral cortex. Thus, there are significant quantitative differences in the enzymatic apparatus for controlling PDHC activity in different parts of the brain.     

Regional Distribution of Glutathione Peroxidase in the Adult Rat Brain

Glutathione peroxidase activity was measured in 10 areas of perfused adult rat brain with the use of a fluorometric assay coupled to NADPH oxidation. The caudate-putamen and the substantia nigra had the highest activities. Cortical areas and several nuclear areas had somewhat lower activity. Activity was lowest in a white matter structure (corpus callosum). High activity of glutathione peroxidase may be related to the need to reduce hydrogen peroxide arising in the course of monoamine metabolism.     

Biochemical Evidence of Selective Nerve Cell Changes in the Normal Ageing Human and Rat Brain

Abstract: Atrophy with ageing of human whole brain, entire temporal lobe, and caudate nucleus was assessed in autopsy specimens, by biochemical techniques. Only the caudate nucleus showed changes. Markers for several neurotransmitter systems were also examined for changes with age. In neocortex and temporal lobe of human brain, small decreases were detected in markers of cholinergic nerve terminals, whereas a large decrease (79%) occurred in the caudate nucleus. Findings were similar in striatum from 3–33-month-old rats. No change occurred in binding of [³H]quinuclidinyl benzilate by human samples. Markers of serotonergic terminals were also unchanged in human and rat brain. By contrast, binding of [³H]lysergic acid diethylamide and [³H]serotonin was decreased (32–81%) in human neocortex and temporal lobe, but not in caudate nucleus. A 43% loss of a marker of γ-aminobutyrate terminals occurred in human neocortex, while [³H]muscimol binding increased (179%). No changes were detected in markers of catecholamine synapses in temporal lobe or rat striatum. Hence, with human ageing there appears to be a loss of markers of γ-aminobutyrate neurones intrinsic to neocortex and acetylcholine cells intrinsic to the caudate nucleus, as well as a change in postsynaptic serotonin receptors in neocortex. These losses are accompanied by relative preservation of markers of ascending projections from basal forebrain and brain stem.     

Resistance of Golden Hamster to l-Methyl-4-Phenyl-1,2,3,6 Tetrahydropyridine: Relationship with Low Levels of Regional Monoamine Oxidase B

Abstract: Effects of acute and chronic administration of 1 -methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were investigated for dopamine (DA) and its metabolites, 3,4-dihydroxyphenylacetic acid and 4-hydroxy-3-methoxyphenylacetic acid, in nucleus caudatus putamen (NCP), limbic system, and substantia nigra (SN) of golden hamster and BALB/c and C57/BL mice to obtain a clue for the variance of MPTP toxicity between the strains and species. Regional differences in the levels of monoamine oxidase (MAO) and the in vitro effects of MAO inhibitors were also determined and correlated with MPTP neurotoxicity. Concentrations of MPTP in the brains of mice and golden hamster at 10 min were comparable. Golden hamster was found to be resistant to the administration of MPTP as indicated by a lack of any alteration from the normal content of DA in NCP, limbic system, and SN. Both strains of mice exhibited >50% and >75% depletion of DA (C57/BL and BALB/c, respectively). The metabolites-to-DA ratios were decreased and increased in golden hamster and mouse strains, respectively, after acute or chronic treatment. Whereas the content of total MAO in golden hamster was one-third to one-sixth of any nuclei or mitochondria of both strains of mice, the ratio of MAO A to B was significantly higher in the former species. A possible involvement of discrete regional MAO activity in determining the extent of susceptibility of a species to MPTP toxicity is indicated from the study because (1) susceptibility as evidenced by DA depletion of a species coincided with high levels of MAO activity in SN and NCP, and (2) a highly positive correlation existed with total MAO and MAO B activity, there was a lack of correlation with MAO A activity, and a negative correlation existed with MAO A-to-B ratio and DA depletion. Hence, we propose that the resistance of a species to MPTP toxicity may depend on the content as well as the ratios of the two forms of MAO in NCP and SN. In other words, a higher MAO activity and a relative dominance of MAO B in these nuclei are critical in determining the susceptibility of a species to MPTP neurotoxicity.     

The Metabolism of Dopamine by Both Forms of Monoamine Oxidase in the Rat Brain and Its Inhibition by Cimoxatone

In the rat brain, dopamine is metabolised by both A and B forms of monoamine oxidase (MAO), although the A form of the enzyme is the major component. The Km of MAO-A toward dopamine (120 microM) is lower than the Km of MAO-B toward this substrate (340 microM). The activity of MAO-A was lower in old rats than in young rats, and the same degree of decrease was found for 5-hydroxytryptamine as for dopamine as substrates for this enzyme form. The activity of MAO-B was higher in the old rats, the degree of increase being the same for dopamine as for beta-phenethylamine as substrates for this enzyme form. The Ki values of the inhibition of MAO-A by cimoxatone and MD770222 (the principal plasma metabolite of cimoxatone) were independent of the substrate used to assay for activity, but were lower than the Ki values for the inhibition of MAO-B by these compounds.     

The Chronic Administration of Nicotine Induces Cytochrome P450 in Rat Brain

Abstract: The objective of these studies was to determine whether chronic administration of nicotine altered the cytochrome P450 (P450) monooxygenase system in rat brain. Male Sprague-Dawley rats received injections of nicotine bitartrate (1.76 mg/kg, s.c, twice daily for 10 days), and total cytochrome P450 content, the activity of N ADPH-cytochrome c reductase, and the activities and relative abundance of P4502B1 and P4502B2 (P4502B1/2) were determined in microsomal fractions from rat brain. The content of P450 increased significantly (p < 0.02) in all brain regions examined from nicotine-injected rats: the largest increase (208% of control) was in frontal cortex and the smallest increase (122% of control) in cerebellum. The activity of NADPH-cytochrome c reductase was unaltered by nicotine administration. Benzyloxyresorufin O-dealkylase (BROD) and pentoxyresorufin O-dealkylase (PROD) activities, mediated by P4502B1/2, increased significantly (p < 0.02) following nicotine administration; the largest increase (213-227% of control) was in frontal cortex. Western blots of microsomal proteins indicated that the increase in enzymatic activity was associated with an increase in content of P4502B1/2 immunoreactive proteins. In contrast to brain, total P450 content, activities of NADPH-cytochrome c reductase, BROD, and PROD, and levels of P4502B1 /2 immunoreactive proteins in liver were unaffected by chronic nicotine administration. Results indicate that chronic nicotine administration regulates the expression of P4502B1/2 in brain and that at the dose schedule used this effect occurs without a demonstrable effect on the hepatic P450 monooxygenase system.     

Regional Heterogeneity of L-Glutamate and L-Aspartate High-Affinity Uptake Systems in the Rat CNS

The high-affinity uptake of L-[3H]glutamate and L-[3H]aspartate into synaptosomes prepared from rat cerebral cortical, hippocampal, and cerebellar tissue was reduced by a number of structural analogues of L-glutamate and L-aspartate. threo-3-Hydroxy-L-aspartic acid was a more potent inhibitor of L-glutamate uptake than of L-aspartate uptake in the cerebral cortex, but not in the hippocampus or cerebellum. A similar pattern of selectivity was observed for cis-1-aminocyclobutane-1,3-dicarboxylic acid. Dihydrokainate was also more potent against L-glutamate than against L-aspartate in the cerebral cortex, but in the hippocampus, it was more potent against L-aspartate than against L-glutamate. By contrast, L-alpha-aminoadipate was significantly more potent in the cerebellum than in the cerebral cortex and hippocampus as an antagonist of both L-glutamate and L-aspartate. These results support other evidence that there is regional heterogeneity in acidic amino acid uptake sites and that the amino acids L-glutamate and L-aspartate may be taken up by a number of transport systems with overlapping substrate specificity but different inhibitor profiles.     

Activator Protein-1 Binding Activities in Discrete Regions of Rat Brain After Acute and Chronic Administration of Methamphetamine

Abstract: The activator protein-1 (AP-1) binding activities increased in three brain regions (striatum, nucleus accumbens, and cingulate cortex) after a single methamphetamine (METH) injection to rats. Pretreatment with SCH 23390, but not (−)-eticlopride, significantly inhibited the enhanced AP-1 binding activities induced by acute METH administration. The magnitude of enhancement of AP-1 binding activities 3 h after the last dose of chronic METH administration (4 mg/kg once daily for 14 days) was significantly attenuated as compared with those 3 h after a single METH administration. The AP-1 binding activities after a 1-, but not 4-, week abstinence from chronic administration of METH were still significantly higher than those of the saline-treated controls. A METH challenge after a 4-week abstinence period induced significantly lower AP-1 binding activities in rats chronically injected with METH than in rats chronically injected with saline. The supershift assay revealed that the levels of Jun family protein, but not Fos-related antigen, increased significantly in the striatum and nucleus accumbens of chronically METH-treated rats after a 1-week abstinence. These results suggest that chronic METH administration leads to delayed decay of the induced AP-1 binding activities and Jun component levels after abstinence for up to 1 week but results in no change in or decreases these activities and attenuates METH challenge-induced AP-1 binding activities after abstinence for 4 weeks.     

Regional and Subcellular Localization of Li+ and Other Cations in the Rat Brain Following Long-Term Lithium Administration

Abstract: Rats were given LiCl in their diet (40 mmol/kg dry weight) for at least 3 months to elucidate the regional and subcellular localization of Li⁺ in the brain as well as the effect of chronic lithium administration on the distribution of other cations. At steady-state the mean concentrations of Li⁺ were 0.66 mmol/kg wet weight in the whole brain and 0.52 mM in plasma. The tissue/plasma concentration ratio exceeded unity in all anatomical regions. No region showed excessive accumulation of Li⁺. Whole brain or regional contents of Na⁺ or K⁺ were unaffected by lithium treatment. Subcellular Li⁺ localization was demonstrated in nuclear, crude mitochondrial, and microsomal fractions of whole brain homogenate. Subfractionation of the crude mitochondrial fraction revealed energy-independent intrasynaptosomal and intramitochondrial Li⁺ and K⁺ localization at 0–4°C. Li⁺ administered in vivo disappeared within 10 min from synaptosomes incubated at 37°C. Li⁺ added in vitro at 1 mM attained a synaptosomal steady-state concentration within 30 min at 37°C. In control rats, synaptosomal concentrations and synaptosomal/medium concentration gradients of cations paralleled their respective in vivo concentrations and gradients. Lithium treatment caused synaptosomal depletion of K⁺ and Mg²⁺ and hence probably partial membrane depolarization. Addition of 1 mM Li⁺ in vitro also caused synaptosomal Mg²⁺ depletion. The results indicate that Li⁺ is “accumulated” in brain sediments and synaptosomes following its long-term treatment. The estimated intracellular and intrasynaptosomal Li⁺ concentrations are lower than predicted by passive distribution according to the Nernst equation, evidencing active extrusion of Li⁺.     

Effects of Chronic Restraint Stress on Feeding Behavior and on Monoamine Levels in Different Brain Structures in Rats

Monoaminergic systems are important modulators of the responses to stress. Stress may influence feeding behavior, and the involvement of monoamines in the control of food intake is well recognized. We investigated the effects induced by chronic-restraint stress, 1 h a day, for 40 days, on eating behavior and on monoamines in distinct brain structures. Increased consumption of sweet pellets, and not of peanuts, was observed. Dopamine (DA), serotonin (5–HT), and their metabolites were measured by HPLC-EC. After chronic restraint, the results observed were decreased 5–HT in hippocampus, with increased 5–HIAA/5–HT; decreased 5–HIAA levels in cortex; reduction in DA in hippocampus, and increased levels in amygdala and hypothalamus; HVA increased in cortex, as well as HVA/DA ratio, while DOPAC/DA decreased. HVA decreased in hypothalamus, as well as HVA/DA, and DOPAC/DA and HVA/DA decreased in the amygdala. These results suggest that restraint stress differentially affects the activity of central dopaminergic and serotonergic neurons, and this may be related to the effects observed in eating behavior.