Circadian variation in the acetylcholinesterase activity of specific rat brain areas

Abstract

Acetylcholinesterase (AChE) activity of the adenohypophysis, cerebellum, cerebral cortex, hypothalamus, amygdala, hippocampus, midbrain, pons, medulla oblongata and caudate nucleus was determined by a spectro‐photometric method in adult, male rats adapted toan LD 12:12cycle. Results of the study show that AChE activity is highest during the light phase and lowest during the dark phase of the cycle in all the brain areas studied except the adenohypophysis, cerebellum, hippocampus and hypothalamus. These findings expand earlier observations on the circadian variation in rat brain AChE activity and suggests a relationship with reported circadian variation in the acetylcholine levels of rat brain.     

Effect of thermal stress and water deprivation on the acetylcholinesterase activity of the pig brain and hypophyses

The effects of direct exposure of boars to thermal stress for 1 h daily for 5 days and to acute water deprivation for 24 or 48 h were studied on the acetylcholinesterase (AChE) activity of porcine brain and hypophysial regions. Mean ambient temperatures, respiratory rates and rectal temperatures in the open were significantly higher than inside the pen. Heat stress induced a rise in AChE activities in the pons, cerebellum, amygdala, hippocampus, hypothalamus, mid-brain and medulla oblongata. However, no significant changes were observed in the cerebral cortex, adenohypophysis and neurohypophysis. Water deprivation significantly (P<0.05) depressed AChE activity to varying extents depending on the duration of water restriction. Thus AChE activity in the amygdala was depressed by water deprivation for 24 h but partially restored at 48 h. The pons and medulla oblongata were comparable to the amygdala in this respect. The adenohypophysis and neurohypophysis were relatively unaffected.     

Influence of intermittent hypoxia and pyrimidinic nucleosides on cerebral enzymatic activities related to energy transduction

The effect of intermittent normobaric hypoxia and of biological pyrimidines (uridine and cytidine) on the specific activities of some enzymes related to cerebral energy metabolism were studied. Measurement were carried out on the following: (a) homogenate in toto; (b) purified mitochondrial fraction; (c) crude synaptosomal fraction, in different areas of rat brain: cerebral cortex, hippocampus, corpus striatum, hypothalamus, cerebellum, and medulla oblongata. Intermittent normobaric hypoxia (12 hours daily for 5 days) caused modifications of the enzyme activities in the homogenate in toto (decrease of hexokinase in cerebellum; increase of pyruvate kinase in medulla oblongata), in the purified mitochondrial fraction (increase of succinate dehydrogenase in the corpus striatum) and in the crude synaptosomal fraction (decrease of cytochrome oxidase activity in cerebral cortex, hippocampus, and cerebellum; decrease of malate dehydrogenase in hippocampus and cerebellum; decrease of lactate dehydrogenase in cerebellum). Daily treatment with cytidine or uridine altered some enzyme activities either affected or unaffected by intermittent hypoxia.     

Phenobarbital and 6-aminonicotinamide effect on cerebral enzymatic activities related to energy metabolism in different rat brain areas

The effect of phenobarbital (100 mg/kg i.p.) and 6-aminonicotinamide (6AN) (35 mg/kg i.p.) on enzyme activities related to energy transduction was investigated on the homogenate in toto, non-synaptic mitochondrial fraction and synaptosomal fraction isolated from different rat brain areas (cerebral cortex, hippocampus, hypothalamus, striatum, and medulla oblongata). 6AN treatment decreased: (a) phosphofructokinase in all the areas tested; (b) lactate dehydrogenase on the homogenate in toto in striatum and hypothalamus, and on the synaptosomal fraction in cerebral cortex and corpus striatum; (c) succinate dehydrogenase on non-synaptic mitochondrial fraction in hippocampus and striatum. Finally, aspartate aminotransferase was increased on non-synaptic mitochondrial fraction in striatum and medulla oblongata. Phenobarbital treatment induced an increase of total NADH cytochrome c reductase on mitochondrial fraction in hippocampus and hypothalamus, and a decrease of cytochrome oxidase activity on non-synaptic mitochondrial fraction in hypothalamus and medulla oblongata.     

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.     

Effects of acute manganese chloride exposure on lipid peroxidation and alteration of trace metals in rat brain

Although manganese (Mn) is an essential element, exposure to excessive levels of Mn and its accumulation in the brain can cause neurotoxicity and extrapyramidal syndrome. We have investigated the differences in the accumulated levels of Mn, the degree of lipid peroxidation, and its effects on the levels of trace elements (Fe, Cu, and Zn) in various regions in the brain of rats having undergone acute Mn exposure. The rats in the dose—effect group were injected intraperitoneally (ip) with MnCl₂ (25, 50, or 100 mg MnCl₂/kg) once a day for 24 h. The Mn significantly accumulated (p<0.05) in the frontal cortex, corpus callosum, hippocampus, striatum, hypothalamus medulla, cerebellum, and spinal cord in each case. The rats in the timecourse group were ip injected with MnCl₂ (50 mg MnCl₂/kg) and then monitored 12, 24, 48, and 72 h after exposure. The Mn accumulated in the frontal cortex, corpus callosum, hippocampus, striatum hypothalamus, medulla, cerebellum, and spinal cord after these periods of time, In both the dose—effect and time-course studies, we observed that the concentration of malondialdehyde, an end product of lipid peroxidation, increased significantly in the frontal cortex, hippocampus, striatum, hypothalamus, medulla, and cerebellum. However, no relationship between the concentrations of Mn in the brain and the extent of lipid peroxidation was observed. In addition, we found that there was a significant increase (p<0.05) in the level of Fe in the hippocampus, striatum, hypothalamus, medulla, and cerebellum, but the Cu and Zn levels had not changed significantly. These findings indicated that Mn induces an increase in the iron level, which provides direct evidence for Fe-mediated lipid peroxidation in the rats' brains; these phenomena might play important roles in the mechanisms of Mn-induced neurotoxicology.     

Neurochemical changes in rats chronically treated with a high concentration of manganese chloride

Several neurochemical parameters were studied in brain regions of rats chronically treated with a high concentration of manganese chloride (20 mg MnCl₂.4H₂O per ml. of drinking water) throughout development until adulthood. Large increases in Mn accumulation were found in all brain regions (hypothalamus, +530%; striatum, +479%; other regions, +152 to +250%) of Mn-treated adult rats. In these animals, Ca levels were decreased (–20 to –46%) in cerebellum, hypothalamus, and cerebral cortex but were increased (+186%) in midbrain. Mg levels were decreased (–12 to –32%) in pons and medulla, midbrain, and cerebellum. Fe levels were increased (+95%) in striatum but were decreased (–28%) in cerebral cortex. Cu levels were increased (+43 to +100%) in pons and medulla and striatum but Zn levels were decreased (–30%) in pons and medulla. Na levels were increased (+22%) in striatum but those of K and Cl remained unchanged. Type A monoamine oxidase activities were decreased (–13 to –16%) in midbrain, striatum, and cerebral cortex, but type B monoamine oxidase activities decreased (–13%) only in hypothalamus. Acetylcholinesterase activities were increased (+20 to +22%) in striatum and cerebellum. The results are consistent with out hypothesis that chronic manganese encephalopathy not only affects brain metabolism of Mn but also that of other metals.We dedicate this paper to Professor Alan N. Davison. Professor Davison has conducted pioneering research in several important areas including: brain development and myelination, aging and Alzheimer's disease, and multiple sclerosis. He encouraged us to investigate the neurochemical mechanisms of neurotoxicity of metal ions, particularly in connection with neurological diseases. His encouragement and continued support facilitated the launching of our multidisciplinary research program in the long-term effects of manganese toxicity on brain development and aging.     

Regional and subcellular distribution of enzymes of branched-chain amino acid metabolism in brains of normal and diabetic rats

Branched-chain-amino-acid:alpha-ketoglutarate transaminase and branched-chain alpha-ketoacid dehydrogenase have been assayed in brains of control and of streptozotocin-induced diabetic rats. Enzyme activities were measured in five distinct regions of the brain: cerebellum, pons + medulla, midbrain, thalamus + hypothalamus, and telencephalon. Subcellular distribution of these enzymes in whole brain was assessed by fractionating brain homogenate into cytoplasm, free mitochondria, and synaptosomes. The following enzymes were used as markers: lactate dehydrogenase for cytoplasm, glutamate dehydrogenase for mitochondria, and glutamate decarboxylase for synaptosomes. The activity of the branched-chain amino acid transaminase in all brain regions was considerably higher than that of the branched-chain alpha-ketoacid dehydrogenase. While the highest activity of the transaminase occurred in brain-stem regions, the highest activity of the dehydrogenase was present in cerebellum and telencephalon. Diabetes did not affect the activity of the transaminase, but it caused a decrease in the total activity of the dehydrogenase in midbrain and in thalamus + hypothalamus. The transaminase was localized in the cytoplasmic fraction of whole brain, while the dehydrogenase was enriched in the free mitochondria.     

Profile of acetylcholinesterase in brain areas of male and female rats of adult and old age

Inhibition of acetylcholinesterase (AChE)-metabolizing enzyme of acetylcholine, is presently the most important therapeutic target for development of cognitive enhancers. However, AChE activity in brain has not been properly evaluated on the basis of age and sex. In the present study, AChE activity was investigated in different brain areas in male and female Sprague-Dawley rats of adult (3 months) and old (18-22 months) age. AChE was assayed spectrophotometrically by modified Ellman's method. Specific activity (micromoles/min/mg of protein) of AChE was assayed in salt soluble (SS) and detergent soluble (DS) fractions of various brain areas, which consists of predominantly G1 and G4 molecular isoforms of AChE respectively. The old male rats showed a decrease (40-55%) in AChE activity in frontal cortex, striatum, hypothalamus and pons in DS fraction and there was no change in SS fraction in comparison to adult rats. In the old female rats the activity was decreased (25-40%) in frontal cortex, cerebral cortex, striatum, thalamus, cerebellum and medulla in DS fraction whereas in SS fraction the activity was decreased only in hypothalamus as compared to adult. On comparing with old male rats, old female rats showed increase in AChE activity in cerebral cortex, hippocampus and hypothalamus of DS fraction and decrease in hypothalamus of SS fraction. There was a significant increase in AChE activity in DS fraction of cerebral cortex, hippocampus, hypothalamus, thalamus and cerebellum in female as compared to male adult rats. However, no significant change in AChE activity was found in the SS fraction, except hypothalamus between these groups. Thus it appears that age alters AChE activity in different brain regions predominantly in DS fraction (G4 isoform) that may vary in male and female. These observations have significant relevance to age related cognitive deficits and its pharmacotherapy.     

Effect of ovariectomy and estrogen supplementation on brain acetylcholinesterase activity and passive-avoidance learning in rats

The effect of ovariectomy and estrogen treatment on the brain acetylcholinesterase activity and cognition in rats was investigated in this study. Ovariectomized and nonovariectomized rats were treated subcutaneously with estradiol dipropionate for 8 d. In the single-trial, passive-avoidance test all the groups showed significant learning and retention of memory as evident by the increase in transfer latency time in trial 2 as compared with trial 1. No-transfer response was significantly increased in the estradiol-dipropionate-treated ovariectomized (80%) and nonovariectomized (60%) group as compared with the ovariectomized (30%) group. Specific activity of acetylcholinesterase was assayed spectrophotometrically in salt-soluble and detergent-soluble fractions of various brain areas: frontal cortex, cerebral cortex, striatum, hippocampus and hypothalamus, thalamus, pons, medulla, and cerebellum. The effect of ovariectomy and estradiol dipropionate was varied in both fractions of these brain areas. Estradiol dipropionate treatment could restore the acetylcholinesterase activity to the control level only in the detergent-soluble fraction of hypothalamus and salt-soluble fraction of hypothalamus, thalamus, and medulla in ovariectomized rats. The results indicate that ovariectomy alters acetylcholinesterase activity in the brain areas but not in a uniform manner and affects only qualitative aspects of cognitive function, which could be improved by estrogen supplementation.     

Developmental Changes in the NGF Content in the Brain of Young,Growing, Low-Birth-Weight Rats

The NGF content in each region of the brain of four-week-old rats was ranked in the decreasing order of cerebral cortex, hippocampus, cerebellum, midbrain/diencephalon, and pons/medulla ob-longata, and the NGF concentration, in the decreasing order of hippocampus, cerebral cortex, cerebellum, midbrain/diencephalon, and pons/medulla oblongata in both AFD and SFD groups. The NGF content and concentration in the cerebral cortex were about the same value at each age between those in the AFD and SFD groups. Those in the hippocampus were a little higher in the SFD group than in the AFD group at the ages of three and four weeks, unlike those in the other regions, where the values for the cerebellum, midbrain/diencephalon and pons/medulla oblongata tended to be somewhat higher in the AFD group than in the SFD group. The NGF concentrations in the hippocampus and cerebral cortex increased with growth: the concentration in the hippocampus at four weeks of age was about 4-fold of that at one week in the AFD group and about 5.7-fold of that at one week in the SFD group; and likewise the concentration in the cerebral cortex at four weeks of age was about 5.3-fold in the AFD group and about 7-fold in the SFD group. The NGF concentrations in the cerebellum decreased, and those in midbrain/diencephalon and pons/medulla oblongata hardly changed with growth in either AFD or SFD group. From these results NGF may have stronger implications for the neuronal growth in the hippocampus compared with those in the lower brain regions of the SFD rats.     

Neonatal 6-hydroxydopamine treatment: noradrenaline levels and in vitro 3Hcatecholamine synthesis in discrete brain regions of adult rats

Endogenous noradrenaline levels are elevated in medulla oblongata, mesencephalon, pons and thalamus of adult rats which had been treated with 6-hydroxydopamine on days 1, 2, 8 and 15 after birth. Levels in spinal cord, cerebellum, hippocampus/amygdala and cortex are depressed, whereas no significant changes are observed in striatum, hypothalamus and medulla spinalis. The rate at which medulla oblongata synthesizes tritiated noradrenaline and dopamine from tritiated tyrosine $\text{in}$$\text{vitro}$ is markedly enhanced. No effect was apparent on catecholamine synthesis in hypothalamus. Tritiated noradrenaline synthesis, but not tritiated dopamine synthesis, in the cortex is depressed. These results support the view that neonatal 6-hydroxydopamine treatment causes a degeneration of noradrenaline nerve terminals in the cortex and induces an increase in noradrenaline terminals in the medulla oblongata.     

Developmental and Regional Studies of the Metabolism of Inositol 1,4,5-Trisphosphate in Rat Brain

Coupling of CNS receptors to phosphoinositide turnover has previously been found to vary with both age and brain region. To determine whether the metabolism of the second messenger inositol 1,4,5-trisphosphate also displays such variations, activities of inositol 1,4,5-trisphosphate 5'-phosphatase and 3'-kinase were measured in developing rat cerebral cortex and adult rat brain regions. The 5'-phosphatase activity was relatively high at birth (approximately 50% of adult values) and increased to adult levels by 2 weeks postnatal. In contrast, the 3'-kinase activity was low at birth and reached approximately 50% of adult levels by 2 weeks postnatal. In the adult rat, activities of the 3'-kinase were comparable in the cerebral cortex, hippocampus, and cerebellum, whereas much lower activities were found in hypothalamus and pons/medulla. The 5'-phosphatase activities were similar in cerebral cortex, hippocampus, hypothalamus, and pons/medulla, whereas 5- to 10-fold higher activity was present in the cerebellum. The cerebellum is estimated to contain 50-60% of the total inositol 1,4,5-trisphosphate 5'-phosphatase activity present in whole adult rat brain. The localization of the enriched 5'-phosphatase activity within the cerebellum was examined. Application of a histochemical lead-trapping technique for phosphatase indicated a concentration of inositol 1,4,5-trisphosphate 5'-phosphatase activity in the cerebellar molecular layer. Further support for this conclusion was obtained from studies of Purkinje cell-deficient mutant mice, in which a marked decrement of cerebellar 5'-phosphatase was observed. These results suggest that the metabolic fate of inositol 1,4,5-trisphosphate depends on both brain region and stage of development.     

Chronic hypoxia in development selectively alters the activities of key enzymes of glucose oxidative metabolism in brain regions

The immature brain is more resistant to hypoxia/ischemia than the mature brain. Although chronic hypoxia can induce adaptive-changes on the developing brain, the mechanisms underlying such adaptive changes are poorly understood. To further elucidate some of the adaptive changes during postnatal hypoxia, we determined the activities of four enzymes of glucose oxidative metabolism in eight brain regions of hypoxic and normoxic rats. Litters of Sprague-Dawley rats were put into the hypoxic chamber (oxygen level maintained at 9.5%) with their dams starting on day 3 postnatal (P3). Age-matched normoxic rats were use as control animals. In P10 hypoxic rats, lactate dehydrogenase (LDH) activity in cerebral cortex, striatum, olfactory bulb, hippocampus, hypothalamus, pons and medulla, and cerebellum was significantly increased (by 100%–370%) compared to those in P10 normoxic rats. In P10 hypoxic rats, hexokinase (HK) activity in hypothalamus, hippocampus, olfactory bulb, midbrain, and cerebral cortex was significantly decreased (by 15%–30%). Neither -ketoglutarate dehydrogenase complex (KGDHC, which is believed to have an important role in the regulation of the tricarboxylic acid [TCA] cycle flux) nor citrate synthase (CS) activity was significantly decreased in the eight regions of P10 hypoxic rats compared to those in P10 normoxic rats. In P30 hypoxic rats, LDH activity was only increased in striatum (by 19%), whereas HK activity was only significantly decreased (by 30%) in this region. However, KGDHC activity was significantly decreased in olfactory bulb, hippocampus, hypothalamus, cerebral cortex, and cerebellum (by 20%–40%) in P30 hypoxic rats compared to those in P30 normoxic rats. Similarly, CS activity was decreased, but only in olfactory bulb, hypothalamus, and midbrain (by 9%–21%) in P30 hypoxic rats. Our results suggest that at least some of the mechanisms underlying the hypoxia-induced changes in activities of glycolytic enzymes implicate the upregulation of HIF-1. Moreover, our observation that chronic postnatal hypoxia induces differential effects on brain glycolytic and TCA cycle enzymes may have pathophysiological implications (e.g., decreased in energy metabolism) in childhood diseases (e.g., sudden infant death syndrome) in which hypoxia plays a role.     

Metallothionein-I induction by stress in specific brain areas

The distribution of metallothionein-I (MT) in several areas of the brain and its induction by immobilization stress has been studied in the rat. MT content was highest in hippocampus and midbrain and lowest in frontal cortex and pons plus medulla oblongata. Immobilization stress for 18 hours (which was accompanied by food and water deprivation) significantly increased MT levels in the frontal cortex, pons plus medulla oblongata and hypothalamus, but not in midbrain and hippocampus. The effect of stress on MT levels was specific as food and water deprivation along had no significant effect on MT levels in any of the brain areas studied. The effect of stress on MT levels was independent of changes in cytosolic Zn content; this was generally unaffected by stress or food and water deprivation but decreased in pons plus medulla oblongata from stressed rats. The results suggest that MT is induced more significantly in the brain areas that are usually involved in the response of animals to stress.     

Regional and Subcellular Calmodulin Content of Rat Brain

Calmodulin contents of cortex, cerebellum, striatum, diencephalon, and medulla + pons and of subcellular fractions of each region were determined by radioimmunoassay. The diencephalon had the highest level of calmodulin (48.87 +/- 4.56 micrograms/mg protein), whereas medulla + pons had the lowest level (8.01 +/- 0.84 micrograms/mg protein). In all brain regions, the mitochondrial fraction was richest in calmodulin (from 71 to 227 micrograms/mg protein) whereas other areas contained from 6 to 66 micrograms/mg protein.     

Extent of RNA editing of glutamate receptor subunit GluR5 in different brain regions of the rat

Summary 1. The structure and function of glutamate receptor subunits GluR2, GluR5, and GluR6 are changed by RNA editing. This reaction produces a base transition in the second transmembrane spanning region. The triplet CAG (coding for glutamine) is changed to CGG (coding for arginine). This transition has a pronounced effect on calcium fluxes through the respective ion channels, because calcium currents decrease with the rate of editing.2. In the present study the extent of RNA editing of the glutamate receptor subunit GluR5 was studied in different brain regions of control rats using a newly developed analysis system. This system is based on restriction analysis of the polymerase chain reaction (PCR) product, derived from reverse-transcribed mRNA as template, with the enzymeBbv1.Bbv1 recognizes the sequence of the nonedited receptor subunit around the edited base (sequence GCAGC) but not that of the edited subunit (sequence GCGGC; A edited to G).3. Total RNA was isolated from the cerebral cortex, striatum, hippocampus, thalamus, hypothalamus, cerebellum, pons/medulla oblongata, and white matter and reverse transcribed into cDNA. The region across the edited sequence was amplified by PCR using GluR5-specific primers and the cDNA as template. PCR products were cleaned by ethanol precipitation, incubated withBbv1, and electrophoresed on an agarose gel together with standards. Gels were photographed and the extent of GluR5 mRNA editing was quantified using an image analysis system. A calibration curve was obtained using PCR products amplified from plasmids with edited and nonedited GluR5 as inserts.4. In the brain of control rats the extent of RNA editing of the GluR5 subunit amounted to 62±6.0% of total (cortex), 43±5.3% (striatum), 52±5.3% (hippocampus), 91±6.3% (thalamus), 85±10.2% (hypothalamus), 82±6.5% (cerebellum), 88±6.8% (pons/medulla oblongata), and 41±2.7% (white matter).5. The extent of RNA editing varied, thus, considerably in different brain regions, being lowest in the white matter and striatum and highest in the thalamus and pons/medulla oblongate. RNA editing of glutamate receptor subunits may play an important role in the control of calcium fluxes through non-N-methyl-D-aspartate receptor channels in different physiological and/or pathological states of the brain.     

Formation of gamma-hydroxybutyrate in brain.

Abstract— Gamma-hydroxybutyric acid is a neuroactive compound which has been found to be a normal constituent of mammalian brain. The present report characterized enzymatic activity in brain forming gamma-hydroxybutyrate (GHB) from succinic semialdehyde (SSA). When NADPH served as cofactor, whole brain homogenate was capable of forming nearly 300 nmol GHB/min/g brain when enzyme activity was measured at 37°C. GHB production was significantly less (50%) when NADH was the cofactor. A regional localization of these activities indicated that the cerebellum and septal area contained the highest capacity to form GHB in the presence of NADPH; intermediate to high activity was found in the cortex, medulla, superior colliculus and corpus striatum; low activity was present in the inferior colliculus, thalamus, pons, hippocampus, substantia nigra and hypothalamus. Activity in the presence of NADH was rather evenly distributed with the exceptions of the cerebellum and inferior colliculus, which contained high and low activity respectively. Both NADPH- and NADH-dependent activities were found primarily in the cytosol. Pentobarbital inhibited enzyme activity and enzyme activity was differentiated from lactic dehydrogenase and alcohol dehydrogenase by use of specific inhibitors. In addition, mixed substrate experiments and kinetic analysis provided evidence for the presence of two reversible NADPH-dependent enzymes capable of producing GHB from SSA.     

Regional brain GABA metabolism and release during hepatic coma produced in rats chronically treated with carbon tetrachloride

Hepatic coma was induced in rats chronically treated with CCl₄, by means of a single injection of ammonium acetate. The activities of glutamate decarboxylase (GAD) and GABA transaminase (GABA-T), as well as the synaptosomal uptake and release of [³H]GABA, were measured in the following brain areas of the comatose rats: cortex, striatum, hypothalamus, hippocampus, midbrain and cerebellum. Hepatic coma was associated with a general decrease of GAD activity, whereas GABA-T activity was diminished only in the hypothalamus, striatum and midbrain. During hepatic coma, the K⁺-stimulated [³H]GABA release was notably diminished in the striatum and cerebellum, whereas a significant increase was observed in the hippocampus. [³H]GABA uptake increased in most regions after CCl₄ treatment, independently of the presence of coma. The results indicate that GABAergic transmission seems to be decreased in most cerebral regions during hepatic coma.     

Highly sensitive assay for phenylethanolamine N-methyl-transferase activity in rat brain by high-performance liquid chromatography with electrochemical detection

This paper describes a new and highly sensitive assay for phenylethanolamine N-methyl-transferase (PNMT) activity with noradrenaline as substrate in various rat brain regions by high-performance liquid chromatography with electrochemical detection. Commercially available noradrenaline contained about 0.27% of contaminating adrenaline, which was removed to reduce the blank value. Enzymatically formed adrenaline was adsorbed on an aluminium oxide column, eluted with 0.5 M hydrochloric acid, separated by high-performance reversed-phase paired-ion chromatography and measured with electrochemical detection. 3,4-Dihydroxybenzylamine was added to the incubation mixture as an internal standard after the reaction. This assay was very sensitive and 0.5 pmol of adrenaline formed enzymatically could be detected. This assay method was applied to measure PNMT activity in various rat brain regions. The highest activity was observed in the hypothalamus, pons plus medulla oblongata, septum, lower brain stem, and cerebral cortex; the lowest activity was in the striatum, hippocampus, cerebellum, and limbic brain.