Glutathione and gamma-glutamyl transpeptidase in the adult female rat brain after intraventricular injection of LHRH and somatostatin

Glutathione content and glutamyl transpeptidase activity in different regions of adult female rat brain were determined at 10 and 30 min following intraventricular injection of LHRH and somatostatin. Hypothalamic glutathione levels were significantly elevated at 10 and 30 min after a single injection of a 0.1 micrograms dose of LHRH. On the contrary, glutathione levels significantly decreased in the hypothalamus, cerebral cortex and cerebellum at 10 and 30 min after 0.5 or 1 microgram dose. However, significant decrease in brain stem glutathione was evident at 30 min after 0.5 microgram and 10 min after the 1 microgram dose. Somatostatin at doses of 0.5 microgram and 1 microgram significantly decreased glutathione levels in all four brain regions both at 10 and 30 min following injection into the 3rd ventricle. Gamma-glutamyl transpeptidase activity in the hypothalamus and cerebral cortex was significantly elevated after intraventricular injection of LHRH. However, a significant increase in gamma-glutamyl transpeptidase activity in cerebellum and brain stem was seen only with 0.5 and 1 micrograms doses of LHRH. Somatostatin also significantly increased gamma-glutamyl transpeptidase activity in hypothalamus, cerebral cortex, brain stem and cerebellum. The decrease in glutathione levels with corresponding increase in gamma-glutamyl transpeptidase activity after intraventricular administration of LHRH and somatostatin suggests a possible interaction between glutathione and hypothalamic peptides.     

An in vivo cholinergic influence on the retention of [3H]noradrenaline in regional brain synaptosomes

Rats were intraventricularly (icv) injected with [³H]noradrenaline and the retention of the amine was determined in synaptosomes obtained from cerebral cortex, hypothalamus and brain stem. Previous icv administration of hemicholinium-3, effective enough to markedly decrease brain acetylcholine levels, increased the retention of synaptosomal [³H]noradrenaline in hypothalamus and cerebral cortex; this increased retention did not occur in the brain stem. The increased retention of [³H]noradrenaline, produced by hemicholinium-3, was reversed by a concomitant icv dose of choline, which in turn reversed the decrease of acetylcholine caused by hemicholinium-3. These results are interpreted as brain cholinergic activity having an influence on the turnover of noradrenaline in some brain regions.     

Comparative evaluation of the biogenic amine level of the brain and the ethanol kinetics in the blood of inbred C57B1/6 and CBA mice

The kinetics of ethanol in blood and the levels of serotonin, noradrenaline, and dopamine in the cortex of cerebral hemispheres, hypothalamus, thalamus, and brain stem were studied in male C57C1/6 and CBA mice characterized by predisposition and non-predisposition to the development of experimental alcoholism. C57B1/6 mice characterized by predisposition to the development of experimental alcoholism demonstrated a high level of serotonin and noradrenaline in the hypothalamus and brain stem and high rate of ethanol elimination from the blood. CBA mice non-predisposed to the development of experimental alcoholism were characterized by a low level of serotonin and noradrenaline in the hypothalamus and brain stem and by low rate of ethanol elimination from blood.     

THE EFFECT OF LIMB ISCHAEMIA ON THE TURNOVER OF NORADRENALINE IN THE HYPOTHALAMUS AND BRAIN STEM OF THE RAT

The effect of ischaemic limb injury on the turnover of noradrenaline in the hypothalamus and brain stem has been studied in rats. There are theoretical reasons for thinking that these regions are activated in trauma and previous work showed that during limb-ischaemia the concentration of noradrenaline in the hypothalamus decreased by 27 per cent. The tourniquets were applied to both hind-limbs 1 h after the injection of [¹⁴C]-tyrosine when the labelling of the noradrenaline was maximal. During 4 h limb ischaemia the endogenous tyrosine concentration in the plasma decreased while that in the hypothalamus first rose and then fell. Changes in a similar direction in the brain stem were not statistically significant. Limb ischaemia did not affect the decline in the specific activity of the plasma or tissue tyrosine. It was concluded that the injury increased the utilization of tyrosine by the body. During the 4 h bilateral hind-limb ischaemia the rate of decline of [¹⁴C]noradrenaline was significantly increased in the brain stem but not in the hypothalamus. Conditions in the brain stem were sufficiently close to ‘steady-state’ to be able to conclude that the injury increased the metabolism of noradrenaline in the brain stem. Conditions in the hypothalamus were too complicated for definite conclusions to be drawn. The possible reasons for this and the limitations of this method for studying noradrenaline turnover are discussed.     

Response of rat brain indoles and motor activity to short light-dark cycles.

Since the vigilance states of the rat can be largely controlled by one hour light-one hour dark cycles, we investigated the effect of this photoperiod on the rat brain indoles and motor activity. Groups of 9 rats were killed 15-20 min or 45-50 min after the onset of the 1-hour light or 1-hour dark period. Serotonin (5HT) and 5-hydroxyindoleacetic acid were determined fluorometrically in cortex, hypothalamus and brain stem. Tryptophan was determined fluorometrically in serum, cortex and hypothalamus. Cortical tryptophan was highest at the end of the dark period, whereas cortical 5-HT and 5-hydroxyindoleacetic. acid were highest at the beginning of the light period. Motor activity was high during darkness and low during light. When the biochemical results were compared to the motor activity records of the individual animals, the decrease in motor activity at the onset of light correlated significantly with the cortical 5-hydroxyin- doleacetic acid/5-HT ratio in the animals killed at the beginning of the light period. The results indicate a rapid response of the cortical indoles to the onset of light and support the hypothesis that the induction of sleep is related to the brain indole metabolism.     

ACETYL- AND BUTYRYLCHOLINESTERASE ACTIVITY OF SELECTED BRAIN AREAS IN DEVELOPING RATS AFTER NEONATAL X-IRRADIATION*

Acetylcholinesterase and butyrylcholinesterase activities in sensori-motor cortex, hypothalamus, cerebellum, and brain stem were compared in normally developing Long-Evans rats and after neonatal whole-body exposure to 450 r X-radiation. Enzyme activities were measured on three postnatal days: day 10, when brain is still immature; day 24, when it has reached functional and morphological maturity; and day 64, after sexual maturation. In controls, acetylcholinesterase and butyrycholinesterase activities increased with age in all areas, especially between 10 and 24 days; e.g., in sensori-motor cortex acetylcholinesterase activity increased 60 per cent from 10 to 24 days and 12 per cent from 24 to 64 days. At all ages acetylcholinesterase activity was highest in the brain stern, followed in decreasing order by the hypothalamus, cerebellum, and sensori-motor cortex. Butyrylcholinesterase activity was higher in subcortical than in cortical areas. In neonatally irradiated rats, acetylcholinesterase activity was significantly decreased in the ontogenetically newer structures at 10, but not at 64, days; in the hypothalamus, it remained normal at 10 days but was significantly decreased at 24 and 64 days. Butyrylcholinesterase activity was significantly decreased in some areas 1 week after radiation but returned to normal at 24 days. Total esterase activity in whole blood was signtficantly decreased at 10 days in irradiated rats but returned to control levels by the end of the experiment. The greatest post-radiation decline in acetylcholinesterase activity (60 per cent below controls) did not result in spontaneous gross behaviour alterations, but may be related to disturbances in functional brain maturation evidenced by specific tests. If the role of acetycholine as a central neurotransmitter is accepted, these data suggest that radiation alters acetycholine/acetylcholinesterase ratios and thereby cholinergie synaptic transmission.     

Evidence for presence of Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) in human brain cortex

Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) was previously isolated from bovine hypothalamus. We have now purified it from the parietal cortex of human brain tissue by gel filtration chromatography and four subsequent high performance liquid chromatographic steps. During isolation, the peptide content was followed by radioimmunoassay and compared with the elution of synthetic Tyr-MIF-1 in identical chromatographic systems. This extends evidence for the presence of Tyr-MIF-1 from bovine to human brain tissue and from hypothalamus to cortex.     

Effects of ethanol on brain monoamine content of spontaneously hypertensive rats (SHR)

Spontaneously hypertensive rats (SHR) were administered either 2.4 g/kg ethanol or an isocaloric glucose daily for 4 weeks and the levels of norepinephrine (NE), epinephrine (EP), dopamine (DA), serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in different brain regions were determined. Results indicated a 3-fold increase in NE level in brain stem and hypothalamus and more than 2-fold increase in DA in corpus striatum in alcohol-treated rats as compared to controls. There was a significant increase in the level of DA in the corpus striatum but the levels in cerebral cortex, brain stem and hippocampus were decreased instead. Decreases in 5-HT levels were found in hypothalamus, brain stem, cortex and cerebellum of alcohol-treated brain as compared to untreated controls. These results indicate alterations of the biogenic amine contents in different regions of the SHR brain after chronic ethanol ingestion. Since stimulated release of biogenic amines in the SHR brain has been implicated in the regulation of blood pressure, changes due to ethanol ingestion may be a risk factor in hypertensive patients.     

Regional cerebral blood flow changes during hypothermia

1. 1. When brain temperature was decreased from 38 to 22 °C using selective hypothermia, tissue blood flow decreased significantly in cerebral cortex, cerebellum, and thalamus, but did not significantly change in hypothalamic or brain stem tissue.

2. 2. A further decrease in brain temperature to 8 °C produced an increase in blood flow in all tissues except cerebral cortex compared to tissue blood flow measured at 22 °C. Compared to normothermic values, blood flow remained significantly decreased at 8 °C in cerebral and cerebellar cortex and was increased in brain stem.

3. 3. After rewarming, tissue blood flow returned to original baseline values in all tissues except cerebral cortex where blood flow was slightly but significantly decreased and brain stem, where blood flow was increased.

4. 4. These results indicate that the cerebrovascular effects of selective brain cooling are regionally specific. These changes appear to be due to both direct and indirect effects of cerebral hypothermia since brain tissue blood flow changes are apparent, compared to control values, after rewarming of the brain.

     

Species differences in epinephrine concentration and norepinephrine N-methyltransferase activity in hypothalamus and brain stem

1. The concentration of epinephrine, norepinephrine and dopamine, and the activity of norepinephrine N-methyltransferase, the epinephrine-forming enzyme, were determined in hypothalamus and brain stem in several species. 2. Epinephrine concentration in hypothalamus, a nerve terminal region, varied in the order frog greater than turtle greater than chicken greater than cat greater than dog greater than pigeon greater than rat greater than ferret greater than hamster greater than mouse, with concentrations being undetectable in rabbits, horses and guinea pigs. 3. Epinephrine concentration was lower than norepinephrine concentration in all species except the frog. 4. NMT activity was detected in all species except guinea pigs. 5. Epinephrine concentration was lower in brain stem, a cell body region, than in hypothalamus in all species. Only in the frog brain stem was there more epinephrine than norepinephrine. 6. No epinephrine or NMT activity was detected in either brain region in guinea-pigs.     

Genetic variations of noradrenaline synthesis and reception in the mouse brain and the animal behavior in the new environment]

C57BL mice were found to have the highest locomotion and the lowest emotionality under novel environment out of three strains of mice. Their brain stem TH activity was increased whereas the density of alpha2-ARs and beta-ARs were decreased in their cortex and hypothalamus. The BALB mice were twice as virulent as the CBA mice whereas the emotionality was the same in both strains. In general, low emotionality and high locomotion in novel environment were found in mice with increased activity of norepinephrine synthesis and decreased amount of adrenergic receptors in the brain.     

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.     

Postnatal ontogeny of uridine kinase in the cerebellum,hypothalamus, and cerebral cortex of the rat

Postnatal developmental patterns of uridine kinase were determined in crude subcellular fractions of the rat cerebellum, hypothalamus and cerebral cortex at ages 3 through 60 days. The highest specific activity and predominant distribution of enzyme was in the 105,000g supernatant of the 3 brain regions. Enzyme activity in hypothalamus and cerebral cortex was maximum at 3 days and decreased with age; in cerebellum it increased through 13 days and decreased thereafter. Thus, the pattern of activity in hypothalamus and cerebral cortex paralleled changes in DNA and RNA synthesis through age 60 days; in cerebellum, it more closely approximated changes in DNA synthesis during early development. Changes inK _m with aging suggest that the brain regions contain more than one form of enzyme. The highest particulate activity was in the microsomal fraction of the cerebellum and hypothalamus at all ages and in the cortex at 35 and 60 days. Relative specific activity for microsomal fractions of the brain regions at 60 days indicate a concentration of the enzyme which may be relevant in the maintenance of RNA activity in adult brain.     

Glutamate decarboxylase and GABA-aminotransferase activity in brain mitochondria under conditions of ethanol exposure in postnatal ontogenesis

The activity of the enzymes of GABA- GDK and GABA-T metabolism in the brain mitochondria in 6 periods of postnatal development under the conditions of high dose of ethanol was studied. It has been revealed that after the impact of high dose of ethanol (3.5 g/kg of 25% solution, intraperitoneally) the enzymes' activities in initial mitochondrial fractions of cerebral cortex, cerebellum, hypothalamus and brain stem increases. The activity of GABA-T in 10- and 21-days animals significantly decreases, while in other periods on the contrary it increases.     

THE EFFECT OF INJURY ON MONOAMINE CONCENTRATIONS IN THE RAT HYPOTHALAMUS

Abstract— The monoamine concentrations have been measured in four regions of the brain (hypothalamus, cortex, cerebellum and brain stem) in rats injured by either hind-limb ischaemia or scald. Both injuries produced a rapid fall in the noradrenaline concentration of the hypothalamus which recovered slowly if the injury was not fatal. This effect of injury was seen after pretreatment with a-methyl-p-tyrosine to inhibit noradrenaline synthesis, indicating an increased rate of utilization of noradrenaline after injury. These injuries did not affect the 5-hydroxytryptamine concentration in the hypothalamus, but changes were found in the concentration of this monoamine and in that of its metabolite, 5-hydroxyindole acetic acid, in the brain stem. It is concluded that these forms of injury had specific effects on the brain monoamines. The hypothalamic changes were not secondary to changes in core temperature or to hypotension or hypovolaemia and they are discussed in relation to the impairment of temperature regulation seen in the injured rat.     

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.     

Alpha-lipoic acid affects the oxidative stress in various brain structures in mice with methionine and choline deficiency

Deficiency in methionine or choline can induce oxidative stress in various organs such as liver, kidney, heart, and brain. This study was to examine the effects of alpha-lipoic acid (LA) on oxidative stress induced by methionine and choline deficiency (MCD) in several brain structures. Male mice C57BL/6 (n = 28) were divided into four groups: (1) control – continuously fed with standard chow; (2) LA – fed with standard chow and receiving LA; (3) MCD2 – fed with MCD diet for two weeks, and (4) MCD2+LA – fed with MCD diet for two weeks and receiving LA (100 mg/kg/day intraperitonealy [i.p.]). Brain tissue (cortex, hypothalamus, striatum and hippocampus) was taken for determination of oxidative stress parameters. MCD diet induced a significant increase in malondialdehyde and NOx concentration in all brain regions, while LA restored their content to normal values. Similar to this, in MCD2 group, activity of total SOD, MnSOD, and Cu/ZnSOD was reduced by MCD diet, while LA treatment improved their activities in all brain structures. Besides, in MCD2 group a decrease in catalase activity in cortex and GSH content in hypothalamus was evident, while LA treatment induced an increase in catalase activity in cortex and striatum and GSH content in hypothalamus. LA treatment can significantly reduce lipid peroxidation and nitrosative stress, caused by MCD diet, in all brain regions by restoring antioxidant enzymes activities, predominantly total SOD, MnSOD, and Cu/ZnSOD, and to a lesser extent by modulating catalase activity and GSH content. LA supplementation may be used in order to prevent brain oxidative injury induced by methionine and choline deficiency.     

Differential distribution of the 5-alpha-reductase in the central nervous system of the rat and the mouse: are the white matter structures of the brain target tissue for testosterone action?

In the brain of several animal species testosterone is converted into a series of 5-alpha-reduced metabolites, and especially into 17-beta-hydroxy-5-alpha-androstan-3-one (DHT), by the action of the enzyme 5-alpha-reductase. The formation of DHT has never been evaluated in the white matter structures of the brain, which are composed mainly of myelinated axons. The experiments here described were performed in order to study, in the rat and the mouse, the DHT forming activity of several white matter structures, in comparison with that of the cerebral cortex and of the hypothalamus. Two sampling techniques were used in the rat: microdissection under a stereo-microscope from frozen brain sections of fragments of corpus callosum, optic chiasm and cerebral cortex; fresh tissue macrodissection of subcortical white matter, cerebral cortex and hypothalamus. Only macrodissection was used in the mice. The data show that, independently from the sampling technique used, there are considerable quantitative differences in the distribution pattern of the 5-alpha-reductase activity within different brain structures. Both in the rat and in the mouse, the enzyme appears to be present in higher concentrations in the white matter structures, than in the cerebral cortex and in the hypothalamus. The present results clearly show that the subcortical white matter and the corpus callosum are at least three times as potent as the cerebral cortex in converting testosterone into DHT. An even higher 5-alpha-reductase activity has been found in the optic chiasm. Further work is needed in order to understand the possible physiological role of DHT formation in the white matter structures.