Effect of adrenaline and triamcinolone on cytochrome oxidase activity in the brain and liver of young and adult rats.

The effect of adrenaline (0.15 i.p./kg b.w.) and of the synthetic glucocorticoid triamcinolone (40 mg i.p./kg b.w.) on cytochrome oxidase activity, the terminal enzyme of the cytochrome system, was studied in homogenates of the cerebral cortex, subcortical formations (including the basal ganglia, the thalamus and the hypothalamus), the medulla oblongata and the liver of 5-day-old and adult rats. Activity in the above mentioned homogenates was measured polarographically 15 and 30 min after administering adrenaline or 48 h after administering triamcinolone. Fifteen minutes after its injection, adrenaline caused a statistically significant drop in cytochrome oxidase activity in the cerebral cortex, subcortical formations and liver of 5-day-old rats. The decrease still persisted 30 min after administration of the hormone, but was intensified only in the liver. In adult rats, on the other hand, a significant increase in activity was observed in the cerebral cortex and liver after adrenaline. Triamcinolone had no effect on cytochrome oxidase activity in any of the given parts of the brain in either young or adult rats. It significantly stimulated cytochrome oxidase activity in the liver of 5-day-old rats, but severely inhibited it in the liver of adult rats.     

Different degrees of lipid peroxidation in the CNS of young and adult rats exposed to short-term hypobaric hypoxia.

The authors studied the effect of short-term (20 min) hypobaric hypoxia at simulated altitudes of 7000 and 9000 m on the peroxidation of lipids in the cerebral cortex, subcortical formations, medulla oblongata and cerebellum of the laboratory rat. In 5- and 21-day-old rats, increased lipoperoxidation was recorded in all the studied regions of the brain. Differences were observed in sensitivity to the degree of hypoxia. In 5-day-old rats the response to both exposures was the same, but in 21-day-old animals exposure at 7000 m stimulated peroxidation in the cerebral cortex only (at 9000 m in all the parts of the CNS examined). In 35-day-old and adult rats, changes in the malondialdehyde concentration were likewise found after exposure at 9000 m, but not in every compartment (in 35-day-old rats in the cerebral cortex and subcortical formations and in adult rats in the cerebral cortex). In young rats, 30 and 60 min after exposure to hypoxia the malondialdehyde concentration was still higher than in older animals.     

Effect of a short-term fasting and altitude hypoxia on the cytochrome oxidase activity of rat brain mitochondria

The effect of short-term fasting and thirst, prolonged fasting and hypoxic hypoxia upon the activity of cytochrome oxidase was studied in mitochondrial fractions obtained from the brain and the liver. The investigation was carried out in two groups of rats, 5 and 60 days old. a) The activity of cytochrome oxidase in mitochondria isolated from the brain cortex, subcortical regions and the medulla oblongata rises, while the changes in liver mitochondrial fractions are reverse. b) A significant increase of mitochondrial cytochrome oxidase was found in 5-day-old rats after both types of fasting and hypoxia in all regions of the brain, as well as in the liver. c) The cytochrome oxidase activity in brain and liver mitochondria of 60-day-old rats was not affected appreciably after 24 h nutritional deprivation, with the exception of a significant rise of activity in the medulla oblongata. Prolonged fasting and hypoxia again markedly increased the activity of this enzyme in all regions of the brain and in the liver.     

Developmental fatty acid changes in different parts of the rat brain

The proportion of 26 fatty acids (FA) in the lipids of the cerebral cortex, subcortical formations (the thalamus, hypothalamus and basal ganglia) and the medulla oblongata was studied in rats aged 5, 10, 14 and 90 days. Very marked developmental changes in the proportion of the various FA were demonstrated in the different parts of the brain. In the cerebral cortex the proportion of 17:1 rose by 285%, 18:3 n-3 by 1820% and 22:6 n-3 by 80%, while the proportion of 14:0, 16:0 and 16:1 fell significantly. In the tissue of subcortical formations we found an increase in the proportion of FA with 18 carbons (18:0 by 40%, 18:1 by 100%, 18:3 n-3 by over 5000%) and a decrease in the proportion of 14:0, 16:0, 16:1 and 20:4 n-6. The situation in medulla oblongata tissue was similar to the one in subcortical formations. On comparing the proportion of FA in individual parts of the CNS in the same age category, we found the smallest number of statistically significant differences in 5-day-old rats. In adult rats we found significant differences chiefly in the proportion of palmitic acid (16:0), oleic acid (18:1), linolenic acid (18:3 n-3) and acids with 20-22 carbons.     

Effect of altitude hypoxia on ATPase activity in the brain of rats of different ages.

In experiments on 5-day-old and adult rats of both sexes, the authors investigated Na+--K+-stimulated and Mg2+-dependent ATPase activity in the cerebral cortex, subcortical formations and the medulla oblongata. They studied the effect of 20 min acute altitude hypoxia corresponding to either 7,000 or 9,000 m, in a thermostable chamber (30 degrees C). ATPase activity was found to increase during ontogenesis -- this being the greatest in cortical tissue and the least in the medulla oblongata. Hypoxia corresponding to 7,000 m altitude significantly depressed total ATPase activity in 5-day-old rats, but significantly stimulated it in adult animals. Changes in Na+--K+-stimulated ATPase activity played the major role in these changes. Hypoxia corresponding to 9,000 m altitude likewise depressed total ATPase activity in 5-day-old rats and to practically the same extent as moderate hypoxia (7,000 m). In adult rats, marked hypoxia (9,000 m) significantly reduced only Mg2+-dependent ATPase activity. Mg2+ activity rose during ontogenesis to a lesser degree than Na+--K+-stimulated ATPase and the reciprocal ratio of these ATPase and the reciprocal ratio of these ATPase activities, in the given parts of the brain, fell progressively in adult animals to values close to 1.     

Arachidonic acid-induced inhibition of (Na+K+)-stimulated ATPase in the cerebral cortex and medulla oblongata of young and adult rats

The effect of arachidonic acid in 5.10(-4) and 5.10(-5) mol.l-1 concentration (as the Na salt, SIGMA) on ouabain-sensitive ATPase (E. C. 3.6.1.3) activity was studied in the cerebral cortex and medulla oblongata of 5-day-old and adult rats. In adult rats, arachidonic acid significantly inhibited ouabain-sensitive ATPase activity in both the cerebral cortex and the medulla oblongata. In 5-day-old rats, only the higher concentration (5.10(-4) mol.l-1) inhibited the enzyme statistically significantly; use of the lower concentration was not followed by any significant changes in Na+-K+-ATPase activity.     

Effect of vanadate on (Na+-K+)-stimulated ATPase activity in the brain of rats of different ages

Experiments were carried out on infant rats aged five days and on adult rats (of both sexes) to investigate vanadate inhibition of (Na+-K+)ATPase activity in various parts of the brain. Vanadate was administered in 10(-5), 10(-7), 10(-8), 10(-9) and 10(-10) mol/l concentration. The enzyme activity and the effect of vanadate were studied in the tissue of the cerebral cortex, subcortical formations and the medulla oblongata. It was demonstrated that an inhibitory effect of vanadate on ouabain-sensitive ATPase could be determined in the brain of very young rats, i.e. in the immature nervous tissue. It was further demonstrated that the inhibitory effect of vanadate (in low concentrations) was significantly more potent in the nervous tissue of adult rats than in the CNS tissue of 5-day-old animals. Lastly, attention is drawn to certain differences in the sensitivity of ouabain-sensitive ATPase to the action of vanadate indifferent parts of the CNS in both the given age groups.     

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.     

Glutamate decarboxylase activity and GABA binding in the brain synaptic membranes in rats with compensatory activation of hypothalamo-pituitary-adrenal system in stress

The reaction of the rat adrenal cortex and medulla to stress was absent under conditions of compensatory activations of function of hypothalamic-pituitary-adrenal system. At the same time, glutamate decarboxylase activity was increased in the hypothalamus, decreased in the hyppocampus, and remained unchanged in medulla oblongata and cortex of cerebral hemispheres of hemiadrenalectomized rats after stress as compared to rats without stress. On the contrary, the intensity of specific GABA binding by synaptic membranes of medulla oblongata of hemiadrenalectomized rats was increased in response to stress effect it was decreased in the cortex of cerebral hemispheres, and remained unchanged in the hyppocampus.     

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.     

Reduced adenosine deaminase activity in the CNS of spontaneously-hypertensive rats

The activity of adenosine deaminase (ADA) has been measured in the hypothalamus, pons medulla and cerebral cortex from 30-day-old and 100-day-old spontaneously-hypertensive rats (SHR) and age-matched WKY controls. At 100 days there was a significant reduction in ADA activity in the hypothalamus (18.0%), pons medulla (20.6%) and cerebral cortex (14.7%). In 30-day-old SHR animals (prior to the development of significant hypertension) no significant changes were seen in the cerebral cortex or pons medulla but there was a small but significant reduction in ADA activity in the hypothalamus (9.2%). There was no significant reduction in the ADA activity in heart or kidney. Extracts of 100-day-old pons medulla which had been briefly heated to destroy endogenous ADA activity did not differentially affect the activity of exogenous purified ADA.     

Regional Development of Glutamate Dehydrogenase in the at Brain

The development of glutamate dehydrogenase enzyme activity in rat brain regions has been followed from the late foetal stage to the adult and through to the aged (greater than 2 years) adult. In the adult brain the enzyme activity was greatest in the medulla oblongata and pons greater than midbrain = hypothalamus greater than cerebellum = striatum = cortex. In the aged adult brain, glutamate dehydrogenase activity was significantly lower in the medulla oblongata and pons when compared to the 90-day-old adult value, but not in other regions. The enzyme-specific activity of nonsynaptic (free) mitochondria purified from the medulla oblongata and pons of 90-day-old animals was about twice that of mitochondria purified from the striatum and the cortex. The specific activity of the enzyme in synaptic mitochondria purified from the above three brain regions, however, remained almost constant.     

Reduction in brain immunoreactive corticotropin-releasing factor (CRF) in spontaneously hypertensive rats

The brain CRF concentration of spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY) was examined by rat CRF radioimmunoassay. Anti-CRF serum was developed by immunizing rabbits with synthetic rat CRF. Synthetic rat CRF was also used as tracer and standard. The displacement of 125I-rat CRF by serially diluted extracts of male Wistar rats hypothalamus, thalamus, midbrain, pons, medulla oblongata, cerebral cortex, cerebellum and neurointermediate lobe was parallel to the displacement of synthetic rat CRF. In both WKY and SHR the highest levels of CRF immunoreactivity were shown by the hypothalamus and neuro-intermediate lobe, and considerable CRF immunoreactivity was also detected in other brain regions. The CRF immunoreactivity in the hypothalamus, neurointermediate lobe, midbrain, medulla oblongata and cerebral cortex was significantly reduced in SHR and it may suggest that CRF abnormality may be implicated in the reported abnormalities in the pituitary-adrenal axis, autonomic response and behavior of SHR.     

Kinetic properties of the 5 alpha-reductase of testosterone in the purified myelin, in the subcortical white matter and in the cerebral cortex of the male rat brain

The 5 alpha-reductase, the enzyme which converts testosterone into dihydrotestosterone (DHT), is present in several CNS structures of the rat. Recent reports from this laboratory indicate that the subcortical white matter and the myelin possess a 5 alpha-reductase activity several times higher than that present in the cerebral cortex. Moreover, previous ontogenetic observations indicate that in all cerebral tissues examined (including the myelin) the 5 alpha-reductase has a higher activity in immature animals. This study was performed in order to verify whether the differences in the 5 alpha-reductase activity on the various brain components might be due to the presence of different concentrations of the same enzyme or to different isoenzymes. To this purpose, the kinetic properties Km and Vmax were measured in the purified myelin as well as in homogenates of the subcortical white matter and of the cerebral cortex, obtained from the brain of adult (60-90-day-old), immature (23-day-old), and aged (greater than 20-month-old) male rats. The results indicate that the enzymes present in the myelin, in the subcortical white matter and in the cerebral cortex of adult male rats possess a very similar apparent Km (1.93 +/- 0.2, 2.72 +/- 0.73 and 3.83 +/- 0.49 microM respectively). On the contrary, the Vmax values obtained in the myelin (34.40 +/- 5.54), in the white matter (19.57 +/- 2.36) and in the cerebral cortex (6.47 +/- 1.03 ng/h/mg protein) of adult animals have been found to be consistently different. Very similar Km values were found in the myelin obtained from the brain of immature and very old rats (2.14 +/- 0.11 and 3.39 +/- 0.75 microM respectively). The Vmax measured in the myelin purified from the immature rat brain (62.25 +/- 4.52) showed a value which was much higher than that found in the myelin of adult animals (34.40 +/- 5.54); a Vmax (34.31 +/- 9.41) almost identical to that of adult animals was found in the myelin prepared from the brain of aged rats.     

Detection of bikunin mRNA in limited portions of rat brain.

Tissue distribution of bikunin mRNA, which encodes a Kunitz-type serine protease inhibitor of the inter-alpha-inhibitor family (IalphaI), was studied in rats and mice by the reverse-transcripsion polymerase chain reaction (RT-PCR). We found that the liver as well as other tissues, such as the kidney, testis and adrenal gland, expressed bikunin mRNA. Although signals of bikunin mRNA were faint in the whole brain of rats and mice, distinct signals were found in limited portions of rat brain, such as the hippocampus, cerebral cortex and pituitary, but undetectable in cerebellum, medulla oblongata, hypothalamus, striatum, midbrain and choroid plexus. In three distinct types of cells, such as neurons, astrocytes and meningeal cells, in primary cultures isolated from the cerebral cortex and meninges of 1-day-old newborn rats, only neurons positively expressed bikunin mRNA. These results suggest that, in addition to peripheral tissues, neurons in the hippocampus and cerebral cortex produce bikunin, suggesting a potential role of bikunin/IalphaI family in these brain regions.     

Glycine,Serine, and Leucine Metabolism in Different Regions of Rat Central Nervous System

We have investigated the glycine, serine and leucine metabolism in slices of various rat brain regions of 14-day-old or adult rats, using [1-¹⁴C]glycine, [2-¹⁴C]glycine, L-[3-¹⁴C]serine and L-[U-¹⁴C]leucine. We showed that the [1-¹⁴C]glycine oxidation to CO₂ in all regions studied occurs almost exclusively through its cleavage system (GCS) in brains of both 14-day-old and adults rats. In 14-day-old rats, the highest oxidation of [1-¹⁴C]glycine was in cerebellum and the lowest in medulla oblongata. In these animals, the L-[U-¹⁴C]leucine oxidation was lower than the [1-¹⁴C]glycine oxidation, except in medulla oblongata where both oxidations were the same. Serine was the amino acid that showed lowest oxidation to CO₂ in all structure studied. In adult rats brains, the highest oxidation of [1-¹⁴C]glycine was in cerebral cortex and the lowest in medulla oblongata. We have not seen difference in the lipid synthesis from both glycine labeled, neither in 14-day-old rats nor in adult ones, indicating that the lipids formed from glycine were not neutral. Lipid synthesis from serine was significantly high than lipid synthesis and from all other amino acids studied in all studied structures. Protein synthesis from L-[U-¹⁴C]leucine was significantly higher than that from glycine in all regions and ages studied.     

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.     

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.     

Aspartate Aminotransferase for Synthesis of Transmitter Glutamate in the Medulla Oblongata: Effect of Aminooxyacetic Acid and 2-Oxoglutarate

The effects of aminooxyacetic acid (AOAA), a transaminase inhibitor, and 2-oxoglutarate, a precursor to glutamate by the activity of aspartate aminotransferase (AAT), on slices of rat medulla oblongata, cerebellum, cerebral cortex, and hippocampus were studied. The slices were superfused and electrically stimulated. There was a Ca2+-dependent stimulus-evoked release of endogenous glutamate, gamma-aminobutyric acid (GABA), and beta-alanine in all regions examined. AOAA (10(-4) and 10(-3) M) decreased the release of glutamate in the medulla oblongata and cerebellum but not in the hippocampus. L-Canaline, a specific inhibitor of ornithine aminotransferase, did not affect the glutamate release in the medulla. 2-Oxoglutarate (10(-3) M) increased the release of glutamate in the medulla oblongata and cerebellum but not in the cerebral cortex and hippocampus. Treatment with AOAA (10(-4) M) almost abolished the activities of AAT in all regions studied. AOAA (10(-4) and 10(-3) M) increased the stimulus-evoked release of GABA in the cerebellum, cerebral cortex, and hippocampus, whereas the stimulus-evoked release of beta-alanine was decreased by this agent in all regions studied. These results suggest the participation of AAT in the synthesis of the transmitter glutamate in the medulla oblongata and cerebellum of the rat.     

Acetylcholinesterase activity in brain slices of rats subjected to cooling

The acetylcholinesterase (AChE) activity is studied in rat slices of the cerebral cortex, corpus striatum, hypothalamus and medulla oblongata of rats during hypothermia (20 degrees C) and also 1 and 7 days after the posthypothermal period. Cooling of animals down to 20 degrees C is accompanied by an increase in the AChE activity in the brain both under incubation temperature of 20 degrees and 37 degrees C. Under prolonged hypothermia the AChE activity in the investigated brain regions, except for corpus striatum, returns to the control level. By the 7th day of posthypothermal period the AChE activity in corpus striatum, hypothalamus and medulla oblongata does not restore completely. The most substantial changes in the AChE activity both under hypothermia and posthypothermal period occur in corpus striatum, which obviously reflects its complicated functional role.