Microglial cells of the rat brain in postnatal period (comparative immunocytochemical analysis)

In the course of the brain’s development, distribution of microglial cells was studied in rats using immunocytochemical detection. To identify the microglial cells, antibodies to lipocortin 1 (LC1) and phosphotyrosine (PT) were used. On postnatal day 1, LC1-positive microglial cells of an ameboid shape were distributed mainly in the subventricular zone; their mean density was 31±8 cells/mm² (counted across the total area of frontal sections). On postnatal day 7, microglial cells of an intermediate type were located throughout the whole brain; their density was 54±15 cells/mm². On the 15th day, LC1-positive cells were of a ramified shape, and their density reached 104±20 cells/mm² (the microglial cell density in the mature normal brain was 103±3 cells/mm²). On postnatal day 7, PT-positive cells were similar in their morphology to LC1-positive cells of an intermediate type, while their mean density was 32 cells/mm². In the mature brain, the density of PT-positive microglia was 53±5 cells/mm²; the shape of the cells in the white and gray matter of the brain was, on the whole, similar to that of LC1-positive microglia. Therefore, LC1 is a specific marker for different types of microglial cells in the developing brain. Our data about 3D distribution and morphological peculiarities of microglial cells at different stages of postnatal development are consistent with the hypothesis on the neuroectodermal origin of microglia.     

Reaction of microglia and astrocytes in the rat brain cortex to free-electron laser irradiation

Reactions of microglia and astrocytes in the sensorimotor cortex of the rat resulting from a cortex tissue lesion made by a free-electron laser were studied with immunohistochemical techniques. Lipocortin-1 (LC1) was used as a microglia marker, while S100-β glycoprotein was used to identify astrocytes. Three days after laser exposure, the quantity of LC1-positive microglial cells observed in the cortex along the edge of the laser lesion was 30% larger than that in the control. There was no reaction of S100-β-positive astrocytes observed within this time interval. Six days after laser exposure, the density of LC1-positive activated microglia along the edge of the laser lesion further increased (210% of the above index), and the density of S100-β-positive astrocytes also slightly increased (by 30%, compared with the control). The data provide evidence that LC1-positive microglia react to a laser-made cortex injury more rapidly and intensively than astrocytes. It can be supposed that namely LC1 plays the role of an anti-inflammatory messenger in cortex microglial cells after laser exposure. In general, the pattern of microglia and astrocyte reactions is indicative of comparatively mild traumatization of the cortex tissue after laser irradiation.     

Assay of 2-Hydroxyputrescine in Various Regions of Rat Brain by Gas Chromatography-Mass Spectrometry

2-Hydroxyputrescine in seven regions of single rat brains was measured with a sensitive, specific assay by gas chromatography-mass spectrometry. The regions were the cerebral cortex, cerebellum, medulla oblongata, hypothalamus, striatum, hippocampus, and midbrain. The level of 2-hydroxyputrescine was very high in the cerebral cortex and cerebellum, high in the medulla oblongata, hypothalamus, and hippocampus, and low in the striatum and midbrain. The level of 2-hydroxyputrescine in the cerebellum was significantly higher than in the striatum and midbrain.     

Increased number of muscarinic binding sites in brain following chronic barbiturate treatment to rat

Rats received as their only drinking fluid a solution of sodium barbital (3.33 mg/ml) for more than 40 weeks. In two groups (A3, A12) the barbital solution was withheld and replaced by water 3 and 12 days before sacrifice. Two other groups consisted of animals drinking barbital until sacrifice (B) and untreated controls (C). Synaptosomes from different parts of the brain were incubated with radioactive quinuclidinyl benzilate (³H-QNB) (0.2 nM) for 60 min. A significantly increased number of ³H-QNB binding sites was found in the striatum and midbrain + medulla oblongata + cerebellum of rats abstinent for 3 days (A3) in comparison with controls (C). Saturation studies indicated that group A3 had significantly more receptors in the midbrain + medulla oblongata + cerebellum than group C, while there was no differences in receptor affinity.     

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.     

REGIONAL BIOSYNTHESIS OF ACETYLCHOLINE IN BRAIN FOLLOWING FORCED ORAL CHRONIC BARBITONE TREATMENT TO RAT

Rats received a solution of sodium barbitone as their only drinking fluid for 33 and 42–44 weeks. In three groups (A3, A12 and A30) the barbitone solution was withheld and replaced by water 3, 12 and 30 days respectively before death. Two other groups consisted of animals drinking barbitone until death (B) and untreated controls (C). Abstinence convulsions were recorded by jiggle cages. Thirty nmol of tritium-labelled choline ([³H]Ch) were injected i.v. and the rats were killed by decapitation 1 min later. A significantly higher content of tritium-labelled acetylcholine ([³H]ACh) was found in the cerebellum + medulla oblongata + midbrain of rats receiving barbitone until death (group B) (+22%) and abstinent for 3 days (+54%) (group A3) compared with group C. The [³H]ACh content was also significantly increased in the hippocampus + cortex of rats abstinent for 3 days (+23%). In the striatum no significant effect on [³H]ACh content was found in any of the groups. The ratio [³H]ACh/[³H]Ch was significantly increased in the cerebellum + medulla oblongata + midbrain of rats in group B and A3 and in the hippocampus + cortex in group A3. These results might indicate an increased turnover of ACh. The effect of long-term barbitone treatment on the enzyme activities of brain choline acetyltransferase and acetylcholinesterase was also studied but no significant effect was found.     

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.     

STEARYL COENZYME A DESATURASE ACTIVITIES IN RAT BRAIN MICROSOMES

—A quantitative assay for the rat brain microsomal stearyl CoA desaturase is described. The method was used to determine the developmental pattern and regional distribution of the desaturase in rat brain. Even though the specific activity of the enzyme decreased to one-third from 8 to 60 days of age, the total activity per brain remained the same. The regional distribution of the activity in adult brain was of the order midbrain > medulla oblongata > cerebral hemispheres > cerebellum, with midbrain activity being about twice as that of the cerebellum.     

Distribution of immunoreactive mesotocin and vasotocin in the brain and pituitary of chickens

The distribution of vasotocin and mesotocin in the pituitary and central nervous system in male chickens was determined using radioimmunoassays. Neither peptide was detected in the pineal. Mesotocin, but not vasotocin, was detected in the cerebellum. Both peptides were found in the septal area, archistriatum, paleostriatum, optic lobe, anterior, medial and posterior hypothalamus, midbrain, pons, medulla oblongata, and the anterior and posterior pituitary. Equal amounts of the 2 peptides were present in the septal area, archistriatum and anterior hypothalamus whereas vasotocin was more abundant (2- to 10-fold) in the paleostriatum, optic lobe, midbrain, and pituitary. The amount of mesotocin was about twice that of vasotocin in the medulla oblongata and the medial and posterior hypothalamus. The wide distribution of vasotocin and mesotocin in extrahypothalamic sites in the central nervous system suggests that the peptides may, as in mammals, have a role in a variety of autonomic and endocrine regulatory processes in chickens.     

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.     

The effects of Δ9-tetrahydrocannabinol Δ9-THC) on the regional concentrations of spermidine in the rat brain

The effects of intravenous administration of Δ⁹-tetrahydrocannabinol (Δ⁹-THC, 2 mg/kg, i.v.) on the regional brain spermidine concentrations of the rat were examined. Thirty minutes after vehicle treatment, the spermidine concentrations were: for the medulla oblongata/pons, 68.2 ± 7.7 μg/g; the hypothalamus, 67.7 ± 2.6 μg/g; the midbrain, 59.1 ± 4.4 μg/g; the cerebellum, 47.3 ± 5.9 μg/g and for the cortex, 13.8 ± 0.8 μg/g. Thirty minutes after Δ⁹-THC, these concentrations were reduced in the midbrain (47.0 ± 8.0% of control, P < 0.0001) and cortex (69.4 ± 7.4% of control, P < 0.009). The spermidine concentrations were not significantly altered in the medulla oblongata/pons (86.5 ± 13.3%, P > 0.36), hypothalamus (107.2 ± 11.8, P > 0.36) or cerebellum (89.0 ± 14.4%, P < 0.48). These results suggest that spermidine within the midbrain and cortex may be involved in the expression of some of the actions of Δ⁹-THC.     

Influence of Proline on Rat Brain Activities of Alanine Aminotransferase,Aspartate Aminotransferase and Acid Phosphatase

Hyperprolinemia type II (HPII) is an autosomal recessive disorder caused by the severe deficiency of enzyme ¹-pyrroline-5-carboxylic acid dehydrogenase leading to tissue accumulation of proline. Chronic administration of Pro led to significant reduction of cytosolic ALT activity of olfactory lobes (50.57%), cerebrum (40%) and medulla oblongata (13.71%) only. Whereas mitochondrial ALT activity was reduced significantly in, all brain regions such as olfactory lobes (73.23%), cerebrum (70.26%), cerebellum (65.39%) and medulla oblongata (65.18%). The effect of chronic Pro administration on cytosolic AST activity was also determined. The cytosolic AST activity from olfactory lobes, cerebrum and medulla oblongata reduced by 75.71, 67.53 and 76.13%, respectively while cytosolic AST activity from cerebellum increased by 28.05%. The mitochondrial AST activity lowered in olfactory lobes (by 72.45%), cerebrum (by 78%), cerebellum (by 49.56%) and medulla oblongata (by 69.30%). In vitro studies also showed increase in brain tissue proline and decrease in glutamate levels. In vitro studies indicated that proline has direct inhibitory effect on these enzymes and glutamate levels in brain tissue showed positive correlation with AST and ALT activities. Acid phosphatase (ACP) activity reduced significantly in olfactory lobes (40.33%) and cerebrum (20.82%) whereas it elevated in cerebellum (97.32%) and medulla oblongata (76.33%). The histological studies showed degenerative changes in brain. Following proline treatment, the animals became sluggish and showed low responses to tail pricks and lifting by tails and showed impaired balancing. These observations indicate influence of proline on AST, ALT and ACP activities of different brain regions leading to lesser synthesis of glutamate thereby causing neurological dysfunctions.     

Homocarnosinase activity in the rat brain areas kidneys under different states of hyperbarooxygenation

The homocarnosinase activity in different brain areas and kidneys of the normal rats and under different conditions of hyperbarooxygenation are determined. The highest activity of this enzyme is observed in cerebellum. The high homocarnosinase activity is typical of kidneys as well. The action of oxygen in a dose of 0.425 MPa for 60 min (in the absence of convulsions) increases the homocarnosinase activity in the cerebral hemispheres by 18.6%, in the midbrain by 18.6%, in midbrain and diencephalon--by 56.5%, and in the medulla oblongata--by 40.6%. The homocarnosinase activity in the cerebellum decreases by 16.7%, in kidneys--by 18.5%. At the convulsive stage of oxygen intoxication caused by the effect of 0.7 MPa dose of oxygen the homocarnosinase activity in cerebral hemispheres rises by 158.5%, in the midbrain and diencephalon--by 141.5%, in the medulla oblongata by--161.1%. Under the same conditions homocarnosinase activity in the cerebellum is unchanged as compared with the control.     

Homocarnosine content and homocarnosine-carnosine synthetase activity in brain areas of hyperoxic rats

The homocarnosine content and homocarnosine synthetase activity were studied in the brain of rats in normal state and under hyperoxia. The homocarnosine content is higher in phylogenetically old brain areas as compared with that in the cerebral hemispheres. Its nonuniform distribution in the brain is associated with different activity of homocarnosine-carnosine synthetase in the corresponding brain areas. At the preconvulsive stage of oxygen poisoning the homocarnosine content in all the brain areas does not change, the homocarnosine-carnosine synthetase activity is 32% lower. At the convulsive stage of hyperoxia the homocarnosine amount in the cerebral hemisphere decreases by 33%, in the midbrain and diencephalon -- by 70, in the medulla oblongata -- by 60, in the cerebellum -- by 58%. The decrease in the homocarnosine content correlates with that in the activity of homocarnosine-carnosine synthetase in the corresponding brain areas; in the cerebral hemispheres -- by 33%, in the midbrain and diencephalon -- by 50, in the medulla oblongata -- by 49, in the cerebellum -- by 40%.     

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.     

应激性高血压大鼠脑干及下丘脑-垂体-肾上腺轴中肾上腺髓质素及其受体mRNA表达的改变

采用逆转录-聚合酶链式反应检测了慢性足底电击结合噪声应激致高血压大鼠下丘脑、延髓、中脑、垂体和肾上腺等组织中编码肾上腺髓质素的肾上腺髓质素前肽原(preproadrenomedullin,ppADM)基因以及ADM的特异性受体组件降钙素受体样受体(calcitonin-receptor-like receptor,CRLR)和受体活性调节蛋白2和3(receptor-activty-modifying proteins,RAMP2和RAMP3)表达的变化.我们观察到:与对照组相比,以3-磷酸甘油醛脱氢酶作为内参照,15 d足底电击结合噪声应激引起下丘脑、垂体和肾上腺中ppADM mRNA表达上调,而在延髓和中脑表达明显下调(P＜0.01或P＜0.05);CRLR基因表达量正常时在下丘脑相对较高,应激15 d后CRLR表达在延髓、中脑和下丘脑下调(P＜0.01或P＜0.05),而在垂体和肾上腺的表达无明显变化;应激后RAMP2基因在延髓和下丘脑表达上调,而在肾上腺表达显著下调(P＜0.01),其他部位无明显变化;RAMP3基因在对照组大鼠的中脑和下丘脑表达较高,在应激性高血压大鼠的下丘脑和垂体表达上调(P＜0.01或P＜0.05),而在中脑和肾上腺表达下调(P＜0.05),在延髓中的表达变化无统计学差异.上述结果提示:慢性足底电击结合噪声应激引起明显的中枢和下丘脑-垂体-肾上腺轴ADM及其受体组件CRLR/RAMP2或CRLR/RAMP3基因的表达变化.但慢性应激后中枢源性ADM及其受体的表达变化对应激和血压的调节以及在应激致高血压中的确切作用及机制尚待进一步研究.     

应激性高血压犬鼠脑干及下丘脑-垂体-肾上腺轴中肾上腺髓质素及其受体mRNA表达的改变

采用逆转录- 聚合酶链式反应检测了慢性足底电击结合噪声应激致高血压大鼠下丘脑、延髓、中脑、垂体和肾上腺等组织中编码肾上腺髓质素的肾上腺髓质素前肽原(preproadrenomedullin, ppADM) 基因以及ADM 的特异性受体组件降钙素受体样受体(calcitonin-receptor-like receptor,CRLR)和受体活性调节蛋白2 和3(receptor-activity-modifying proteins, RAMP2 和RAMP3)表达的变化。我们观察到:与对照组相比,以 3- 磷酸甘油醛脱氢酶作为内参照,15 d 足底电击结合噪声应激引起下丘脑、垂体和肾上腺中ppADM mRNA表达上调,而在延髓和中脑表达明显下调(P<0.01 或 P<0.05); CRLR基因表达量正常时在下丘脑相对较高,应激15 d 后CRLR 表达在延髓、中脑和下丘脑下调(P<0.01 或 P<0.05), 而在垂体和肾上腺的表达无明显变化;应激后RAMP2 基因在延髓和下丘脑表达上调,而在肾上腺表达显著下调(P <0.01), 其他部位无明显变化;RAMP3 基因在对照组大鼠的中脑和下丘脑表达较高,在应激性高血压大鼠的下丘脑和垂体表达上调(P<0.01 或P<0.05), 而在中脑和肾上腺表达下调(P<0.05), 在延髓中的表达变化无统计学差异。上述结果提示:慢性足底电击结合噪声应激引起明显的中枢和下丘脑- 垂体-肾上腺轴ADM 及其受体组件CRLR/RAMP2 或CRLR/R     

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.     

Effect of thyroxine and thiourea on cholesterol total lipid and glycogen contents of brain of Singi fish (Heteropneustes fossilis bloch)

Three consecutive days injections of thyroxine of different doses (1, 2 and 4 μg/g of body weight) caused significant increase in cholesterol content of cerebrum of Singi fish at 25°C in comparison to the control. The cholesterol content of cerebellum, midbrain and medulla oblongata was enhanced significantly with higher doses of 2 and 4 μg of thyroxine per g of body weight. The lipid and glycogen contents of whole brain were also found to increase with different doses of thyroxine after three consecutive days injections. These cellular constituents decreased with hypothyroid condition induced by thiourea treatment.The results indicate the thyroid hormonal regulation of lipid and carbohydrate metabolism in brain of Singi fish.