SUSTAINED DRUG-INDUCED ELEVATION OF BRAIN GABA IN THE RAT1

Abstract— The contents of GABA, homocarnosine, and β-alanine can be raised in rat brain for long periods of time by the continued administration of phenelzine, aminooxyacetic acid (AOAA), or isonicotinic acid hydrazide (INH). These 3 compounds apparently act by preferential inhibition of the enzyme GABA aminotransferase (GABA-T). Oral administration of phenelzine (20 mg/kg per day) caused a 25–50 per cent increase in GABA levels in rat brain, but produced appreciable toxic side effects. A similar increase in GABA levels in brain resulted from oral administration to rats of INH in a dosage of 60 mg/kg per day, without production of any obvious toxic effects. Simultaneous administration of large doses of pyridoxine did not abolish the GABA-elevating effect of INH. Brain GABA levels in the rat were increased by approx. 50 per cent by daily injections of AOAA (2.5 mg/kg per day). At this low dosage, AOAA injections in rats could be continued for at least 6 weeks without producing evident toxic effects. Oral administration of large amounts of GABA, on the other hand, failed to increase the content of GABA in the brains of rats not treated with GABA-T inhibitors, and failed to produce any further increase of brain GABA levels in rats treated with AOAA.     

微波辐射对小鼠脑内乙酰胆碱含量及胆碱乙酸转移酶活性的影响

用频率为2450MHz功率密度为10mW/Cm~2(WBASAR约11.4W/kg)的微波(连续波)对置于微波暗室内的昆明种雄性小鼠急性全身照射1小时后,立即按常规方法断头,取脑,制成样品,然后用放射免疫测定法测量小鼠脑内乙酰胆碱(ACh)含量及胆碱乙酰转移酶(ChAT)活性。结果表明:照射组的ACh含量为11.6±1.4pmol/mg(脑鲜重),ChAT活性为45.4±8.7pmolACh/min.mg(脑鲜重);而对照组的分别为16.0±2.1pmol/mg和61.0±13.8pmolACh/min.mg。证明微波照射后可引起动物脑内ACh水平和ChAT活性下降,提示微波辐射对中枢胆碱能系统确有不利影响。     

HISTAMINE AND MAST CELLS IN DEVELOPING RAT BRAIN

The number and distribution of mast cells in rat brain were determined at different postnatal ages. The number of brain mast cells was found to change during ontogenic development following the same pattern as brain histamine (HA) levels. The calculated HA content of brain mast cells was close to the HA content of the crude nuclear fraction at every age studied. Since most of the brain HA in the newborn sediments with the crude nuclear fraction, these results suggest that the developmental pattern of brain HA reflects changes in the number of brain mast cells, that is, in the size of the mast cell HA pool. The HA content of the supernatant of the crude nuclear fraction corrected for mast cell HA contamination, on the other hand, follows a developmental pattern similar to that of other known neurotransmitters.     

微波辐射对小鼠脑内乙酰胆碱含量及胆碱乙酸转移酶活性的影响

用频率为2450MHz功率密度为10mW/Cm~2(WBASAR约11.4W/kg)的微波(连续波)对置于微波暗室内的昆明种雄性小鼠急性全身照射1小时后,立即按常规方法断头,取脑,制成样品,然后用放射免疫测定法测量小鼠脑内乙酰胆碱(ACh)含量及胆碱乙酰转移酶(ChAT)活性。结果表明:照射组的ACh含量为11.6±1.4pmol/mg(脑鲜重),ChAT活性为45.4±8.7pmolACh/min.mg(脑鲜重);而对照组的分别为16.0±2.1pmol/mg和61.0±13.8pmolACh/min.mg。证明微波照射后可引起动物脑内ACh水平和ChAT活性下降,提示微波辐射对中枢胆碱能系统确有不利影响。     

ELEVATED CONCENTRATIONS OF HISTAMINE IN THE RAT BRAIN

—Whole brain concentrations of histamine (Hm) in the rat were measured with a sensitive and specific single-isotope enzymatic microassay and found to be elevated about 2-fold in rats killed by focused microwave irradiation compared with rats killed by decapitation. However, regardless of the method of killing, whole brain Hm concentrations were elevated about 4-fold when the brains were homogenized in 12.5 vol vs 2.5 vol of water or phosphate buffer. Homogenization of brain regions in 12.5 vol of water resulted in Hm concentrations that were about 10 times greater than previously reported values (except in the hypothalamus). Since (1) the‘extra’Hm could be extracted from pellets of low volume homogenates, and (2) synthesis of Hm is not likely to occur through an increase in the volume of water or buffer used for homogenization, we suggest that the elevated concentrations of Hm seen after high volume homogenization of brain tissue are due to a more complete extraction of Hm from brain tissue, and not to an artifactual production of this amine from its precursor (histidine) or some other compound(s).     

REGIONAL γ-AMINOBUTYRIC ACID LEVELS IN RAT BRAIN DETERMINED AFTER MICROWAVE FIXATION

—GABA levels in rat whole brain were compared following three methods of sacrifice: rapid microwave fixation, decapitation into liquid nitrogen, and decapitation at 20°C. Levels were shown to be identical in animals sacrificed by microwave fixation and decapitation into liquid nitrogen. In contrast, rats decapitated at 20°C had 18 per cent higher GABA levels when determined immediately post-mortem and 48 per cent higher levels after 30 min at 20°C. Microwave treatment prevented these post-mortem increases. The increase in GABA after decapitation at 20°C was even greater in hypothalamus than in whole brain. A comparison of 3 GABA extraction methods following microwave fixation demonstrated that sodium acetate was 88 per cent as effective as 80 per cent ethanol and more effective than 0·5 n -perchloric acid in extracting GABA. Fifteen brain regions were dissected from microwave-treated brains and the GABA levels determined.     

THE GLUTAMATE AND GLUTAMINE CONTENT OF RAT BRAIN AFTER PORTOCAVAL ANASTOMOSIS

Abstract— Rats have been subjected to portocaval anastomosis and the ammonium ion in plasma and the glutamate and glutamine levels in plasma, red cells and brain have been estimated up to 6 weeks after operation. The glutamine, but not the glutamate, levels in brain were consistently raised, being about 2.5 times greater than normal and the level can be correlated with the level of plasma ammonium ion. Consideration is given to the possibility that the glutamine may be in the greatly enlarged neuroglial compartment in this abnormal metabolic state.     

FORMATION OF γ-GLUTAMYLHISTAMINE FROM HISTAMINE IN RAT BRAIN

Abstract– γ-Glutamyl amides of histamine, serotonin and dopamine were formed from these amines by the transfer of the γ-glutamyl moiety from γ-glutamyl peptides in the presence of γ-glutamyl transpeptidase. [^I4C]Histamine was injected intraventricular into rats, and the formation of γ-glutamyl-[¹⁴C] histamine in the brain was confirmed by purification and identification with the authentic compound. The radioactivity was highest 30 min after the injection. The possible significance of γ-glutamyl amides in nerve transmission is discussed.     

METABOLISM OF MALONIC ACID IN RAT BRAIN AFTER INTRACEREBRAL INJECTION

Labeled malonic acid ([1-¹⁴C] and [2-¹⁴C]) was injected into the left cerebral hemisphere of anesthetized adult rats in order to determine the metabolic fate of this dicarboxylic acid in central nervous tissue. The animals were allowed to survive for 2, 5, 10. 15 or 30min. Blood was sampled from the torcular during the experimental period and labeled metabolites were extracted from the brain after intracardiac perfusion. There was a very rapid efflux of unreacted malonate in the cerebral venous blood. Labeled CO₂ was recovered from the venous blood and the respired air after the injection of [1-¹⁴C]malonate but not after [2-¹⁴C]malonate. The tissue extracts prepared from the brain showed only minimal labeling of fatty acids and sterols. Much higher radioactivity was present in glutamate, glutamine, aspartate, and GABA. The relative specific activities (RSA) of glutamine never rose above 1.00. Aspartate was labeled very rapidly and revealed evidence of ¹⁴CO₂ fixation in addition to labeling through the Krebs cycle. GABA revealed higher RSA after [1-¹⁴C]malonate than after [2-¹⁴C]malonate. Sequential degradations of glutamate and aspartate proved that labeling of these amino acids occurred from [1-¹⁴C] acetyl-CoA and [2-¹⁴C] acetyl-CoA, respectively, via the Krebs cycle. Malonate activation and malonyl-CoA decarboxylation in vivo were similar to experiments with isolated mitochondria. However, labeled malonate was not incorporated into the amino acids of free mitochondria. The results were compared to data obtained after intracerebral injection of [1-¹⁴C]acetate and [2-¹⁴C]acetate.     

QUANTITATIVE CHANGES WITH AGE IN THE DNA CONTENT OF METHYLAZOXYMETHANOL-INDUCED MICROENCEPHALIC RAT BRAIN1

Abstract— DNA synthesis in methylazoxymethanol (MAM)-treated foetal brain was reduced during the first 3 days after the injection of the compound into the mother rat. The MAM-treated brain grew at almost the normal rate after this period, but the reduction in DNA persisted through maturity of the animal. This difference in DNA content between normal and microencephalic brain was restricted to the cerebral hemispheres. The major increase in DNA content of prenatal brain occurred in the cerebrum, whereas the postnatal increase took place in the cerebellum. jH-Labelled MAM was incorporated more extensively into foetal brain DNA than into RNA. The half-life of the MAM-modified nucleic acids was 4–5 days. We suggest that removal of necrotic cells from the brain may account for the rapid loss of label from nucleic acids.     

CHOLINE ACETYLTRANSFERASE CONTENT IN DISCRETE REGIONS OF THE RAT BRAIN STEM

—Choline acetyltransferase (ChAc) content of 50 separate rat brain stem nuclei and cerebellum removed by microdissection was determined using a sensitive radiometric assay. The distribution of ChAc activity is uneven, with extremely high levels in the cranial motor nuclei and the nucleus salivatorius. Low ChAc concentrations were observed in the cranial sensory nuclei, the nuclei of the reticular formation, the raphe nuclei and the nuclei of the acoustic system. The lowest ChAc levels were measured in the cerebellum. Comparison of the distribution of ChAc with histochemical localization of acetylcholinesterase revealed generally good agreement, and notable exceptions are discussed.     

THE LEVELS OF LABILE INTERMEDIARY METABOLITES IN MOUSE BRAIN FOLLOWING RAPID TISSUE FIXATION WITH MICROWAVE IRRADIATION1

The levels of several labile glycolytic and organic phosphate metabolites in mouse brain were determined following rapid inactivation with 2450 MHz microwave irradiation. The levels of ATP in mouse brain following a 0·25 s exposure in a 6 kW microwave oven was found to be 2·416 ± 0·061. Whole brain levels of 8 labile intermediary metabolites in 0·4 s irradiated samples were comparable to those reported using the previously-described methods of freeze-blowing or whole-animal immersion. Analysis of these same metabolites in 4 gross areas of brain did not reveal any anoxic changes betwen superficial and deeper brain areas. The advantages of the mcrowave irradiation inactivation technique for regional brain studies of labile intermediary metabolites is discussed.     

PROTEIN SYNTHESIS IN VITRO IN RAT BRAIN AFTER SHORT-TERM PROTEIN STARVATION AND REFEEDING

Rats were fed a protein-free diet for 4 or 6 days. They were compared with rats kept on the same diet for 3 or 5 days and on adequate protein for one additional day. The incorporation of ¹⁴C-labelled amino acid into protein was studied in systems containing ATP, GTP, phosphoenolpyruvate, pyruvate kinase and if required, a mixture of unlabelled amino acids and either the 6000 g supernatant fraction of a brain homogenate or microsomes and soluble enzymes. The 6000 g supernatant fraction showed variation in amino acid incorporating activity as well as in RNase activity as measured by breakdown of labelled polyuridylic acid. There was no difference in RNase activity in isolated microsomes, but the amino acid incorporating activity was significantly higher in preparations obtained from rats fed one meal of protein after 5 days of protein-starvation.     

电磁脉冲辐照大鼠海马区细胞凋亡与形态学变化

以体外原代培养的大鼠海马神经元和Wistar大鼠为研究对象,探讨电磁脉冲(场强为6× 104 V/m)辐照后早期海马区细胞凋亡和病理形态学的变化.在照射后1h、6h、12h、24h和48h分别采用MTT法和流式细胞仪测定死亡细胞和凋亡细胞的比例,用光镜和电镜分别进行形态学观察.结果显示在电磁脉冲辐照后,海马神经细胞不仅发生快速的坏死,而且还发生凋亡,同时在早期即可见到血管、胶质细胞和神经元等组织的形态学异常.表明大鼠大脑受电磁脉冲辐照后早期海马区可发生神经细胞坏死和凋亡,以及各组织成分的病理形态学改变,上述变化可能与电磁脉冲致细胞DNA损伤有关.     

ALTERATIONS IN GABA METABOLISM AND MET-ENKEPHALIN CONTENT IN RAT BRAIN FOLLOWING REPEATED ELECTROCONVULSIVE SHOCKS

The effects of electroconvulsive shock (ECS; 120 V for 1 s through ear-clip electrodes) or sub-convulsive shocks (70 V for 1 s) on rat brain GABA and met-enkephalin concentration and GABA turnover has been examined 24 h after a single treatment (×1) or once daily for 10 days (×10). ECS × 10 increased GABA concentrations in the N. caudatus and N. accumbens and decreased the synthesis rate of GABA by 40% and 50% respectively in these regions. Sub-convulsive shocks (× 10 × 10) or ECS × 1 had no effect. No consistent changes were seen in the substantia nigra. Met-enkephalin concentrations increased by 50% in the N. caudatus after ECS × 10 but were unchanged in the cortex and pons/medulla. No other shock regimen had any effect on the concentration of this peptide. The results are discussed in relation to the enhanced monoamine-induced responses seen only after ECS × 10.     

FORMAMIDASE IN RAT BRAIN

Kynurenine formamidase (aryl-formylamine amidohydrolase, EC 3.5.1.9) was found to be present in rat brain and was partially purified and characterized. The partially purified enzyme catalysed the hydrolysis of 5-hydroxyformyl-dl -kynurenine to 5-hydroxy-dl -kynurenine and that of formyl-l -kynurenine to l -kynurenine at similar rates. The apparent K_m values of the enzyme for 5-hydroxyformyl-dl -kynurenine and formyl-l -kynurenine were 4.0 ± 10^?4 and 1.8 ± 10^?4m , respectively. The enzyme was active over a wide pH range (5.5–8.5). The activity was inhibited by low concentrations of Ag⁺ and Hg²⁺. The physiological significance of the enzyme is discussed.     

REGIONAL CHANGES IN BRAIN CATECHOLAMINES AFTER PROTON IRRADIATION OF THE STRIATE CORTEX IN THE SQUIRREL MONKEY

To establish possible functional relationships between anatomically distinct cortical centres in primates, we compared changes in visual acuity, nucleic acids, protein, enzymes and catecholamines in cortical and subcortical areas six months after 10,000 and 20,000 rd of proton irradiation of striate area 17 of the visual cortex of the squirrel monkey. Minimum separable acuity was reduced from 1 minute of arc in controls to 4 minutes in the 20,000-rd group. DNA, RNA and protein contents were not altered, but the activity of acetylcholinesterase was increased significantly in area 17. A decrease of norepinephrine occurred in the hypothalamus, hippocampus, caudate nucleus, putamen and brain stem of the irradiated brains. Since no ultrastructural basis has been found to account for these changes, we assume that a sustained chemical change occurred at the irradiated site and that the effect was transmitted to non-irradiated regions of the brain.     

射线照射后大鼠骨髓、十二指肠、小脑微血管通透性的改变

本文观察了r线全身照射后,大鼠骨髓、十二指肠和小脑微血管通透性的改变。结果表明:正常时骨髓微血管通透性最高,十二指肠次之,小脑最低。照后骨髓微血管通透亢进发生早、增加快、变化大、恢复慢,主要发生在血窦;照后十二指肠微血管通透亢进发生较早、增加较快、变化较大、恢复较快,主要发生在肠绒毛的毛细血管和细静脉;照后小脑微血管通透性变化较小,在400.0Gy组可见通透亢进,主要发生在小脑皮质的毛细血管。所有这三个脏器微血管通透亢进部位,实质细胞的退变明显加重。说明微血管邇透亢进可明显加重实质细胞的损伤,而实质细胞的再生修复,就发生在微血管通透恢复,基底背景较清净地方。说明:照后骨髓、十二指肠、小脑微血管通透性的改变,在造血型、胃肠型和脑型急性放射病的发病机理上有重要意义。     

EARLY CHANGES IN ACETYLCHOLINE POOLS IN THE HIPPOCAMPUS OF THE RAT BRAIN AFTER SEPTAL LESIONS

—After placement of lesions in the hippocampus by electrocoagulation, an increase in bound acetylcholine above control levels was shown. There was no concomitant rise in free acetylcholine. The rise in bound acetylcholine was not due to changes in levels of either acetylcholinesterase or choline acetyltransferase and it is suggested that the interruption of septal impulses and/or alterations in uptake or availability of substrates is responsible.