Diurnal alterations of catecholamines, indoleamines and their metabolites in specific brain regions of the mouse

1. The diurnal variations of regional brain concentrations of dopamine (DA), norepinephrine (NE), serotonin (5-HT) and metabolites were determined in unperturbed male CD-1 mice. Determinations were made every 4 hr for 24 hr. 2. The most striking and significant variations in biogenic amines were seen in the hypothalamus, where concentrations of NE, DA and 5-HT varied in a rhythmic pattern and as much as two-fold during this period. 3. In some cases, daily alterations in parent biogenic amines were reflected by concurrent changes in their metabolites. 4. Since concentrations of neurotransmitters in the brain are often used as an indicator of stress and/or toxicity, these data should provide an accurate data base allowing for more accurate interpretation of results.     

THE STABILITY OF VITAMIN B6 ACCUMULATION AND PYRIDOXAL KINASE ACTIVITY IN RABBIT BRAIN AND CHOROID PLEXUS

The effects of changes in the concentrations of pyridoxal phosphate and blogenic amines in brain on: (I) pyridoxal kinase (EC 2.7.1.35) activity in brain and choroid plexus; and (2) vitamin B₆ accumulation by brain slices and isolated, intact choroid plexuses were studied. New Zealand white rabbits were treated parenterally with 200 mg/kg pyridoxine-HCl for 3 days or 120 mg/kg 4-deoxypyridoxine HCI or 5 mg/kg reserpine I day before death. After these treatments the mean concentration of pyridoxal phosphate in brain was elevated by 39% by pyridoxine and decreased by 57% by 4-deoxypyridoxine. Reserpine had no effect. However, the ability of brain slices and isolated, intact choroid plexuses from the treated rabbits to accumulate [³H] vitamin B₆ (with [³H]pyridoxine in the medium) was not different from untreated controls. Also, the specific activity of pyridoxal kinase in brain and choroid plexus of treated rabbits was not different from controls. These results show that vitamin B₆ accumulation and pyridoxal kinase activity in brain and choroid plexus are independent of both pyridoxal phosphate and reserpine-sensitive biogenic amine concentrations in brain. In vitro studies with pyridoxal kinase showed that. in both choroid plexus and brain. pyridoxal kinase was a single enzyme with a molecular weight of 43.000 and a K_m , for pyridoxine of 2.0 μM Crude and partially-purified pyridoxal kinase from brain was not inhibited by biogenic amines (1 mM) or pyridoxal phosphate (5 μM). These in vitro data are consistent with the lack of effect of changes in pyridoxal phosphate and biogenic amine concentrations (in brain) on pyridoxal kinase activity in brain in vivo.     

EFFECTS OF AMINES ON THE LEVEL OF N-ACETYL-ASPARTATE AND N-ACETYL- ASPARTYL-GLUTAMATE IN MOUSE BRAIN TISSUE SLICES

The effect of some biogenic amines and amino acids on the level of N-acetyl-asparticacid and N-acetyl-aspartyl-glutamic acid has been investigated in mouse brain tissue slices. The amines all caused a significant decrease in the levels of N-acetyl-aspartic acid and N-acetytl-aspartyl-glutamic acid within 5 min of incubation, while the amino acids, in spite of being possible transmitter candidates, had no such effect.     

Diurnal patterns in brain biogenic amines of rats exposed to 60-Hz electric fields

Levels of brain neurotransmitters and their metabolites, as well as concentrations of enzymes associated with their synthesis and metabolism, fluctuate during the day in patterns defined as circadian. The present study examined these rhythms in albino rats exposed to 60-Hz electric fields. Thirty-six animals were exposed to a 39 kV/m field for 4 weeks, 20 h/day, in a parallel-plate electrode system. A group of 36 sham animals was similarly handled and housed in a nonenergized exposure system. On the sampling day, animals were sacrificed at 4-h intervals throughout the 24-h day. Brains were removed, dissected, and kept frozen until chemically analyzed. The levels of biogenic amines and their acidic metabolites in the striatum, hypothalamus, and hippocampus were determined by high-performance liquid chromatography with electrochemical detection (HPLC-ECD) methods. Repeated exposure to 60-Hz electric fields produced significant alterations in the diurnal rhythms of several biogenic amines: dihydroxyphenylacetic acid (DOPAC, the primary metabolite of dopamine in the rat) in the striatum, and norepinephrine, dopamine, and 5-hydroxyindoleacetic acid (5-HIAA; serotonin metabolite) in the hypothalamus. Levels of serotonin in the striatum and hypothalamus showed clear circadian patterns that was not affected by the field. No diurnal or field-related changes were observed in the hippocampal amines.     

Tissue concentration changes of amino acids and biogenic amines in the central nervous system of mice with experimental autoimmune encephalomyelitis (EAE)

We have characterized the changes in tissue concentrations of amino acids and biogenic amines in the central nervous system (CNS) of mice with MOG_35-55-induced experimental autoimmune encephalomyelitis (EAE), an animal model commonly used to study multiple sclerosis (MS). High performance liquid chromatography was used to analyse tissue samples from five regions of the CNS at the onset, peak and chronic phase of MOG_35-55 EAE. Our analysis includes the evaluation of several newly examined amino acids including d-serine, and the inter-relations between the intraspinal concentration changes of different amino acids and biogenic amines during EAE. Our results confirm many of the findings from similar studies using different variants of the EAE model as well as those examining changes in amino acid and biogenic amine levels in the cerebrospinal fluid (CSF) of MS patients. However, several notable differences were observed between mice with MOG_35-55-induced EAE with findings from human studies and other EAE models. In addition, our analysis has identified strong correlations between different amino acids and biogenic amines that appear to change in two distinct groups during EAE. Group I analyte concentrations are increased at EAE onset and peak but then decrease in the chronic phase with a large degree of variability. Group II is composed of amino acids and biogenic amines that change in a progressive manner during EAE. The altered levels of these amino acids and biogenic amines in the disease may represent a critical pathway leading to neurodegenerative processes that are now recognized to occur in EAE and MS.     

Biogenic amines in the two-spotted spider mite

Whole body extracts of the two-spotted spider mite (Tetranychus urticae Koch) were analyzed using a 16-channel electrochemical array high performance liquid chromatography (HPLC)-based detection system that allows the simultaneous isolation and identification of a variety of biogenic amines. The spider mite extracts were found to contain the biogenic amines octopamine, dopamine, and 5-hydroxytryptamine (5-HT), as well as several precursors and metabolites including tyrosine, tyramine, tryptophan, and N-acetyl octopamine. Differences in the levels of biogenic amines were observed between eggs and the adult stages and between males and females. This is the first direct determination of biogenic amines in the Tetranychidae and the first demonstration of 5-HT in any mite species. © 1994 Wiley-Liss, Inc.     

The inverse relationship between the activity of pyridoxal kinase and the level of biogenic amines in rabbit brain

In a series of experiments, an inverse relationship was found to exist between the activity of pyridoxal kinase and the concentrations of brain norepinephrine, dopamine, and serotonin. When the biogenic amine content of the brain is lowered, the activity of pyridoxal kinase rises. Conversely, when the concentration of brain biogenic amines is elevated, a depression occurs in the activity of pyridoxal phosphokinase. This relationship, which resembles an in vivo allosteric inhibition phenomenon, not only is highly suggestive of a definite relation between the brain vitamin B₆ content and the metabolism of the biogenic amines, but also suggests that the concentration of brain biogenic amines might control the production of coenzyme pyridoxal phosphate.     

Analysis of endogenous pyrrolidine levels by mass fragmentography

Pyrrolidine, one of biogenic volatile amines, possesses nicotine-like synaptotropic actions on the nervous systems. In the present study, pyrrolidine levels in the tissues were examined by using mass fragmentographic technique. High concentrations of pyrrolidine were found in the seminal vesicle and lung of rabbits. Only trace amounts of pyrrolidine existed in the brain of mice and rats, although higher concentrations were detected in the brain of rabbits. In the rat brain, however, high levels of pyrrolidine were found in the pineal gland, pituitary gland and corpus striatum.     

PRODUCTS OF BIOGENIC AMINE METABOLISM IN THE LOBSTER: SULFATE CONJUGATES

Octopamine, dopamine and serotonin, the three biogenic amines found in the lobster nervous system, are each converted by lobster tissues into two principal classes of products, A and B metabolites. In this paper, evidence is presented that the B metabolites are sulfate conjugates of the amines and their A metabolites. Two double-labelled conjugates were formed from each of the three amines during incubations of lobster nerves with tritiated amine and ³⁵SO₄. When the two octopamine conjugates were hydrolyzed by mild acid, one of the conjugates was converted to a mixture of ³⁵SO₄ and [³H]-octopamine, and the other to a mixture of ³⁵SO₄, [³H]octopamine, and [³H]metabolite A. [³H]Metabolite A was also converted to octopamine by acid hydrolysis. The results indicated that one of the double-labelled conjugates was octopamine-sulfate, and the other metabolite-A-sulfate. An enzyme fraction prepared from nerve homogenates catalyzed the synthesis of double-labelled sulfate conjugates from the tritiated amines and [³⁵S]3′-phosphoadenosine-5′-phospho-sulfate. Double-labelled conjugates formed in this way contained 1 mol of sulfate per mol of amine. Indirect evidence suggested that the sulfate was in ester linkage with the ring hydroxyls of the amines. Neither monoamine oxidase, nor catechol-O-methyl transferase is found in lobster tissues; therefore, in these animals, sulfation may be a major means of inactivation of the biogenic amines following their release from nerve endings.     

METABOLIC CHANGES IN THE BRAINS OF MICE FROZEN IN LIQUID NITROGEN

Abstract— Autolytic changes in the mouse brain, occurring during immersion of the animal in liquid nitrogen, were evaluated by measuring the tissue concentrations of glucose, lactate, pyruvate, α-oxoglutarate, phosphocreatine, creatine, ATP, ADP and AMP. The values thus obtained were compared with those obtained in paralysed mice under nitrous oxide anaesthesia, the brains of which were frozen in such a way that arterial blood pressure and oxygénation were upheld during the freezing. Immersion of unanaesthetized mice in liquid nitrogen gave rise to significant alterations in phosphocreatine, creatine, lactate, lactate/pyruvate ratio, ADP and AMP. A comparison with values obtained in paralysed and anaesthetized mice that were frozen by immersion in liquid nitrogen showed that the metabolic changes observed in the unanaesthetized animals could not be caused by an anaesthetic effect on the metabolic pattern. It is concluded that autolysis in the mouse brain occurs during immersion of the animal in a coolant, mainly because arterial hypoxia develops before the tissue is frozen. A comparison with previous results on rat cerebral cortex indicates that mice offer no advantage for studies of cerebral metabolites in unanaesthetized animals. In both species, accurate analyses of labile cerebral metabolites require that the brain is frozen in a way that prevents arterial hypoxia during the fixation of the tissue.     

DEVELOPMENTAL AND REGIONAL VARIATIONS IN NEUROTRANSMITTER-SENSITIVE ADENYLATE CYCLASE SYSTEMS IN CELL-FREE PREPARATIONS FROM RAT BRAIN

Investigations have been carried out on regional and developmental variations in the properties of adenylate cyclase systems in participate preparations from rat brain. EGTA was routinely included in the assay medium to minimize differences in the state of activation of these systems resulting from variations in their exposure to endogenous Ca²⁺. At birth, adenylate cyclase activity was much higher in the hindbrain-medullary preparations than in comparable fractions from cerebellum, cerebral cortex or subcortex (including midbrain, corpus striatum, hypothalamus and hippocampus). Adenylate cyclase activity increased during early development in preparations from all areas of the brain. Maximal levels were reached at 14 days of age or later. These levels were not greatly altered in the young adult animal, except in the hindbrain-medullary area, where a decrease in activity was observed. Adenylate cyclase systems in cerebral cortical and subcortical preparations were activated by norepinephrine and dopamine throughout development. Serotonin also stimulated adenylate cyclase activity in these preparations from young animals but was much less effective in comparable fractions from adult rats. The response to dopamine was diminished with age in cerebral cortical preparations, but not in subcortical fractions. The responses to norepinephrine increased in both brain regions during early development. Adenylate cyclase systems in particulate preparations from the cerebellum and hindbrain-medullary areas exhibited relatively poor responses to the biogenic amines. Detailed studies of the properties of the cerebral cortical adenylate cyclase systems revealed enhancement of activity by Ca²⁺ and F^? at all stages of development with the maximal activation at 2–3 weeks of age. The results suggest that developmental differences in hormonal sensitivity of adenylate cyclase systems from diverse areas of the brain are related to changes in the proportions of the receptor-enzyme complexes responsive to the different biogenic amines.     

The fever reaction of the polecat Mustela putorius x Mustela putorius furo to a bacterial pyrogen: the hypo- and hyperthermic phases]

It has been demonstrated that the ferret (Mustela putorius x Mustela putorius furo) responds to intramuscular injection of Salmonella typhi lipopolysaccharide (30 ng/kg-100 micrograms/kg) by biphasic change in the body temperature (Tb): the initial decrease in the latter is followed by hyperthermia. Maximum rise in Tb (1.6 +/- 0.1 degrees C) was observed after the injection of lipopolysaccharide in the highest dose. Rabbit leucocytic pyrogen/interleukin-1 (1 ml from 3.5 x 10(7) peritoneal phagocytes, 3 ml/kg) induces a pronounced (1.1 +/- 0.3 degrees C) decrease in Tb. Mechanisms of hypothermic effects of pyrogens are discussed. The described pattern (hypothermia-hyperthermia) of Tb response to lipopolysaccharide in the ferret presumably reflects the central thermoregulatory process which is the same for different changes in Tb during fever.     

Mice Transgenic for Copper/Zinc Superoxide Dismutase Exhibit Increased Markers of Biogenic Amine Function

Abstract: Mice that are transgenic for and overexpress human copper/zinc superoxide dismutase were used to investigate the role of this enzyme in the pathophysiology of Down's syndrome (DS; trisomy 21). Previous studies have indicated that overexpression of copper/zinc superoxide dismutase leads to deficits in peripheral markers of neurochemical function, which are consistent with the hypothesis that this enzyme plays a role in the pathophysiology of DS. We have measured concentrations of amino acids and biogenic amines (catecholamines, serotonin, and their metabolites), uptake of biogenic amines into crude synaptosomes, and activities of synthetic enzymes in both control mice and mice transgenic for human copper/zinc superoxide dismutase that overexpress it by two- to fivefold above control values. We find that these transgenic mice exhibit higher concentrations of the biogenic amines in specific brain regions, with little or no change in amino acid concentration. Furthermore, tyrosine hydroxylase activity is increased in the striatum of the transgenics, whereas glutamic acid decarboxylase and choline acetyltransferase activities are unchanged in all but one brain region. These findings indicate that over-expression of copper/zinc superoxide dismutase, by itself, is not sufficient to cause the synaptic neurochemical deficits reported in DS.     

Changes in the balance of biogenic monoamines and their metabolites in the organs of rats with oxygen-induced epilepsy

The content of some biogenic monoamines and their metabolites in rat brain and heart in different periods of oxygen epilepsia was studied using high performance liquid chromatography with electrochemical detection. It was shown that already at the 5th minute of exposure to oxygen adrenaline, DOPA and some noradrenaline metabolites disappeared in the brain and noradrenaline level reduced. At this period in rat heart the reduction of catecholamine content was the most distinct and serotonin level was unchanged. At the beginning of convulsive period the modifications of biogenic amines content were nonparallel in brain regions: in the heart the reduction of catecholamine level went on, especially in right ventricle. In the terminal phase of oxygen epilepsia brain biogenic amines increased, however, not up to normal meaning, heart catecholamines at this period were at the same level as at the beginning of the convulsive period.     

Variations of biogenic amine levels in the brain of Pieris brassicae pupae during nondiapausing and diapausing development

The post-embryonic development of Pieris brassicae can either be continuous (under a long photoperiod) or interrupted at the pupal stage (induced by a short photoperiod); this phenomenon is termed facultative diapause. Several studies have indicated that certain brain mechanisms could be directly involved in the perception of variations in the photoperiod and could mediate some physiological effects particular to dormancy. Biogenic amines have been particularly implicated in the response to photoperiod variations and also in the regulation of development, especially in diapause induction and termination. High performance liquid chromatography with dual electrochemical detection has therefore been used to measure several biogenic amines in pupal nervous tissues at various stages of nondiapausing and diapausing development. During direct development, the levels of dopamine (DA) and N-acetyldopamine (NADA: a DA metabolite) in brain were relatively high in 3-day-old pupae and at the end of pupal life (on the 8th day). Dihydroxyphenylacetic acid (another metabolite of DA) showed no variation. Serotonin was mainly observed in 2–3-day-old pupae but 5-hydroxyindoleacetic acid was never detected. In young diapausing insects, similar variations of DA levels were observed even though a slight decrease of DA metabolites was noted. Serotonin appeared somewhat later (4–5 days) and attained higher levels. In late diapausing pupae, a marked increase in DA levels was observed, especially when pupae were kept at low temperature (4°C). During diapause, serotonin levels were reduced or even absent.     

BIOGENIC AMINES AND THEIR METABOLITES AS SUBSTRATES FOR PHENOL SULPHOTRANSFERASE (EC 2.8.2.1) OF BRAIN AND LIVER

Phenol sulphotransferase activity in homogenates of rat liver and brain was determined spectrophotometrically. Rat liver had about 100-fold more phenol sulphotransferase activity than brain; however, both tissues showed about the same spectrum of activity towards the phenolic compounds tested. Dopamine and its acidic and neutral metabolites and the neutral metabolites of norepinephrine were the compounds most readily sulphury-lated in vitro. They were also the compounds most readily sulphurylated in vivo when they were injected intraventricularly together with labelled Na₂SO₄. When labelled Na₂SO₄ was injected alone, we detected conjugation of endogenous phenols. One of the compounds formed was identified by its chromatographic characteristics as 3-methoxy-4-hydroxy-phenylethyleneglycol sulphate. We detected other conjugates which appeared to be the sulphate esters of 3,4-dihydroxyphenylethyleneglycol; 3,4-dihydroxyphenylacetic acid; and homovanillic acid. In brain, sulphate conjugation may be a major route of metabolism for many of the phenolic compounds related to the biogenic amines and possibly for phenolic drugs which enter the brain.     

Reverse-phase high-performance liquid chromatographic separation and electrochemical detection of norepinephrine,dopamine, serotonin,and related major metabolites

A convenient method for the determination of biogenic amines and their metabolites in small samples of brain tissue weighing from 0.5 to 5 mg, based on reverse-phase chromatography, is described. The biogenic amines, norepinephrine, dopamine, and serotonin, and the metabolites normetanephrine and 3-methoxytyramine were separated by ion-pair chromatography on a μBondapak phenyl column with an aqueous eluant, while the metabolites 3,4-dihydroxyphenylacetic acid, homovanillic acid, 3-methoxy-4-hydroxyphenylglycol, and 5-hydroxyindoleacetic acid were separated on a μBondapak C18 column with a methanol aqueous mobile phase. The sensitivity of the method is in the picogram range, from 20 to 100 pg, depending on the substances analyzed.     

Analysis of the biogenic amines in the central nervous system of the tobacco hornworm by high-performance liquid chromatography with 16-sensor electrochemical detection.

A method was developed to analyze biogenic amines in extracts of the central nervous system of the tobacco hornworm, Manduca sexta by high-performance liquid chromatography with 16-sensor electrochemical detection (n-EC-HPLC). The amines, precursors, and metabolites were separated in two dimensions. The first dimension involves separation based upon retention time by reversed-phase HPLC, while the second dimension involves separation based upon the characteristic oxidation potentials achieved by n-EC. Biogenic amine identification was based upon maximum oxidation potential and peak height ratios in addition to retention time. The improved resolving power of this method allows for a simplified sample preparation procedure and simultaneous determination of a wide range of compounds, including phenylethylamine, catecholamines, indoleamines, and some of their precursors and metabolites.     

常用大鼠发热模型研究

目的探讨干酵母、2,4-二硝基酚、脂多糖（LPS）、细菌内毒素引起SD大鼠实验性发热的过程和特点,比较不同浓度外致热原对大鼠发热过程的影响。方法建立大鼠干酵母（2 g/kg、1 g/kg）、2,4-二硝基酚（30mg/kg、15 mg/kg）、LPS（100μg/kg、20μg/kg）、细菌内毒素（120 EU/kg、60 EU/kg）发热模型,记录不同时间点大鼠升温值,绘制各模型平均升温曲线,比较不同大鼠发热模型的发热特点。结果皮下注射干酵母混悬液,注射后2～3 h开始升温,6～7 h达峰值,升温持续20 h;皮下注射2,4-二硝基酚溶液,注射后20 min开始升温,1～1.5 h达峰值,升温持续3～5 h;腹腔注射LPS、细菌内毒素,注射后30 min开始升温,此后升温曲线表现为双相热或三相热,升温持续5～8 h。结论不同外致热原所致SD大鼠发热的过程和特点不同;外致热原浓度不同,所致大鼠发热过程和特点不同。解热试验中,应根据受试药物本身特点选用合适的大鼠发热模型。     

Responses of baboons to traditionally pyrogenic agents

It is not clear whether baboons develop fever in response to endotoxin or other pyrogens. We injected various pyrogens intravenously in 12 unrestrained baboons (Papio ursinus) and measured their body temperature using intra-abdominal radiotelemeters. Serum iron concentration was also measured. The baboons developed fever after injection of killed Staphylococcus aureus (5 X 10(7) organisms/kg). No significant fever was measured after injection of lipopolysaccharide (Salmonella typhosa) (0.1, 8, 40, and 100 micrograms/kg), bovine serum albumin (4 mg/kg), killed Salmonella minnesota (5 X 10(7) organisms/kg), and killed Salmonella typhi (5 X 10(7) organisms/kg). A significant decrease in serum iron concentration was found only after injection of S. aureus and lipopolysaccharide, 100 micrograms/kg. The phagocytic synthesis of interleukin-1 following pyrogen stimulation in baboons and some other primates appears to differ from that in man and in nonprimates.