Differential cellular distribution of two sulphur-containing amino acids in rat cerebellum

Summary An antiserum to homocysteic acid was raised in rabbits. Immunogens were prepared by coupling this amino acid to bovine serum albumin by means of glutaraldehyde and paraformaldehyde. When applied to semithin or ultrathin sections of rat cerebellum, the antiserum produced selective labelling of glial cells and processes, including the Bergmann fibers. No enrichment of immunoreactivity was detected in nerve terminals of the major excitatory fiber systems. The distribution of homocysteic acid-like immunoreactivity was very different from that of taurine (another sulphur-containing amino acid), as judged from consecutive semithin sections labelled with a postembedding immunoperoxidase procedure and from ultrathin sections labelled with a postembedding double immunogold procedure. Taurine-like immunoreactivity was concentrated in Purkinje cells and was low in glial elements. Our data suggest that the cerebellum contains a glial pool of homocysteic acid (and/or precursors that may undergo spontaneous oxidation to homocysteic acid) and that this amino acid is unlikely to act as a cerebellar transmitter.     

Differential sensitivity of selected brain areas to excitatory amino acids

The 22Na+ efflux stimulated by selected agonists of the 4 excitatory amino acid receptors, previously detected in the striatum, has been studied on 22Na+-preloaded slices prepared from 10 major areas of the rat brain. All brain areas were found to be sensitive, albeit to varying extents, to excitatory amino acids. The cerebellum was exceptional in its high sensitivity to kainate and quisqualate and in the absence of effect of N-methyl-D-aspartate. These results support the suggestion that excitatory amino acids interact with heteregenous receptors which differ from each other not only in their pharmacological properties but also in their regional distribution.     

Age-related alteration in excitatory amino acid neurotransmission in rat brain

The excitatory amino acids as neurotransmitters in the neocortex, hippocampus, striatum, thalamus, amygdala, nucleus basalis of Meynert and cerebellum from rats aged 4 months, 12 months and 24 months have been examined by measuring sodium-dependent high affinity uptake of D-[3H]-aspartate into preparations containing synaptosomes. Calcium-dependent K(+)-stimulated release of endogenous glutamate from the nucleus basalis was also measured. The hippocampus and cerebellum failed to show significant age-related changes in uptake of D-[3H]-aspartate. D-[3H]-aspartate uptake decreased significantly in the neocortex (29%), striatum (29%), nucleus basalis (26%), amygdala (19%) and thalamus (16%) in the middle-aged rats as compared to the young rats, but the changes were not progressive with age. The release of glutamate from the nucleus basalis was unaltered during the aging process.     

Receptors for excitatory amino acids on neurons in rat pyriform cortex

The actions of a variety of agonists and antagonists of the excitatory amino acids on rat pyriform cortex pyramidal neurons were studied in a submerged, perfused brain slice. The order of potency for the agonists, applied by ionophoresis, was kainate greater than quisqualate greater than N-methyl-D-aspartate greater than aspartate = glutamate. The endogenous monosynaptic excitation of pyramidal neurons upon stimulation of the lateral olfactory tract was blocked post-synaptically by DL-2-amino-4-phosphonobutyric acid, although this drug did not consistently block any of the exogenous responses. The synaptic excitation was not blocked, however, by antagonists presumed specific for the quisqualate (glutamate diethyl ester), kainate, (gamma-D-glutamylglycine), or N-methyl-D-aspartate (DL-2-amino-5-phosphonovaleric acid, DL-2-amino-7-phosphonohetaonic acid) receptors. Several antagonists blocked N-methyl-D-aspartate responses at lower concentrations than those to aspartate, and other antagonists distinguished between kainate and quisqualate responses. These results suggest that 1) pyriform neurons have a variety of receptors that have properties somewhat different from those found in other preparations and 2) the endogenous transmitter activates a receptor distinct from those activated by kainate, quisqualate, and N-methyl-D-aspartate.     

含硫氨基酸对糖尿病大鼠窦房结损伤的保护作用

目的　探讨补充半胱氨酸或牛磺酸对糖尿病大鼠窦房结损伤的作用。 方法　SD大鼠随机分为正常对照组（C）组、糖尿病（DM）组、糖尿病+半胱氨酸（DM+Cys）组和糖尿病+牛磺酸（DM+Tau）组。检测SOD和MDA含量;测定窦房结功能SNRT、CSNRT、SACT和IHR;检测窦房结SOD2、HO-1、NOX2、NOX4、HCN2、HCN4表达。 结果　与C组比较,DM组血SOD降低,MDA升高;SNRT、CSNRT和SACT延长,IHR减慢;窦房结SOD2、HO-1、HCN2和HCN4表达降低,NOX2和NOX4表达升高（P<0.05）。与DM组比较,DM+Cys和DM+Tau组血SOD升高;IHR增加;窦房结SOD2、HO-1、HCN2和HCN4表达升高,NOX2和NOX4表达降低（P<0.05）。 结论　补充Cys或Tau能降低STZ大鼠窦房结氧化应激损伤,增加HCN4和HCN2表达,改善窦房结功能障碍。     

Developmental assembly of calcium-mobilizing systems for excitatory amino acids in rat cerebellum.

The postnatal development of calcium-mobilizing systems was studied by both microfluorometric imaging analysis of Ca2+ on living rat cerebellar slices and immunohistochemical labeling of phosphatidylinositol 4,5-bisphosphate (PIP2) and inositol 1,4,5-trisphosphate binding protein (IP3BP) in fixed rat cerebellum. Stimulation with quisqualate (QA) or N-methyl-D-aspartate (NMDA) enhanced the Ca2+ level only diffusely on postnatal day (PND) 3, but more discretely on PNDs 7 and 15. On PND 21, QA-induced responses were localized in the molecular layer especially, but not in the granular layer. By contrast, NMDA mobilized Ca2+ prominently in the granular layer, but only weakly in the molecular layer. Localized expression of PIP2 in the molecular layer paralleled QA-induced Ca2+ mobilization, but IP3BP was expressed more diffusely. The present study offers the first direct evidence that PIP2, but not IP3BP, is essential for QA-induced Ca2+ mobilization in the cerebellar cortex.     

Delayed neurotoxicity of excitatory amino acids in vitro

The acute neurotoxicity produced by glutamate and related excitatory amino acids is probably caused by depolarization leading to excessive anionic and cationic fluxes and osmotic lysis. Recently, a more delayed form of glutamate neurotoxicity, which is critically dependent upon calcium influx, has been described in cultured neocortex. We investigated this phenomenon in cultures of dispersed rat hippocampal neurons. When these cultures were briefly incubated with various excitatory amino acids in low extracellular chloride, there was no acute toxicity, but a gradual drop-out of neurons occurred over the next day. When calcium was removed from the extracellular medium during amino acid incubation, this late neuronal loss was not seen. Interestingly, blocking excitatory amino acid receptors in cultures after the amino acid exposure also prevented this delayed neuronal death. In addition, these treated cultures contained neurons with normal physiological properties, and had concentrations of adenosine triphosphate that were close to control values. The findings suggest an amino acid-induced calcium influx may elevate the release of endogenous excitatory transmitter, likely glutamate, and/or increase the sensitivity of these neurons to glutamate. These in vitro observations may partially explain the delayed neuronal loss seen in some pathological conditions affecting man.     

Sensitivity of rubrospinal neurons to excitatory amino acids in the rat red nucleus in vivo.

Responses of rubrospinal neurons (RSNs) to iontophoretic applications of L-glutamate (L-Glu), L-aspartate (L-Asp), quisqualate (Quis) and N-methyl-D-aspartate (NMDA) have been studied in the rat red nucleus (RN) in vivo. All agonists produced a dose-dependent increase of the firing rate and Quis was found to be the most efficient. The responses to NMDA and to a lesser extent to L-Asp were abolished by steady application of 2-amino-5-phosphonovalerate (2APV) whereas responses to Quis were unaffected and those to L-Glu poorly antagonized. On the other hand, NMDA-mediated excitations were insensitive to steady application of 6,7-dinitroquinoxaline-2,3-dione (DNQX) which abolished responses to Quis and to a lesser extent to L-Glu while those to L-Asp were less affected. These results show the presence of both NMDA and non-NMDA receptors on RSNs in the rat. A specific localization of the NMDA receptors on distal dendrites of these neurons is suggested.     

兴奋性氨基酸抑制胎鼠神经干细胞的增殖

目的：研究兴奋性氨基酸对体外培养胎鼠神经干细胞（NSCs）增殖的影响。 方法:体外分离、培养与鉴定SD胚胎大鼠脑室下带(SVZ)神经干细胞，利用MTT比色法测定兴奋性氨基酸N-甲基-D-天冬氨酸（NMDA）和谷氨酸（Glu）对细胞增殖的作用。 结果:成功分离出具有胚胎源性的神经干细胞，NMDA和Glu均能导致神经干细胞的增殖活性下降。 结论:培养的SVZ区域细胞具有自我更新和多分化潜能，是中枢神经系统干细胞，兴奋性氨基酸能有效地抑制神经干细胞的增殖。     

Effect of aromatic acids on the influx of aromatic amino acids in rat brain slices

Summary The influx of [³H] phenylalanine, [³H]tyrosine and [³H]tryptophan into brain cells was studied using brain slices from adult rats. Each aromatic amino acid inhibited the influx of the others into the brain cells. Tryptophan inhibited non-competitively the influx of phenylalanine, and phenylalanine similarly that of tyrosine and tryptophan. On the other hand, tyrosine inhibited competitively the influx of phenylalanine, and similarly tryptophan that of tyrosine, and tyrosine that of tryptophan. Among the aromatic organic acids tested, only phenylpyruvate and homogentisate had any inhibitory effect on the influx of the aromatic amino acids. These effects were generally competitive, non-competitive inhibition being obtained only in the inhibition of phenylalanine influx by homogentisate. The existence of only one common transport system for aromatic amino acids appears to be unlikely.     

The regional distribution of sulphonylurea binding sites in rat brain

Sulphonylureas such as glibenclamide, which are used in the treatment of Type-2 diabetes, are inhibitors of ATP-sensitive potassium channels. These channels link cellular metabolism to membrane electrical activity and it is likely that they are closely associated with glibenclamide binding sites. Quantitative autoradiography was used to localize high-affinity [3H]glibenclamide binding sites in coronal sections of rat brain. The relative density of binding sites was found to correlate well with the relative capacity of sites determined in homogenate assays. There was no evidence of any variation of affinity between brain regions. The highest levels of binding were found in the substantia nigra with high levels in the globus pallidus, cerebral cortex, hippocampus and caudate-putamen, intermediate levels in the cerebellum, and low levels in the hypothalamus and pons. The density of [3H]glibenclamide binding sites was low in glucose-responsive brain regions, known to contain ATP-sensitive potassium channels that are inhibited by sulphonylureas. However, higher densities were associated with brain regions (often limbic structures) active during temporal lobe epilepsy. In at least two of these structures, the CA3 region of the hippocampus and the substantia nigra, it is probable that these sites are coupled to ATP-sensitive potassium channels. These results are discussed with reference to the reported actions of ATP-sensitive potassium channels on CNS function.     

Voltammetrically monitored brain ascorbate as an index of excitatory amino acid release in the unrestrained rat

To discover the significance of changes in the extracellular concentration of brain ascorbate, we used linear sweep voltammetry to monitor the ascorbate signal. Recordings were made with carbon paste electrodes implanted in the striatum and hippocampus of anaesthetised and unanaesthetised rats under a variety of conditions. Intraperitoneal administration of excitatory amino acid transmitters, but not tyrosine or glycine, increased extracellular striatal ascorbate; similarly, microinfusion of L-glutamate beside striatal electrodes enhanced the ascorbate signal. Electrical stimulation of the perforant path increased the extracellular concentration of dentate ascorbate in the unanaesthetised, but not in the anaesthetised, rat. These results support our hypothesis that changes in the extracellular concentration of brain ascorbate monitored by voltammetry reflect the release of excitatory amino acids.     

Turning behaviour and catalepsy after injection of excitatory amino acids into rat substantia nigra

Unilateral injection of AMPA ((R,S)-α-3-hydroxyl-5-methyl-4-isoxazolepropionic acid), 4-bromohomoibotenic acid, kainic acid and N-methylaspartic acid into the caudal part of substantia nigra induced ipsilateral turning behaviour. AMPA and kainic acid were most potent. Ibotenic acid produced short-lasting ipsilateral turning followed by long-lasting contralateral turning behaviour which was antagonized by the GABA antagonist bicuculline methiodide. The turning induced by AMPA was not blocked by previous 6-hydroxydopamine lesion of the nigrostriatal dopaminergic pathway and was increased by systemic apomorphine. High doses of bilateral intranigral AMPA induced rigid catalepsy. The results are consistent with an involvement of glutamate or aspartate receptors in the regulation of dopamine output systems.     

An aversive diet as thiamine-free food blocks food-induced release of excitatory amino acids in the accumbens

AIMS: As the nucleus accumbens shell plays an important role in the control of eating behaviour, the aim of this study was to evaluate the changes in: (a) the level of aspartic and glutamic acids in the accumbens shell of conditioned rats after the presentation of an aversive diet containing thiamine-free food; (b) the temperature of interscapular brown adipose tissue, effector of thermogenesis related to food intake. METHODS: The concentration of aspartic and glutamic acids in the accumbens shell, and brown adipose tissue temperature were monitored in conditioned male Sprague-Dawley rats before and after the presentation of thiamine-free food or standard laboratory food. The aspartic and glutamic acids were collected using a microdialysis probe and quantified by HPLC. Food intake was also measured. RESULTS: The results indicated that an intake of standard laboratory food induced an increase in the level of aspartic and glutamic acids, and an elevation in temperature of brown adipose tissue; whereas an intake of thiamine-free food blocks these increases in the conditioned animals. CONCLUSION: The thiamine-free diet modifies the release of excitatory amino acids in the nucleus accumbens of conditioned animals. This diet also affects thermogenesis.     

The regional distribution and cellular localization of iron in the rat brain

The regional distribution and cellular localization of iron throughout the rat brain was determined with iron histochemistry. Densitometry was used to measure the intensity of stain of 51 iron-concentrating sites. Among the areas of highest iron content are the circumventricular organs, islands of Calleja, globus pallidus, ventral pallidum, substantia nigra pars reticulata, interpeduncular nucleus, dentate nucleus, and interpositus nucleus. Iron occurs most commonly in oligodendrocytes and in the fibrous network of the neuropil, but is also found in the interstitial spaces of circumventricular organs and in the tanycytes of the organum vasculosum of the lamina terminalis, median eminence, and walls of the third ventricle. In diverse areas throughout the brain—among them, the islands of Calleja, dentate gyrus of the hippocampal formation, lateral septal nucleus, and central amygdala—iron is found in association with the perikarya and neuronal processes of nerve cells.The overlapping distribution patterns of iron and γ-aminobutyric acid, enkephalin, and luteinizing hormone-releasing hormone suggest that the distribution of iron is related to its association with the metabolism of one or more neurotransmitters or neuroactive compounds.     

Microdialysis in the study of extracellular levels of amino acids in the rat brain

Microdialysis has been used for in vivo studies of extracellular amino acids in rat brain. We describe a method where a probe was designed to be implanted vertically. This probe is suitable for regional stereotaxic studies of the rat brain. The dialysis probe was perfused with Ringer's solution and the perfusates were analysed for their amino acid content with a high performance liquid chromatography (HPLC) technique. An orthophthaldialdehyde derivative of the amino acids was formed before the sample was injected onto the column. In vitro studies of the dialysis probe show that the relative recovery of substances outside the membrane is dependent on perfusion speed and length of dialysing membrane but not on the concentration outside. We were also able to show that the probe was within the blood-brain barrier (BBB) when implanted into the brain since after intravenous injection of Na99mTcO4, a substance that cannot pass through the intact BBB, it was not possible to recover any isotope from the perfusate. We conclude that microdialysis is a unique method of studying regional neurochemical events within the BBB, for example, changes in putative amino acid neurotransmitters and their metabolites.     

The action of six antagonists of the excitatory amino acids on neurones of the rat spinal cord

Summary The differential susceptibility to blockade by six antagonists of the excitatory actions produced by a number of acidic amino acids has been determined. Four of these, DL--aminoadipate, 2-amino-5-phosphonovalerate, 2-amino-6-phosphonocaproate and -D-glutamylglycine block the action of N-methyl-D-aspartate (NMDA) most effectively and those of L-glutamate, trans-1-amino-1,3-dicarboxy-cyclopentane (trans-ADCP) and kainate the least. Cis-2,3-piperidine dicarboxylate resembles the other four, save in that kainate excitations are also powerfully blocked. L-glutamate diethylester was most effective against L-glutamate and least against NMDA: kainate and trans-ADCP excitations were also relatively unaffected.The data support the hypothesis of the existence of at least three classes of amino acid receptor (NMDA-, L-glutamate- and kainate-excited), and the possibility of a fourth category activated by trans-ADCP must be considered.     

Structure-activity relations of dipeptide antagonists of excitatory amino acids

Structure-activity relations of dipeptides related to γ-d-glutamylglycine have been investigated with respect to the ability of these substances to antagonize depolarizing responses of frog motoneurones in vitro to N-methyl-d-aspartate, kainate and quisqualate. A terminal phosphono group was optimal for N-methyl-d-aspartate antagonist activity in relation to both potency and selectivity. Substances containing a terminal phosphono group were relatively poor antagonists of kainate or quisqualate responses. Terminal car?ylic and sulphonic groups were both effective with respect to kainate and/or quisqualate antagonism. Sulphonic compounds were the more selective in this type of action because of their low affinity for N-methyl-d-aspartate receptors. Optimum chain length for N-methyl-d-aspartate antagonism was between one and two carbon atoms shorter than for optimum kainate/quisqualate antagonist activity. Bulky groups within the N-acylated amino acid moiety generally, but differentially, reduced the ability of the substance to antagonize responses to each of the three agonists. Glutamyl peptides were generally more effective than aspartyl peptides of the same overall chain length. However, the most potent dipeptide (selective for N-methyl-d-aspartate antagonism) was the aspartyl derivative, >β-d-aspartylaminomethyl phosphonate, for which there was no glutamyl equivalent. Other useful substances to emerge from this study include the relatively selective kainate/quisqualate antagonists, γ-d-glutamylaminomethyl sulphonate and γ-d-glutamyltaurine. If similar selectivity is shown in other preparations also, these substances may prove preferable to γ-d-glutamylglycine as antagonists of synaptic excitation mediated by kainate or quisqualate receptors. Abbreviations: γDGG, γ-d-glutamylglycine; NMDA, N-methyl-d-aspartate     

Regulation of the post-translational conjugation of amino acids to rat brain proteins

Post-translational conjugation of arginine (but not other amino acids) to proteins has been reported to occur in a high speed supernatant fraction of rat brain homogenates from which molecules of less than 5000 mol. wt have been removed. In the present study we report that removal of molecules of less than 1000 mol. wt by dialysis, does not result in incorporation of arginine into protein in amounts significantly different than in the undialysed supernatant. The addition of molecules with molecular weights greater than 1000 and less than 5000 to the active fraction, inhibits the incorporation of arginine into proteins in a concentration dependent manner suggesting that the post-translational incorporation of arginine into brain is regulated by a molecule(s) of greater than 1000 and less than 5000 mol. wt. Incorporation of lysine into proteins did not occur following removal of molecules of less than 5000 mol. wt, but did occur in the void volume fraction of a Sephacryl S-200 column (molecular weight cut-off 125,000), suggesting that the incorporation of lysine into proteins is regulated by molecules retained by the S-200 column but greater than 5000 mol. wt. When experiments were repeated using the void volume of a Sephacryl S-300 column (molecular weight exclusion, approximately 200 k), leucine and proline were incorporated in amounts similar to arginine and lysine and serine, alanine, valine, phenylalanine and histidine were incorporated at lower but measurable levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sound-evoked efflux of excitatory amino acids in the guinea-pig cochlea in vitro

We have used the perfused guinea-pig temporal-bone preparation to study the sound-evoked efflux of aspartate and glutamate, which are putative afferent transmitters in the cochlea. The cochlea was stimulated with white noise at 89, 95, and 101 dB SPL. Cochlear function was monitored by recording the endocochlear potential, the cochlear microphonic, and the summating potential. In silence, there was a low basal efflux of both amino acids. A significant and intensity dependent sound-evoked efflux of aspartate was observed at all levels, whereas a significant efflux of glutamate was found only at the 101 dB SPL level. Immunohistochemistry of sections from the organ of corti showed an ubiquitous distribution of glutamate-like immunoreactivity in the sensory organ and ganglion, whereas aspartate-like immunoreactivity was found in the region of the inner hair cells and in the spiral ganglion. In view of these findings, we suggest that not only glutamate, but also aspartate may have a neurotransmitter role in the afferent pathway of the cochlea. Received: 7 November 1997 / Accepted: 3 March 1998