Glutamate stimulates inositol phosphate formation in striatal neurones

The major excitatory amino acids, glutamate (Glu) and aspartate (Asp), are thought to act at three receptor subtypes in the mammalian central nervous system (CNS). These are termed quisqualate (QA), N-methyl-D-aspartate (NMDA) and kainate (KA) receptors according to the specific agonist properties of these compounds revealed by electrophysiological studies. Although Glu has been shown to stimulate cyclic GMP formation in brain slices, direct regulation of second messenger systems (cyclic AMP, Ca2+ or inositol phosphates) subsequent to activation of excitatory amino-acid receptors, has not been extensively studied. Here we demonstrate that in striatal neurones, excitatory amino acids, but not inhibitory or non-neuroactive amino acids, induce a three- to fourfold increase in inositol mono-, di- and triphosphate (IP, IP, IP) formation with the relative potency QA greater than Glu greater than NMDA, KA. The Glu-evoked formation of inositol phosphates appears to result principally from actions at QA as well as NMDA receptors on striatal neurones. Our results suggest that excitatory amino acids stimulate inositol phosphate formation directly, rather than indirectly by the evoked release and subsequent actions of adenosine or acetylcholine.     

Mediation of thalamic sensory input by both NMDA receptors and non-NMDA receptors

Excitatory amino acids such as L-glutamate and L-aspartate are well established as neurotransmitter candidates in the mammalian central nervous system, and three types of receptor for these substances have been proposed, characterized by the agonists N-methyl-D-aspartate (NMDA), kainate and quisqualate. All these receptors have been suggested to have synaptic roles in excitatory transmission in the brain. Here I demonstrate that NMDA receptors play a crucial role in the observed response of ventrobasal thalamus (VB) neurones to natural stimulation of somatosensory afferents, but do not appear to be responsible for the short-latency excitation seen on electrical stimulation of the afferents which is apparently mediated by excitatory amino-acid receptors of the non-NMDA type. This result indicates an involvement of NMDA and non-NMDA receptors in the responses of VB neurones to stimulation of somatosensory somatosensory afferents, depending on the mode of stimulation of the pathway.     

Regulation of glutamate receptors by cations.

Current evidence suggests that glutamate is a major excitatory neurotransmitter in the mammalian central nervous system (CNS); particularly, glutamate excites most neurones in the CNS. Until recently this effect was widely used to study glutamate receptors and to distinguish them from those of other excitatory amino acids. The development of ligand binding studies for many neurotransmitters has facilitated the study of receptors at the molecular level and using these methods we recently reported the existence in hippocampal membranes of pharmacologically distinct sodium-dependent and sodium-independent glutamate binding sites, the former related to high-affinity uptake and the latter exhibiting several characteristics of postsynaptic receptor sites. We now report that, as with other neurotransmitters, several ions regulate the Na-independent binding of glutamate; the monovalent cations induce a decreased binding while certain divalent cations enhance this Na-independent binding. Additionally, since some of these effects appear to be irreversible, we propose that the regulation of glutamate binding by cations might account for the extremely long-lasting potentiation of synaptic responses found in the hippocampus following bursts of repetitive electrical stimulation (see ref. 9 for a review).     

NMDA-receptor activation increases cytoplasmic calcium concentration in cultured spinal cord neurones

Excitatory amino acids act via receptor subtypes in the mammalian central nervous system (CNS). The receptor selectively activated by N-methyl-D-aspartic acid (NMDA) has been best characterized using voltage-clamp and single-channel recording; the results suggest that NMDA receptors gate channels that are permeable to Na+, K+ and other monovalent cations. Various experiments suggest that Ca2+ flux is also associated with the activation of excitatory amino-acid receptors on vertebrate neurones. Whether Ca2+ enters through voltage-dependent Ca2+ channels or through excitatory amino-acid-activated channels of one or more subtype is unclear. Mg2+ can be used to distinguish NMDA-receptor-activated channels from voltage-dependent Ca2+ channels, because at micromolar concentrations Mg2+ has little effect on voltage-dependent Ca2+ channels while it enters and blocks NMDA receptor channels. Marked differences in the potency of other divalent cations acting as Ca2+ channel blockers compared with their action as NMDA antagonists also distinguish the NMDA channel from voltage-sensitive Ca2+ channels. However, we now directly demonstrate that excitatory amino acids acting at NMDA receptors on spinal cord neurones increase the intracellular Ca2+ activity, measured using the indicator dye arsenazo III, and that this is the result of Ca2+ influx through NMDA receptor channels. Kainic acid (KA), which acts at another subtype of excitatory amino-acid receptor, was much less effective in triggering increases in intracellular free Ca2+.     

Cloning of cDNA for the glutamate-binding subunit of an NMDA receptor complex.

The amino acids L-glutamic and L-aspartic acids form the most widespread excitatory transmitter network in mammalian brain. The excitation produced by L-glutamic acid is important in the early development of the nervous system, synaptic plasticity and memory formation, seizures and neuronal degeneration. The receptors activated by L-glutamic acid are a target for therapeutic intervention in neurodegenerative diseases, brain ischaemia and epilepsy. There are two types of receptors for the excitatory amino acids, those that lead to the opening of cation-selective channels and those that activate phospholipase C (ref. 11). The receptors activating ion channels are NMDA (N-methyl-D-aspartate) and kainate/AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate)-sensitive receptors. The complementary DNAs for the kainate/AMPA receptor and for the metabotropic receptor have been cloned. We report here on the isolation and characterization of a protein complex of four major proteins that represents an intact complex of the NMDA receptor ion channel and on the cloning of the cDNA for one of the subunits of this receptor complex, the glutamate-binding protein.     

Frequency-dependent involvement of NMDA receptors in the hippocampus: a novel synaptic mechanism

Acidic amino acids, such as l-glutamate, are believed to be excitatory neurotransmitters in the mammalian brain and exert effects on several different receptors named after the selective agonists kainate, quisqualate and N-methyl-D-aspartate (NMDA). The first two receptors collectively termed non-NMDA receptors, have been implicated in the mediation of synaptic transmission in many excitatory pathways in the central nervous system (CNS), whereas NMDA receptors, with few exceptions do not appear to be involved; this is typified in the hippocampus where there is a high density of NMDA receptors yet selective NMDA receptor antagonists, such as D-2-amino-5-phosphonovalerate (APV), do not affect synaptic potentials. NMDA receptors have, however, been shown to be involved in long-term potentiation (LTP) in the hippocampus, a form of synaptic plasticity which may be involved in learning and memory. NMDA receptors have also been found to contribute to epileptiform activity in this region. We now describe how NMDA receptors can participate during high-frequency synaptic transmission in the hippocampus, their involvement during low-frequency transmission being greatly suppressed by Mg2+. A frequency dependent alleviation of this blockade provides a novel synaptic mechanism whereby a single neurotransmitter can transmit very different information depending on the temporal nature of the input. This mechanism could account for the involvement of NMDA receptors in the initiation of LPT and their contribution, in part, to epileptic activity.     

Potassium conductances in hippocampal neurons blocked by excitatory amino-acid transmitters

Excitatory amino acids mediate fast synaptic transmission in the central nervous system through the activation of at least three distinct ionotropic receptors: N-methyl-D-aspartate (NMDA), the alpha-amino-3-hydroxy-5-methyl-isoxasole-4-propionate (AMPA)/quisqualate (QUIS) and the kainate subtypes (for reviews, see refs 1, 2). They also activate the additional QUIS 'metabotropic' receptor (sensitive to trans-1-amino-cyclopentyl-1,3-dicarboxylate, ACPD) linked to inositol phospholipid metabolism. We have used hippocampal slice cultures to study the electrophysiological consequences of the metabotropic response. We find that activation of an ACPD-sensitive QUIS receptor produces a 'slow' excitation of CA3 pyramidal cells, resulting from depression of a Ca2(+)-dependent K+ current and a voltage-gated K+ current. Combined voltage-clamp and microfluorometric recordings show that, although these receptors can trigger an increase in intracellular Ca2+ concentration, suppression of K+ currents is independent of changes in intracellular Ca2+. These effects closely resemble those induced by activating muscarinic acetylcholine receptors in the same neurons and suggest that excitatory amino acids not only act as fast ionotropic transmitters but also as slow neuromodulatory transmitters.     

Aspartate and glutamate as possible neurotransmitters of cells in layer 6 of the visual cortex

Earlier work has suggested that aspartate, glutamate and gamma-aminobutyric acid (GABA) act as transmitters in the cerebral cortex. There is reasonable evidence for the identity of the cell population responsible for GABA release but until now there has been little evidence concerning the sources for release of aspartate and glutamate. Here we have used two approaches to identify possible neurotransmitters used by cells in the visual cortex: measurement of the efflux of endogenous compounds in conditions of synaptic release and localization of these compounds to particular cell classes using neurotransmitter-specific histochemical techniques. Our results suggest that the acidic amino acids aspartate and glutamate may be cortical neurotransmitters, as shown by calcium-dependent release from endogenous stores and by uptake specific to pyramidal cells in layer 6 of the cortex. These substances may therefore have a role in the function of layer 6 cells, which are responsible for the recurrent projection from the cortex to the lateral geniculate nucleus and for the projection within the cortex from layer 6 to layer 4.     

Hyperpolarization of fish retinal horizontal cells by kainate and quisqualate

Kainic (KA) and quisqualic (QA) acids have a potent depolarizing action on a variety of neurones of the central nervous system, including retinal horizontal cells. We now report the novel finding that at low concentrations (1-3 microM), these 'excitatory' amino acids hyperpolarize horizontal cells of the fish retina. We show that the hyperpolarizing effects of both KA and QA are reversed by the gamma-aminobutyric acid (GABA) antagonist bicuculline, whereas a second GABA antagonist, picrotoxin, reverses the effects of KA, but not of QA. Neither GABA antagonist influences horizontal cell depolarization by 50 microM KA or 50 microM QA, thus the excitatory (depolarizing and inhibitory (hyperpolarizing) effects of the amino acids involve independent mechanisms. We provide evidence that the hyperpolarizing effects are not mediated by the dopaminergic pathways associated with retinal horizontal cells.     

JWA, a novel microtubule－associated protein, regulates homeostasis of intracellular amino acids in PC12 cells

Our previous studies demonstrated that JWA,a novel retinoic acids responsive and cytoskeleton related gene, is associated with cell differentiation and apoptosis. In the present study, to elucidate if the JWA is a novel kind of microtubnle-associated proteins (MAPs) and functiona llylink to microtubule, we first successfully identified JWA from the physically purified MAPs complex of rat braint issues. The results of co-immunoprecipitation, gene transfection and immunofluorescence microscopy assays from HBE and NIH3T3 cells provide strong evidence for a linkage between JWA and β-tubulin. In general, JWA is stably binding to β-tubulin whenever microtubule is polymerized or not,and it may be critical to the mitosis process. In addition, by use of the antisense oligonucleotides technique, we also showed that JWA is a negative modulator on intracellular amino acids in PC12 cells. Further analysis indicated that JWA selectively regulates both taurine, an inhibitory aminoacid, and glutamate, an excitatory amino acid. In conclusion,JWA is not only structurally associated, but also a novel functional MAP.     

低频调制磁场对脑电节律的影响机理

神经细胞Ca2 内流及脑内5 HT、NE、DA等神经递质的变化影响了神经细胞放电速率,从而影响脑电节律.为揭示低频调制磁场对脑电节律产生影响的微观作用机理,检测了Wistar大鼠脑内神经递质的释放,电镜下观察了其神经突触形态的变化.实验结果显示,低频调制磁场作用引起大鼠脑内去甲肾上腺素(NE)、多巴胺(DA)、5 羟色胺(5 HT)释放显著增加(P<0.05),并观察到大鼠神经细胞内钙颗粒及释放神经递质的突触小泡数明显增加,提示低频调制磁场对人脑电节律的影响机理是其改变了Ca2 内流及脑内神经递质的释放量.     

Polyamine-dependent facilitation of postsynaptic AMPA receptors counteracts paired-pulse depression.

At many glutamatergic synapses in the brain, calcium-permeable alpha - amino - 3 - hydro - 5 - methyl - 4 - isoxazolepropionate receptor (AMPAR) channels mediate fast excitatory transmission. These channels are blocked by endogenous intracellular polyamines, which are found in virtually every type of cell. In excised patches, use-dependent relief of polyamine block enhances glutamate-evoked currents through recombinant and native calcium-permeable, polyamine-sensitive AMPAR channels. The contribution of polyamine unblock to synaptic currents during high-frequency stimulation may be to facilitate currents and maintain current amplitudes in the face of a slow recovery from desensitization or presynaptic depression. Here we show, on pairs and triples of synaptically connected neurons in slices, that this mechanism contributes to short-term plasticity in local circuits formed by presynaptic pyramidal neurons and postsynaptic multipolar interneurons in layer 2/3 of rat neocortex. Activity-dependent relief from polyamine block of postsynaptic calcium-permeable AMPARs in the interneurons either reduces the rate of paired-pulse depression in a frequency-dependent manner or, at a given stimulation frequency, induces facilitation of a synaptic response that would otherwise depress. This mechanism for the enhancement of synaptic gain appears to be entirely postsynaptic.     

Sensory transmitters regulate intracellular calcium in dorsal horn neurons

Primary afferent terminals in the dorsal horn of the spinal cord release excitatory amino acid and peptide transmitters that initiate the central processing of nociceptive information. The postsynaptic actions of amino acid transmitters on spinal neurons have been well characterized, but the cellular basis of peptide actions remains unclear. Substance P is the best characterized of the peptides present in sensory neurons and has been shown to depolarize dorsal horn neurons and to facilitate nociceptive reflexes. To determine the mechanisms by which substance P contributes to afferent synaptic transmission, we have monitored the levels of intracellular calcium in single isolated rat dorsal horn neurons and report that substance P can produce a prolonged elevation in calcium concentration by mobilizing its release from intracellular stores. This elevation may contribute to the long-term changes in the excitable properties of dorsal horn neurons that occur following afferent fibre stimulation. We have also found that L-glutamate elevates intracellular calcium in substance P-sensitive dorsal horn neurons by increasing calcium influx. These results provide a direct demonstration of intracellular calcium changes in response to neuropeptides in mammalian central neurons. They also indicate that there is convergent regulation of intracellular calcium in dorsal horn neurons by two different classes of sensory transmitters that are co-released from the same afferent terminals.     

Auto-inhibition of brain histamine release mediated by a novel class (H3) of histamine receptor

Although histaminergic neurones have not yet been histochemically visualized, there is little doubt that histamine (HA) has a neurotransmitter role in the invertebrate and mammalian central nervous system. For example, a combination of biochemical, electrophysiological and lesion studies in rats have shown that histamine is synthesized in and released from a discrete set of neurones ascending through the lateral hypothalamic area and widely projecting in the telencephalon. Histamine acts on target cells in mammalian brain via stimulation of two classes of receptor (H1 and H2) previously characterized in peripheral organs and probably uses Ca2+ and cyclic AMP, respectively, as second messengers. It is well established that several neurotransmitters affect neuronal activity in the central nervous system through stimulation not only of postsynaptic receptors, but also of receptors located presynaptically which often display distinct pharmacological specificity and by which they may control their own release. Such 'autoreceptors' have been demonstrated (or postulated) in the case of noradrenaline, dopamine, serotonin, acetylcholine and gamma-aminobutyric acid (GABA) neurones but have never been demonstrated for histamine. We show here that histamine inhibits its own release from depolarized slices of rat cerebral cortex, an action apparently mediated by a class of receptor (H3) pharmacologically distinct from those previously characterized, that is, the H1 and H2 receptors.     

Modulation of brain polyphosphoinositide metabolism by ACTH-sensitive protein phosphorylation

Phosphorylation of membrane components is thought to be an important process in membrane function. Phosphorylated proteins and a special class of phospholipids, the (poly)phosphoinositides (poly PI), are implicated in the regulation of membrane permeability and synaptic transmission in neurones. For many years, protein phosphorylation and poly PI metabolism have been studied in parallel without knowledge of their possible interaction. We report here that the ACTH-sensitive protein kinase/B-50 protein complex which we recently isolated in soluble form from rat brain synaptosomal plasma membranes has lipid phosphorylating activity. Exogenously added phosphatidylinositol 4-phosphate (DPI) is phosphorylated to phosphatidylinositol 4,5-diphosphate (TPI), and this DPI-kinase activity is dependent on the state of phosphorylation of the protein kinase/B-50 protein complex. The results imply that phosphorylation of protein may affect the metabolism of (poly) PI in brain cell membranes.     

Pertussis toxin reverses adenosine inhibition of neuronal glutamate release

Adenosine and its analogues are potent inhibitors of synaptic activity in the central and peripheral nervous system. In the central nervous system (CNS), this appears to arise primarily by inhibition of presynaptic release of transmitters, including glutamate, which is possibly the major excitatory transmitter in the brain. In addition, postsynaptic effects of adenosine have been reported which would also serve to reduce neurotransmission. The mechanism by which adenosine inhibits CNS neurotransmission is unknown, although it appears to exert its effect via an A1 receptor which in some systems is negatively coupled to adenylate cyclase. In an attempt to elucidate the mechanism of inhibition, we have examined the effect of pertussis toxin (PTX) on the ability of the stable adenosine analogue (-)phenylisopropyladenosine (PIA) to inhibit glutamate release from cerebellar neurones maintained in primary culture. PTX, by ADP-ribosylating the nucleotide-binding protein Ni, prevents coupling of inhibitory receptors such as the A1 receptor to adenylate cyclase. As reported here, we found that PTX, as well as preventing inhibition of adenylate cyclase by PIA, also converts the PIA-induced inhibition of glutamate release to a stimulation. Our results suggest strongly that purinergic inhibitory modulation of transmitter release occurs by inhibition of adenylate cyclase.     

猪血浆巯基蛋白酶抑制剂的分离纯化及性质研究

用(NH_4)_2SO_4分段盐析,DEAE-纤维素离子交换和Papain—Sepharose亲和层析,从猪血浆纯化了一种巯基蛋白酶抑制剂(SUlfhydryl proteinase inhibitor;简称SPI);纯化倍数为54.36倍.经高pH、低PH不连续电泳呈一条区带.含糖量为7.77％;PI为4.80.氨基酸组成分析表明,猪血SPI富含酸性氨基酸、芳香族氨基酸和含硫氨基酸.用SDS-PAGE和SephadexG-100层析,测得分子量为130000d和131000d,由两个分子量略有差别的亚基组成.对木瓜蛋白酶有强烈抑制作用,对胰蛋白酶无作用。α-螺旋结构含量高。     

Synaptic excitation produces a long-lasting rebound potentiation of inhibitory synaptic signals in cerebellar Purkinje cells.

Persistent changes in synaptic efficacy are thought to underlie the formation of learning and memory in the brain. High-frequency activation of an afferent excitatory fibre system can induce long-term potentiation, and conjunctive activation of two distinct excitatory synaptic inputs to the cerebellar Purkinje cells can lead to long-term depression of the synaptic activity of one of the inputs. Here we report a new form of neural plasticity in which activation of an excitatory synaptic input can induce a potentiation of inhibitory synaptic signals to the same cell. In cerebellar Purkinje cells stimulation of the excitatory climbing fibre synapses is followed by a long-lasting (up to 75 min) potentiation of gamma-aminobutyric acid A (GABAA) receptor-mediated inhibitory postsynaptic currents (i.p.s.cs), a phenomenon that we term rebound potentiation. Using whole-cell patch-clamp recordings in combination with fluorometric video imaging of intracellular calcium ion concentration, we find that a climbing fibre-induced transient increase in postsynaptic calcium concentration triggers the induction of rebound potentiation. Because the response of Purkinje cells to bath-applied exogenous GABA is also potentiated after climbing fibre-stimulation with a time course similar to that of the rebound potentiation of i.p.s.cs, we conclude that the potentiation is caused by a calcium-dependent upregulation of postsynaptic GABAA receptor function. We propose that rebound potentiation is a mechanism by which in vivo block of climbing fibre activity induces an increase in excitability in Purkinje cells. Moreover, rebound potentiation of i.p.s.cs is a cellular mechanism which, in addition to the long-term depression of parallel fibre synaptic activity, may have an important role for motor learning in the cerebellum.     

Different voltage-dependent thresholds for inducing long-term depression and long-term potentiation in slices of rat visual cortex

In the hippocampus and neocortex, high-frequency (tetanic) stimulation of an afferent pathway leads to long-term potentiation (LTP) of synaptic transmission. In the hippocampus it has recently been shown that long-term depression (LTD) of excitatory transmission can also be induced by certain combinations of synaptic activation. In most hippocampal and all neocortical pathways studied so far, the induction of LTP requires the activation of N-methyl-D-aspartate (NMDA) receptor-gated conductances. Here we report that LTD can occur in neurons of slices of the rat visual cortex and that the same tetanic stimulation can induce either LTP or LTD depending on the level of depolarization of the postsynaptic neuron. By applying intracellular current injections or pharmacological disinhibition to modify the depolarizing response of the postsynaptic neuron to tetanic stimulation, we show that the mechanisms of induction of LTD and LTP are both postsynaptic. LTD is obtained if postsynaptic depolarization exceeds a critical level but remains below a threshold related to NMDA receptor-gated conductances, whereas LTP is induced if this second threshold is reached.     

稀有鮈鲫铜蓝蛋白基因 cDNA 克隆及组织表达分析

铜蓝蛋白(Ceruloplasmin,Cp)是一种重要的铜转运蛋白,合成于肝脏并参与生物体铁的代谢,在医学上是各种炎症、感染、中毒及癌症疾病的标志性蛋白.铜蓝蛋白的研究已在多种真骨鱼类中被报道,文中第一次在稀有鮈鲫(Gobiocypris rarus)中报道此基因.采用cDNA末端快速扩增技术(rapid amplification of cDNA ends,RACE)克隆了稀有鮈鲫铜蓝蛋白基因,使用荧光定量PCR的方法构建了该基因组织表达谱.序列分析表明稀有鮈鲫铜蓝蛋白基因包含3 264bp全长编码序列,该序列编码1 087个氨基酸,其核苷酸和氨基酸序列与斑马鱼同源性最高(分别为88.1%和90.3%).理论相对分子质量和等电点分别为124 429.1D和6.41.荧光定量PCR检测表明该基因在肝脏和脾脏中相对表达量最高,在肌肉和鳃中相对表达量最低.使用氨基酸序列进行蛋白结构保守域分析,结果表明铜蓝蛋白基因在脊椎动物中是相对保守的,推测其功能也与其他物种相似.这为进一步研究稀有鮈鲫该基因的功能及其应用奠定了基础.