Rho激酶对大鼠海马神经元骨架相关蛋白mRNA表达的影响

目的:探讨Rho激酶对大鼠海马神经元突起生长和骨架相关蛋白mRNA表达的影响.方法:用Rho激酶的抑制剂Y-27632和激动剂溶血磷脂酸(LPA)干预海马神经元,观察突起的生长并用RT-PCR检测大鼠海马神经元神经丝结合蛋白(Dbn1)、微丝肌动蛋白(F-actin)、微管相关蛋白(Tau)、α-微管蛋白(α-tubulin) mRNA的表达.结果:用LPA干预2 h后突起从远端逐渐退缩,长度缩短,细小突起退缩明显;Y-27632干预2 h后细胞胞体和突起的远侧端新生出细小突起.RT-PCR实际扩增长度与设计长度相吻合.内参照β-actin电泳条带在不同分组灰度较均一,呈高表达.Dbn1呈较高水平表达,激动剂LPA组表达下降(P<0.05),抑制剂Y-27632组表达量增多(P<0.05);F-actin和Tau表达呈低水平,激动剂作用后均使表达量相应的减少(P<0.05),抑制剂组表达增多(P<0.05);α-tubulin表达量最高,激动剂组表达量出现明显下降(P<0.05),抑制剂组表达增加(P<0.05).结论:激活Rho激酶下调Dbn、F-actin、Tau、α-tubulin mRNA的表达并诱导突起缩短,抑制则增加其表达并促进突起生长.     

NMDA receptor agonists selectively block N-type calcium channels in hippocampal neurons.

The modulation of voltage-dependent calcium channels by various neurotransmitters has been demonstrated in many neurons. Because of the critical role of Ca2+ in transmitter release and, more generally, in transmembrane signalling, this modulation has important functional implications. Hippocampal neurons possess low-threshold (T-type) Ca2+ channels and both L- and N-type high voltage-activated Ca2+ channels. N-type Ca2+ channels are blocked selectively by omega-conotoxin and adenosine. These substances both block excitatory synaptic transmission in the hippocampus, whereas dihydropyridines, which selectively block L-type channels, are ineffective. Excitatory synaptic transmission in the hippocampus displays a number of plasticity phenomena that are initiated by Ca2+ entry through ionic channels operated by N-methyl-D-aspartate (NMDA) receptors. Here we report that NMDA receptor agonists selectively and effectively depress N-type Ca2+ channels which are involved in neurotransmitter release from presynaptic sites. The inhibitory effect is eliminated by the competitive NMDA antagonist D-2-amino-5-phosphonovalerate, does not require Ca2+ entry into the cell, and is probably receptor-mediated. This phenomenon may provide a negative feedback between the liberation of excitatory transmitter and entry of Ca2+ into the cell, and could be important in presynaptic inhibition and in the regulation of synaptic plasticity.     

Dependence on pH of polarized sorting of secreted proteins

The plasma membranes of epithelial cells are divided into apical and basolateral domains. These two surfaces are characterized by markedly different protein compositions, reflecting the ability of the cell to target newly synthesized membrane proteins to specific regions of the cell surface. This targeting capability is also apparent in the polarized release of secretory products. Recent studies using canine renal tubule (MDCK) cells have suggested that distinct sets of secretory proteins are released from their apical and basolateral poles. We report experiments designed to examine secretory protein sorting by MDCK cells. We have shown that secretion of basement membrane components (laminin and heparan sulphate proteoglycan (HSPG] takes place from the basolateral cell surface and that this polarized release results from active sorting. The sorting process which mediates this polarized secretion requires an acidic intracellular compartment. MDCK cells treated with NH4Cl to raise the pH of their intracellular compartments, secrete laminin and HSPG by a default pathway which leads to their release in roughly equal quantities into the medium of both the apical and basolateral compartments.     

Selective dendritic transport of RNA in hippocampal neurons in culture

Typical neurons of the central nervous system (CNS) elaborate tens of thousands of membrane specializations at sites of synaptic contacts on their dendrites. To construct, maintain, and modify these specializations, neurons must produce and deliver the appropriate molecular constituents to particular synaptic sites. Previous studies have revealed that polyribosomes are selectively positioned beneath postsynaptic sites, suggesting that in neurons, as in other cell types, protein synthetic machinery is located at or near the sites where particular proteins are needed. The mechanisms that deliver ribosomes and messenger RNA to their specific destinations in cells are therefore of considerable interest. Here we describe a system for RNA transport in dendrites that could provide a mechanism for the delivery of ribosomes and mRNA to synaptic sites in dendrites. Hippocampal neurons grown in culture incorporate 3H-uridine in the nucleus, then selectively transport the newly synthesized RNA into dendrites at a rate of about 0.5 mm day-1. The transport is inhibited by metabolic poisons, suggesting that it is an active, energy-dependent process. The RNA may be transported in association with the cytoskeleton.     

GABAA responses in hippocampal neurons are potentiated by glutamate

In the mammalian cortex, glutamate and gamma-aminobutyric acid (GABA) are the principal transmitters mediating excitatory and inhibitory synaptic events. Glutamate activates cation conductances that lead to membrane depolarization whereas GABA controls chloride conductances that produce hyperpolarization. Here we report that the GABAA-activated conductance in hippocampal pyramidal cells is enhanced by glutamate at concentrations below that required for its excitatory action. The GABA-potentiating effect can be induced, with comparable potency, by several glutamate analogues such as quisqualate, N-methyl-D-aspartate (NMDA), kainate and, surprisingly, by D-2-amino-5-phosphonovalerate (APV), an antagonist for NMDA receptors. Data from dose-response curves show that glutamate enhances the GABAA conductance without significantly changing GABA binding affinity. The low concentration of glutamate needed to enhance GABAA responses raises the possibility that glutamate modulates the strength of GABA-mediated transmission in the cortex.     

Glutamate activates multiple single channel conductances in hippocampal neurons

There is considerable evidence that glutamate is the principal neurotransmitter that mediates fast excitatory synaptic transmission in the vertebrate central nervous system. This single transmitter seems to activate two or three distinct types of receptors, defined by their affinities for three selective structural analogues of glutamate, NMDA (N-methyl-D-aspartate), quisqualate and kainate. All these agonists increase membrane permeability to monovalent cations, but NMDA also activates a conductance that permits significant calcium influx and is blocked in a voltage-dependent manner by extracellular magnesium. Fast synaptic excitation seems to be mediated mainly by kainate/quisqualate receptors, although NMDA receptors are sometimes activated. We have investigated the properties of these conductances using single-channel recording in primary cultures of hippocampal neurons, because the hippocampus contains all subtypes of glutamate receptors and because long-term potentiation of synaptic transmission occurs in this structure. We find that four or more distinct single-channel currents are evoked by applying glutamate to each outside-out membrane patch. These conductances vary in their ionic permeability and in the agonist most effective in causing them to open. Clear transitions between all the conductance levels are observed. Our observations are compatible with the model that all the single channel conductances activated by glutamate reflect the operation of one or two complex molecular entities.     

Glutamate-induced long-term potentiation of the frequency of miniature synaptic currents in cultured hippocampal neurons.

Glutamate application at synapses between hippocampal neurons in culture produces long-term potentiation of the frequency of spontaneous miniature synaptic currents, together with long-term potentiation of evoked synaptic currents. The mini frequency potentiation is initiated postsynaptically and requires activity of NMDA receptors. Although the frequency of unitary quantal responses increases strongly, their amplitude remains little changed with potentiation. Tests of postsynaptic responsiveness rule out recruitment of latent glutamate receptor clusters. Thus, postsynaptic induction can lead to enhancement of presynaptic transmitter release. The sustained potentiation of mini frequency is expressed even in the absence of Ca2+ entry into presynaptic terminals.     

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.     

Regulation of NMDA receptor desensitization in mouse hippocampal neurons by glycine

Responses to the excitatory amino acid N-methyl-D-aspartate (NMDA) are markedly potentiated by nanomolar concentrations of glycine. This is due to the action of glycine at a novel strychnine-resistant binding site with an anatomical distribution identical to that for NMDA receptors, suggesting that the NMDA receptor channel complex contains at least two classes of amino-acid recognition site. Antagonists at the glycine-binding site associated with NMDA receptors act as potent non-competitive antagonists, but do not alter the mean open time or conductance, as estimated by fluctuation analysis. The mechanisms by which glycine acts on NMDA receptors are unknown, but single-channel recording experiments show an increase in opening frequency with no change in mean open time or conductance, suggesting that glycine could regulate transitions to states that are intermediate between binding of NMDA receptor agonists and ion-channel gating. It has been suggested that glycine acts as a co-agonist at the NMDA receptor, and that responses to NMDA cannot be obtained in the complete absence of glycine, but in these experiments the response to NMDA was measured at equilibrium, and it is unlikely that sufficient temporal resolution was achieved to detect rapid alterations in receptor gating. Using a fast perfusion system we find that glycine regulates desensitization at NMDA receptors; this has a major effect on the response to NMDA measured at equilibrium, as would occur with slower applications of agonist. Reduction of NMDA receptor desensitization by glycine provides an example of a novel mechanism for regulation of ion-channel activity.     

Temporal integration by a slowly inactivating K+ current in hippocampal neurons

A central aspect of neuronal function is how each nerve cell translated synaptic input into a sequence of action potentials that carry information along the axon, coded as spike frequency. When transduction from a graded depolarizing input to spikes is studied by injecting a depolarizing current, there is often a remarkably long delay to the first action potential, both in mammalian and molluscan neurons. Here, I report that the delayed excitation in rat hippocampal neurons is due to a slowly inactivating potassium current, ID. ID co-exists with other voltage-gated K+ currents, including a fast A current and a slow delayed rectifier current. As ID activates in the subthreshold range, and takes tens of seconds to recover from inactivation, it enables the cell to integrate separate depolarizing inputs over long times. ID also makes the encoding properties of the cell exceedingly sensitive to the prevailing membrane potential.     

Differential modulation of three separate K-conductances in hippocampal CA1 neurons by serotonin

The hippocampus receives a dense serotonin-containing innervation from the divisions of the raphe nucleus. Serotonin applied to hippocampal neurons to mimic the action of endogenous transmitter often produces complex and variable responses (see for example ref. 3). Using voltage-clamp methods and new ligands that are selective for subtypes of serotonin receptors, we have been able to clarify the mechanism of serotonin action on CA1 cells in rat hippocampal slices. We describe three distinct actions of serotonin (or 5-HT) on identified K-conductances in these cells. First, it activates a Ca-independent K-current which is responsible for neuronal hyperpolarization and is inhibitory. Second, it simultaneously suppresses the slow Ca-dependent K-conductance that is largely responsible for the accommodation of cell firing in CA1 neurons: this produces a paradoxical increase in neuronal discharge in response to a depolarizing input. Third, serotonin produces a more slowly developing and long-lasting suppression of an intrinsic voltage-dependent K-conductance, Im (ref. 9), leading to neuronal depolarization and excitation. The hyperpolarizing response is mediated by class 1A serotonin receptors, whereas the other responses are not. Modulation of these different conductances by endogenously released serotonin could therefore change the probability or the duration (or both) of neuronal firing in the mammalian brain in different ways to give inhibitory, excitatory or mixed effects.     

Noradrenaline and beta-adrenoceptor agonists increase activity of voltage-dependent calcium channels in hippocampal neurons

The predominance of unconventional transmitter release sites at noradrenaline-containing synapses and the diffuse projections of noradrenaline-containing fibres originating in locus coeruleus have led to speculation that noradrenaline may act as a neuromodulator in the central nervous system. Evidence suggests that it has a modulatory function in the plasticity of the developing nervous system, in controlling behavioural states of an organism, and in learning and memory. Recently, Hopkins and Johnston demonstrated that noradrenaline enhances the magnitude, duration and probability of induction of long-term potentiation (LTP) at mossy fibre synapses in the hippocampal formation, and LTP is widely believed to be a cellular substrate for aspects of memory. To investigate the membrane effects of noradrenaline on central neurons, we used a newly developed preparation in which patch-clamp techniques can be applied to exposed adult cortical neurons. We report here that noradrenaline produces an enhancement in the activity of voltage-dependent calcium channels in granule cells of the hippocampal dentate gyrus. This action appears to be mediated by beta-adrenoceptors and can be mimicked by cyclic AMP.     

Effect of ytterbium on sodium current and its kinetics in rat hippocampal neurons

This study addressed the effects of Yb3＋ on voltage-gated sodium currents in rat hippocampal neurons using the whole-cell patch-clamp technique. Voltage-clamp recordings in single neurons were filtered and stored in a computer. Yb3＋ increased the amplitude of sodium currents in a concentration-dependent and voltage-dependent man- ner. The 50 % enhancement concentration of Yb3＋ on sodium currents was about 8.97 μmol/L, which was dif- ferent from the inhibitory effects of Yb3＋ on potassium current. The analysis on the activation and inactivation kinetics of Na＋ current showed that 100 μmol/L Yb3＋ did not change the process of activation and inactivation. In addition, the times reaching the peak of current （t） and inactivated time constant （τ） were voltage dependent. 100 μmol/L Yb3＋ significantly prolonged the time to peak at -70 and -80 mV. The effect disappeared at the positive direction of -70 mV. Furthermore, Yb3＋ decreased r val- ues to more positive values than -80 mV. In total, Yb3＋ did not change the process of activation, but impelled inacti- vated process. Yb3＋ mainly increased the Na＋ current through changing its conductance. It might be one of the mechanisms that Yb3＋ affected the hippocampal neurons.     

大鼠海马神经元neurobasal无血清的原代培养方法

目的:建立纯度和活力较高的无血清原代培养海马神经元的方法。方法:新生SD大鼠海马,用neuro-basal培养基培养,免疫荧光鉴定神经元纯度,MTT法检测其活力。结果:神经元接种12~24 h后贴壁,并长出细小突起,3 d具有典型神经元形态特征,4 d突起形成稀疏的神经纤维网络,8 d后神经元5~10个聚集成团,突起密集,生长稳定,12 d后出现细胞碎片。Tubulin荧光染色显示清晰的神经元,突起绵长且相互交织,占细胞总数的66.7%;GFAP荧光染色的细胞数量少,突起短粗,占33.7%。培养1~5 d MTT代谢率逐渐上升,6~11 d处于平台期,11 d后下降。结论:neurobasal无血清培养获得的神经元纯度大于60%,6~11 d的细胞适于细胞学实验。     

Polarized synchronous light scattering characterization of the interaction of proteins with sodium dodecyl sulfonate

In acid buffer solution, proteins with positive charge can react with anion surfactant and result in a great enhancement of synchronous light scattering (SLS) signals. In this contribution, the correlative experiment was made to compare the interaction of human serum albumin (HSA) and immunoglobulin G (IgG) with sodium dodecyl sulfonate (SDS). Based on the measurements of the polarization light scattering signals, a new method of scattering polarization was constituted to distinguish these two interaction systems with molecular weight difference (HSA 66 kDa; IgG 150 kDa). The results were con- sistent with the data measured by dynamic light scattering (DLS) technique.     

Rho激酶影响大鼠海马神经元突起生长的实时成像

目的:观察Rho激酶对大鼠海马神经元突起生长的影响.方法:体外培养新生大鼠海马神经元5 d后,连续动态观察溶血性磷脂酸(lysophosphatidic acid,LPA)及Rho激酶抑制剂Y-27632对神经元突起生长的影响.结果:对照组神经元一级突起迅速生长、延伸,不断发分支,形成丰富的二级、三级突起;用LPA处理后,神经元大部分突起进行性塌陷,一级突起逐渐缩短并且变细,二级、三级突起数量减少,且较细小;而预先用Y-27632处理后再加LPA,细胞突起塌陷的现象减少,神经元的突起不断分支、延伸,一级、二级、三级突起数量较LPA组增多且长度也增加.结论:Rho激酶参与LPA诱导海马神经元突起回缩的过程,抑制Rho激酶的活性可抑制LPA诱导的突起回缩.     

Achyranthes bidentata Blume extract promotes neuronal growth in cultured embryonic rat hippocampal neurons

We have prepared an aqueous extract of Achyranthes bidentata Blume, a commonly prescribed Chinese medicinal herb, and reported, in previous studies, that A. bidentata extract benefits nerve growth and prevents neuron apoptosis. In this study, we investigated the actions of A. bidentata extract on survival and growth of primarily cultured rat hippocampal neurons. The morphological observation revealed that neurite growth from hippocampal neurons was significantly enhanced by A. bidentata extract with similar effects to those induced by nerve growth factor (NGF), and the greatest neurite growth appeared on treatment with A. bidentata extract at 1 μg/ml for 24 h. DNA microarray analysis indicated that there were 25 upregulated genes and 47 downregulated genes exhibiting significantly differential expression in hippocampal neurons treated with A. bidentata extract at 1 μg/ml for 6 h when compared to those in untreated hippocampal neurons. Real-time quantitative RT-PCR and Western blot analysis demonstrated that the expression of growth-associated protein-43 in hippocampal neurons was upregulated at both mRNA and protein levels after treatment with A. bidentata extract, and the optimal dosage of the extract was also 1 μg/ml. These data confirm that A. bidentata extract could promote in vitro hippocampal neuronal growth in a dose- and time-dependent manner.     

偏振光与偏振器件的矩阵分析

讨论了光的偏振态的矩阵表示和偏振器件的矩阵表示方法以及它们的应用。利用坐标变换关系，能快捷地求出偏振器件的琼斯矩阵；而当偏振光依次通过几个偏振器件时，只要连续应用矩阵相乘，就可以方便地求出透射光的偏振状态。矩阵表示法对于讨论偏振光偏振态的变化非常快捷方便。     

CRMPs对神经元突起生长的调控

神经元突起是建立神经网络的物质基础,其生长为生长信号启动胞内信号促使神经元不断极化的过程.作为Rho GTPases的下游信号,CRMPs富集于神经系统,参与神经元的发育过程,可作为不同信号通路的共同受体后分子,通过改变细胞骨架的运动调控突起生长.其不同亚基的功能分化、不同亲和性特点显示其具有突起生长调控的分子开关特征...