Dendritic I h Selectively Blocks Temporal Summation of Unsynchronized Distal Inputs in CA1 Pyramidal Neurons

The active dendritic conductances shape the input-output properties of many principal neurons in different brain regions, and the various ways in which they regulate neuronal excitability need to be investigated to better understand their functional consequences. Using a realistic model of a hippocampal CA1 pyramidal neuron, we show a major role for the hyperpolarization-activated current, Ih, in regulating the spike probability of a neuron when independent synaptic inputs are activated with different degrees of synchronization and at different distances from the soma. The results allowed us to make the experimentally testable prediction that the Ih in these neurons is needed to reduce neuronal excitability selectively for distal unsynchronized, but not for synchronized, inputs.     

On the integration of subthreshold inputs from Perforant Path and Schaffer Collaterals in hippocampal CA1 pyramidal neurons

Using a realistic model of a CA1 hippocampal pyramidal neuron, we make experimentally testable predictions on the roles of the non-specific cation current, I _h , and the A-type Potassium current, I _A , in modulating the temporal window for the integration of the two main excitatory afferent pathways of a CA1 neuron, the Schaffer Collaterals and the Perforant Path. The model shows that the experimentally observed increase in the dendritic density of I _h and I _A could have a major role in constraining the temporal integration window for these inputs, in such a way that a somatic action potential (AP) is elicited only when they are activated with a relative latency consistent with the anatomical arrangement of the hippocampal circuitry.     

乌拉坦对大鼠海马CA1区锥体神经元自发放电的抑制作用

为了深入研究麻醉药乌拉坦对大鼠海马CA1锥体神经元自发放电的作用及其机制,分析了10 mmol/L乌拉坦对自发放电、电压门控钠通道、电压门控钾通道的作用．从自发放电信号中计算了放电频率、提取了峰峰间隔序列(ISI)并利用样品熵和去趋势波动法对ISI进行了非线性分析．结果表明,乌拉坦不仅抑制了自发放电的频率,而且降低了自发放电ISI序列的复杂度并弱化了其长时程相关性．离子通道研究结果表明,乌拉坦显著地抑制了钠通道电流(I_Na),对延迟整流钾通道电流(I_K)和瞬时外向钾通道电流(I_A)虽然也有抑制作用但无统计学意义．由于乌拉坦不影响突触传递,因此它可能通过抑制I_Na使自发放电的阈值升高而降低放电频率,同时,由于参与的通道数量或活性降低而使得ISI的复杂度下降,长时程相关性弱化．     

Role of an A-Type K+ Conductance in the Back-Propagation of Action Potentials in the Dendrites of Hippocampal Pyramidal Neurons

Action potentials elicited in the axon actively back-propagate into the dendritic tree. During this process their amplitudes can be modulated by internal and external factors. We used a compartmental model of a hippocampal CA1 pyramidal neuron to illustrate how this modulation could depend on (1) the properties of an A-type K⁺ conductance that is expressed at high density in hippocampal dendrites and (2) the relative timing of synaptic activation. The simulations suggest that the time relationship between pre- and postsynaptic activity could help regulate the amplitude of back-propagating action potentials, especially in the distal portion of the dendritic tree.     

The Nootropic Drug Vinpocetine Inhibits Veratridine-Induced [Ca2+]i Increase in Rat Hippocampal CA1 Pyramidal Cells

The alkaloid derivative vinpocetine (14-ethoxycarbonyl-(3,16-ethyl)-14,15-eburnamine; Cavinton) has a well known beneficial effect on brain function in hypoxic and ischemic conditions. While it increases CNS blood flow and improves cellular metabolism, relatively little is known about vinpocetine's underlying molecular mechanisms on the single cell level. Since apoptotic and necrotic cell damage is always preceded by an increase in [Ca²⁺]_i, this study investigated the effect of vinpocetine on [Ca²⁺]_i increases in acute brain slices. Sodium influx is an early event in the biochemical cascade that takes place during ischemia. The alkaloid veratridine can activate this Na⁺ influx, causing depolarization and increasing [Ca²⁺]_i in the cells. Therefore, it can be used to simulate an ischemic attack in brain cells. Using a cooled CCD camera-based ratio imaging system and cell loading with fura 2/AM, the effect of vinpocetine on [Ca²⁺]_i changes in single pyramidal neurons in the vulnerable CA1 region of rat hippocampal slices was investigated. Preperfusion and continuous administration of vinpocetine (10 M) significantly inhibited the elevation in [Ca²⁺]_i induced by veratridine (10 M). When the drug was administered after veratridine, it could accelerate the recovery of cellular calcium levels. Piracetam, another nootropic used in clinical practice, could attenuate the elevation of [Ca²⁺]_i only at a high, 1 mM, concentration. We have concluded that vinpocetine, at a pharmacologically relevant concentration, can decrease pathologically high [Ca²⁺]_i levels in individual rat hippocampal CA1 pyramidal neurons; this effect might contribute to the neuroprotective property of the drug.     

大鼠海马CA1区锥体细胞上一种Ca2+依赖性KATP通道

K_ATP通道在细胞的新陈代谢与膜兴奋性的耦联中起重要作用.采用膜片钳的内面向外式记录方法,在成年大鼠海马CA1区锥体细胞上记录到一种被胞浆侧ATP和甲糖宁（tolbutamide,一种K_ATP通道阻断剂）抑制的Ca²⁺依赖性钾离子通道.在细胞膜内外的K⁺浓度均为140 mmol/L时,通道的电导为(204±21) pS,翻转电位为(3.57±1.13) mV,通道无整流性.通道开放概率及ATP对通道的抑制作用均呈现电压依赖性.该K_ATP通道与以往报道的“经典”K_ATP通道有显著不同,其活动受膜电位、胞内Ca²⁺和ATP三重调节,表明这是一种新型的K_ATP通道.上述结果表明在海马神经元上至少有两种性质不同的K_ATP通道,提示神经元可能通过不同性质的K_ATP通道感受细胞内的代谢状态,进而调节细胞膜的兴奋性.     

The Serotonin 1A A Receptor: A Representative Member of the Serotonin Receptor Family

1. Serotonin is an intrinsically fluorescent biogenic amine that acts as a neurotransmitter and is found in a wide variety of sites in the central and peripheral nervous system. Serotonergic signaling appears to play a key role in the generation and modulation of various cognitive and behavioral functions.2. Serotonin exerts its diverse actions by binding to distinct cell surface receptors which have been classified into many groups. The serotonin_1A (5-HT_1A) receptor is the most extensively studied of the serotonin receptors and belongs to the large family of seven transmembrane domain G-protein coupled receptors.3. The tissue and sub-cellular distribution, structural characteristics, signaling of the serotonin_1A receptor and its interaction with G-proteins are discussed.4. The pharmacology of serotonin_1A receptors is reviewed in terms of binding of agonists and antagonists and sensitivity of their binding to guanine nucleotides.5. Membrane biology of 5-HT_1A receptors is presented using the bovine hippocampal serotonin_1A receptor as a model system. The ligand binding activity and G-protein coupling of the receptor is modulated by membrane cholesterol thereby indicating the requirement of cholesterol in maintaining the receptor organization and function. This, along with the reported detergent resistance characteristics of the receptor, raises important questions on the role of membrane lipids and domains in the function of this receptor.     

Identification of gibberellin A4 in Pisum sativum L. and the effects of applied gibberellins A9, A4, A5 and A3 on the le mutant

Gibberellin A₄ (GA₄) was identified for the first time in the garden pea (Pisum sativum) L.), by gas chromatography-mass spectrometry. However, in wild-type shoots the level of GA₄ was only about 6% of the level of GA₁, and it is therefore unlikely that GA₄ plays a major role per se in the control of pea stem elongation. In shoots of the le mutant, GA₄ was not detected, while the level of GA₉ was approximately twice that found in the wild-type. The le mutation also markedly reduced the elongation response to applied GA₉. It appears, therefore, that in Pisum the le mutation blocks the 3-hydroxylation of GA₉ to GA₄, in addition to the 3-hydroxylation of GA₂₀ to GA₁. In contrast, the le mutation did not reduce the response to applied GA₅, suggesting the step GA₅ to GA₃ is not catalysed by the enzyme controlled by the Le gene. The step GA₅ to GA₃ was confirmed in peas by metabolite analysis after treatment with deuterated GA₅.     

The behavior of R-ovalbumin and its individual components A1, A2, and A3 in urea solution: kinetics and equilibria

Procedures are described for the isolation of the individual components A₁, A₂, and A₃ of native R-ovalbumin from freshly laid domestic hen eggs. Because heavy metal ion contaminants result in spurious irreproducible kinetics, particularly at high pH, considerable care is taken to avoid their presence. Kinetics studies are made of the behavior of whole R-ovalbumin and its individual components in urea solution over the pH range 3.7–9.6 following the reaction by determining absorbance differences at 233, 287, and 293 nm and ORD and CD changes at 350 and 221 nm, respectively. Reaction is rapid at low pH, slowing with increasing pH. Except under limited conditions, the reaction is not simple first order. Equations are presented for describing the reactions, and the nature of the reaction products is considered. Unfolding equilibrium profiles were also determined by ORD at several wavelengths and were not stigmoidal in shape and the normalized curves were not superimposed.Deceased December 8, 2001     

Ligand Binding and G-protein Coupling of the Serotonin1A Receptor in Cholesterol-enriched Hippocampal Membranes

The serotonin_1A receptor is the most extensively studied member of the family of seven transmembrane domain G-protein coupled serotonin receptors. Since a large portion of such transmembrane receptors remains in contact with the membrane lipid environment, lipid–protein interactions assume importance in the structure-function analysis of such receptors. We have earlier reported the requirement of cholesterol for serotonin_1A receptor function in native hippocampal membranes by specific depletion of cholesterol using methyl- β-cyclodextrin. In this paper, we monitored the serotonin_1A receptor function in membranes that are enriched in cholesterol using a complex prepared from cholesterol and methyl-β-cyclodextrin. Our results indicate that ligand binding and receptor/G-protein interaction of the serotonin_1A receptor do not exhibit significant difference in native and cholesterol-enriched hippocampal membranes indicating that further enrichment of cholesterol has little functional consequence on the serotonin_1A receptor function. These results therefore provide new information on the effect of cholesterol enrichment on the hippocampal serotonin_1A receptor function.     

心室细胞生成的生物起搏器的仿真研究

植入电子起搏器可以治疗因窦房结功能失常引起的猝死等心脏疾病.但是,电子起搏器存在很多弊端,比如电池寿命有限、容易感染等.因此,生物起搏器被期待能够取代电子起搏器.为了探讨在心室内诱导心室细胞生成生物起搏器的可行性,我们首先抑制内向整流钾电流(I_(K1)),使心室肌细胞产生起搏行为,然后基于理想心室组织和真实人体心室切片数据,构建2D生物起搏器模型.基于该模型,我们研究细胞间的电偶联和起搏电流I_f对起搏器功能的影响.发现起搏电流I_f对起搏器功能有增强作用,但细胞间的弱电偶联对起搏器的起搏有更为关键的影响.     

Nucleotide P2Y1 receptor agonists are in vitro and in vivo prodrugs of A1/A3 adenosine receptor agonists: implications for roles of P2Y1 and A1/A3 receptors in physiology and pathology

Rapid phosphoester hydrolysis of endogenous purine and pyrimidine nucleotides has challenged the characterization of the role of P2 receptors in physiology and pathology. Nucleotide phosphoester stabilization has been pursued on a number of medicinal chemistry fronts. We investigated the in vitro and in vivo stability and pharmacokinetics of prototypical nucleotide P2Y₁ receptor (P2Y₁R) agonists and antagonists. These included the riboside nucleotide agonist 2-methylthio-ADP and antagonist MRS2179, as well as agonist MRS2365 and antagonist MRS2500 containing constrained (N)-methanocarba rings, which were previously reported to form nucleotides that are more slowly hydrolyzed at the α-phosphoester compared with the ribosides. In vitro incubations in mouse and human plasma and blood demonstrated the rapid hydrolysis of these compounds to nucleoside metabolites. This metabolism was inhibited by EDTA to chelate divalent cations required by ectonucleotidases for nucleotide hydrolysis. This rapid hydrolysis was confirmed in vivo in mouse pharmacokinetic studies that demonstrate that MRS2365 is a prodrug of the nucleoside metabolite AST-004 (MRS4322). Furthermore, we demonstrate that the nucleoside metabolites of MRS2365 and 2-methylthio-ADP are adenosine receptor (AR) agonists, notably at A₃ and A₁ARs. In vivo efficacy of MRS2365 in murine models of traumatic brain injury and stroke can be attributed to AR activation by its nucleoside metabolite AST-004, rather than P2Y₁R activation. This research suggests the importance of reevaluation of previous in vitro and in vivo research of P2YRs and P2XRs as there is a potential that the pharmacology attributed to nucleotide agonists is due to AR activation by active nucleoside metabolites.

     

Lymphocyte subsets and T(h)1/T(h)2 immune responses in patients with adenocarcinoma of the oesophagus or oesophagogastric junction: relation to pTNM stage and clinical outcome

Introduction Recent studies have indicated that the cytokines produced by CD4⁺ T helper type 1 (T_h1) and type 2 (T_h2) cells are critically important in antitumour immunity and perhaps clinical outcome. From this perspective, we investigated the immunocompetence of patients with previously untreated cancer of the oesophagus or oesophagogastric junction (OGJ) in relation to stage of disease and postoperative survival.Methods Blood samples were taken prior to surgery from 32 patients with adenocarcinoma of the oesophagus or OGJ. Ten healthy volunteers served as normal controls. T-cell and monocyte subpopulations were determined using flow cytometry. Monocyte as well as T_h1- and T_h2-lymphocyte cytokine levels were assessed in stimulated whole blood cultures.Results Absolute T-cell and monocyte (subset) counts as well as monocyte cytokine levels were similar among patients and controls. Production of T_h1-type cytokines was higher in patients than in controls (IFN-, p=0.01; IL-2, p=0.05), whereas T_h2-type cytokine levels were comparable (IL-4, p=0.5; IL-13, p=0.3). T-cell CD4⁺/CD8⁺ ratios decreased as pTNM stage worsened (stage I/II vs stage III/IV, p=0.009). Of all measured immunological parameters, only IL-2 production significantly affected both overall survival (p=0.015) and disease-free survival (p=0.0062). High IL-2 levels corresponded with a favourable prognosis.Conclusions Patients awaiting surgery for adenocarcinoma of the oesophagus or oesophagogastric junction demonstrated a shift in the T_h1/T_h2 balance—in favour of T_h1—compared with healthy volunteers. The ability of T cells to produce IL-2 was related to survival indicating a crucial role of T_h1-type cells in antitumour immunosurveillance.     

磁耦合谐振式无线电能传输磁场对海马齿状回区神经元K+通道的影响

目的 磁耦合谐振式无线电能传输(magnetically-coupled resonant wireless power transfer,MCR-WPT)具有传输距离较大、传输效率高、穿透性好等优点,为人们生活带来便利的同时其引起的生物电磁效应安全问题备受人们关注。本文通过研究MCR-WPT电磁环境对小鼠海马齿状回(DG)区神经元K⁺通道特性的影响,为无线电能传输(WPT)技术的发展及其合理开发应用提供实验依据。方法 每天对小鼠不间断辐射5 h,持续30 d,对比分析对照组和磁场暴露5 d、15 d、30 d组小鼠的学习记忆能力、海马DG区神经元的瞬时外向K⁺通道电流(I_A)和延迟整流K⁺通道电流(I_K)的变化。结果 磁场暴露组瞬时外向K⁺通道的激活过程受到抑制、延迟整流K⁺通道的激活特性向去极化方向移动,减少K⁺的外流,增强了神经兴奋性,但磁场暴露组小鼠行为上无显著性变化。结论 长期处于MCR-WPT电磁环境...     

Ontogenetic variation in levels of gibberellin A1 in Pisum

The levels of the biologically active gibberellin (GA), GA₁, and of its precursor, GA₂₀, were monitored at several stages during ontogeny in the apical portions of isogenic tall (Le) and dwarf (le) peas (Pisum sativum L.) using deuterated internal standards and gas chromatography-selected ion monitoring. The levels of both GAs were relatively low on emergence and on impending apical arrest. At these early and late stages of development the internodes were substantially shorter than at intermediate stages, but were capable of large responses to applied GA₃. Tall plants generally contained 10–18 times more GA₁ and possessed internodes 2–3 times longer than dwarf plants. Further, dwarf plants contained 3–5 times more GA₂₀ than tall plants. No conclusive evidence for the presence of GA₃ or GA₅ could be obtained, even with the aid of [²H₂]GA₃ and [²H₂]GA₅ internal standards. If GA₃ and GA₅ were present in tall plants, their levels were less than 0.5% and 1.4% of the level of GA₁, respectively. Comparison of the effects of gene le on GA₁ levels and internode length with the effects of ontogeny on these variables shows that the ontogenetic variation in GA₁ content was sufficient to account for much of the observed variation in internode length within the wild-type. However, evidence was also obtained for substantial differences in the potential length of different internodes even when saturating levels of exogenous GA₃ were present.Abreviations GA_n gibberellin A_n We thank Noel Davies, Omar Hasan, Leigh Johnson, Katherine McPherson and Naomi Lawrence for technical help, Professor L. Mander (Australian National University, Canberra) for deuterated GA standards and the Australian Research Council for financial assistance.     

Influence of A1 cytoplasmic substitution on the downy-mildew incidence of pearl millet

Large-scale cultivation of pearl millet [Pennisetum glaucum (L.) R. Br. F₁ hybrids in India has led to increased incidence of downy-mildew (Sclerospora graminicola). There is concern that the A₁ male-sterile cytoplasm used in all the hybrids released so far is responsible for this increase. The influence of A₁ malesterile cytoplasm on downy-mildew incidence in pearl millet was studied by comparing the disease reaction of 40 pairs of F₁ hybrids, each pair carrying respectively a₁ male-sterile and normal B cytoplasm. Mean downy-mildew incidence was similar in the hybrids carrying either A₁ male-sterile or B cytoplasm. The general combining ability of lines with and without A₁ cytoplasm was found to be similar for downy-mildew incidence. These results indicated that in pearl millet A₁ cytoplasm is not associated with increased downymildew incidence. The possible danger of using only one source of cytoplasm has been briefly discussed.     

Structural and functional analysis of the coupling subunit F in solution and topological arrangement of the stalk domains of the methanogenic A1AO ATP synthase

The first low-resolution shape of subunit F of the A₁A_O ATP synthase from the archaeon Methanosarcina mazei Gö1 in solution was determined by small angle X-ray scattering. Independent to the concentration used, the protein is monomeric and has an elongated shape, divided in a main globular part with a length of about 4.5 nm, and a hook-like domain of about 3.0 nm in length. The subunit-subunit interaction of subunit F inside the A₁A_O ATP synthase in the presence of 1-ethyl-3-(dimethylaminopropyl)-carbodiimide EDC was studied as a function of nucleotide binding, demonstrating movements of subunits F relative to the nucleotide-binding subunit B. Furthermore, in the intact A₁A_O complex, crosslinking of subunits D-E, A-H and A-B-D was obtained and the peptides, involved, were analyzed by MALDI-TOF mass spectrometry. Based on these data the surface of contact of B-F could be mapped in the high-resolution structure of subunit B of the A₁A_O ATP synthase.     

The Human Serotonin 1A Receptor Expressed in Neuronal Cells: Toward a Native Environment for Neuronal Receptors

1. The serotonin_1A (5-HT_1A) receptor is an important representative of G-protein coupled family of receptors. It is the most extensively studied among the serotonin receptors, and appears to be involved in various behavioral and cognitive functions.2. We report here the pharmacological and functional characterization of the human serotonin_1A receptor stably expressed in HN2 cell line, which is a hybrid cell line between hippocampal cells and mouse neuroblastoma.3. Our results show that serotonin_1A receptors in HN2-5-HT_1AR cells display ligand-binding properties that closely mimic binding properties observed with native receptors. We further demonstrate that the differential discrimination of G-protein coupling by the specific agonist and antagonist, a hallmark of the native receptor, is maintained for the receptor in HN2-5-HT_1AR cells. Importantly, the serotonin_1A receptor in HN2-5-HT_1AR cells shows efficient downstream signalling by reducing cellular cyclic AMP levels.4. We conclude that serotonin_1A receptors expressed in HN2-5-HT_1AR cells represent a useful model system to study serotonin_1A receptor biology, and is a potential system for solubilization and purification of the receptor in native-like membrane environment.     

CNS Adenosine A1 Receptors Are Altered After the Administration of Convulsant 3-Mercaptopropionic Acid and Cyclopentyladenosine: An Autoradiographic Study

Rat CNS adenosine A₁ receptors were studied by quantitative autoradiography after the administration of convulsant 3-mercaptopropionic acid (MP) and an adenosine analogue cyclopentyladenosine (CPA), using 2-chloro-N⁶-[cyclopentyl-2,3,4,5-³H adenosine]-([³H]CCPA) as radioactive ligand. Specific binding was quantified in hippocampus, cerebellum, cerebral cortex, thalamic nuclei, superior colliculus and striatum, and the highest densities were found in CA1, CA2, and CA3 hippocampus subareas and the lowest levels in superior colliculus and striatum. MP administration (150 mg/kg, i.p.) produced significant increases in [³H]CCPA binding in CA1 subarea at seizure (15%) and postseizure (21%) and in CA2 at seizure (15%) but a tendency to decrease in dentate gyrus. There was an increase in cerebellum at seizure (18%) but no significant changes in the other studied regions. CPA injection (2 mg/kg, i.p.) enhanced [³H]CCPA binding in CA1 and CA2 areas (17–18%) but not in CA3 area of the hippocampus. When CPA was administered before MP, which delayed seizure onset, an increase in [³H]CCPA binding in CA1 hippocampus subarea (19%) and cerebellum (28%) was also observed. Results showed that the administration of convulsant MP and adenosine analogue CPA exerts differential effects on adenosine A₁ receptors in CNS areas; hippocampus is the most affected area with all treatments, specially CA1 subarea, supporting an essential role in convulsant activity as well as in seizure prevention.