Effects of a spider toxin (JSTX) on hippocampal CA1 neurons in vitro

The effect of a toxin (JSTX) obtained from Nephila clavata (Joro spider) on the CA1 pyramidal neurons of the hippocampus was studied using slice preparations. JSTX blocked the excitatory postsynaptic potentials (EPSPs) in the pyramidal neuron evoked by Schaffer collateral stimulation but was without effect on the antidromic action potentials or on the resting conductance. Depolarization induced by ionophoretic application of glutamate was readily suppressed by JSTX but aspartate-induced depolarization was much less sensitive to the toxin. Among preferential agonists activating 3 receptor subtypes for excitatory amino acids, quisqualate responses were most effectively suppressed by JSTX. Kainate responses were similarly suppressed but in some cells higher concentration of the toxin was needed to block the responses. N-methyl-D-aspartate (NMDA) responses were the least sensitive to JSTX but they were suppressed by +/- 2-amino-5-phosphonovaleric acid (APV). Long term potentiation (LTP) once it had taken place was not completely inhibited by APV. In the presence of JSTX, however, LTP was blocked and tetanic stimuli produced only a short-lived potentiation. In Mg2+ free solution, an orthodromic stimulation evoked repetitive spike responses which were superimposed on the depolarization following the initial spike. APV suppressed the depolarization and associated spikes leaving an orthodromic response which was sensitive to JSTX. The results suggest that JSTX blocks EPSPs in CA1 pyramidal neurons which are mediated by non-NMDA type receptors.     

螺旋CT的重建间隔对三维重建图像质量的影响

目的：通过对使用不同重建间隔处理螺旋CT的原始数据进行三维重建得到的图像技术的评价，分析重建间隔对图像质量的影响及其临床实用价值和局限性。材料与方法：收集2000．1～2002．6我院螺旋CT扫描的颈、胸、腰椎检查结果为正常的病例15例(扫描层厚分别为2mm、3mm、5mm)，利用计算机后处理技术，对同一容积性原始数据使用不同的重建间隔(0．5～7mm)处理后三维成像，并对图像技术的各个指标进行客观的评价，探讨重建间隔与图像层厚的关系及对三维重建的影响。结果：2mm扫描层厚病例的原始数据不同重建间隔重建三维图像质量有差异，但非显著性差异；3mm、5mm扫描层厚病例的原始数据不同重建间隔重建三维图像质量有显著性差异，且在一定范围内重建间隔越小，重建图像质量越好。结论：将容积性原始数据装三维软件前对数据进行必要的处理对提高图像质量是有作用的，是对传统三维重建方法的补充，具有一定的临床使用价值。     

64排螺旋CT仿真结肠镜在结肠癌诊断中的应用

目的探讨64排螺旋CT仿真结肠镜(CT virtual colonoscopy,CTVC)及其二维三维重建在结肠占位的CT表现及临床应用。方法回顾分析52例行64排螺旋CT双体位结肠扫描的结肠癌病例,所有患者均进行结肠充气仰卧位及俯卧位增强扫描,其中20例增加了左或右侧位三期扫描,将MSCT扫描原始数据传送至ADW4.4工作站采用CT仿真结肠镜(CTVC)、多平面重建(MPR)、表面遮盖显示(SSD)、透明重建显示(Raysum)4种方法进行结肠重建处理,对病灶的形态、大小、密度、结肠壁受损的程度、范围以及结肠外情况、淋巴结转移情况进行综合分析,做出准确判断。结果结肠、直肠癌52例,全部病例均经过手术及纤维结肠镜检查活检病理证实。结论 MSCT仿真结肠镜可显示结肠占位病变的形态、大小及肠壁、肠周侵犯情况,多体位扫描能使病变肠管扩张充分,更好地显示病灶的细节,提高病变检出的敏感性,为临床选择合理的治疗提供有价值的依据,是诊断结肠占位的一种有效检查方法。     

Seletracetam (ucb 44212) inhibits high-voltage–activated Ca2+ currents and intracellular Ca2+ increase in rat cortical neurons in vitro

Purpose: We analyzed the effects of seletracetam (ucb 44212; SEL), a new antiepileptic drug candidate, in an in vitro model of epileptic activity. The activity of SEL was compared to the effects of levetiracetam (LEV; Keppra), in the same assays. Methods: Combined electrophysiologic and microfluorometric recordings were performed from layer V pyramidal neurons in rat cortical slices to study the effects of SEL on the paroxysmal depolarization shifts (PDSs), and the simultaneous elevations of intracellular Ca²⁺ concentration [Ca²⁺]_i. Moreover, the involvement of high‐voltage activated Ca²⁺ currents (HVACCs) was investigated by means of patch‐clamp recordings from acutely dissociated pyramidal neurons. Results: SEL significantly reduced both the duration of PDSs (IC₅₀ = 241.0 ± 21.7 nm ) as well as the number of action potentials per PDS (IC₅₀ = 82.7 ± 9.7 nm ). In addition, SEL largely decreased the [Ca²⁺]_i rise accompanying PDSs (up to 75% of control values, IC₅₀ = 345.0 ± 15.0 nm ). Furthermore, SEL significantly reduced HVACCs in pyramidal neurons. This effect was mimicked by ω‐conotoxin GVIA and, to a lesser extent, by ω‐conotoxin MVIIC, blockers of N‐ and Q‐type HVACC, respectively. The combination of these two toxins occluded the action of SEL, suggesting that N‐type Ca²⁺ channels, and partly Q‐type subtypes are preferentially targeted. Conclusions: These results demonstrate a powerful inhibitory effect of SEL on epileptiform events in vitro. SEL showed a higher potency than LEV. The effective limitation of [Ca²⁺]_i influx might be relevant for its antiepileptic efficacy and, more broadly, for pathologic processes involving neuronal [Ca²⁺]_i overload.     

Huwe1-shRNA 靶向沉默L2.3神经干细胞的Huwe1表达

【摘要】 目的 探讨Huwe1 shRNA通过慢病毒载体转染L23神经干细胞,是否可以靶向沉默L23神经干细胞的Huwe1基因。方法 将体外培养的L23神经干细胞分成3组：huwe1 shRNA未转染组（control 组）、阴性序列转染组（control shRNA组）、huwe1 shRNA转染组（Huwe1 shRNA组）。Control组：仅常规体外培养L23神经干细胞。不将阴性序列和Huwe1 shRNA通过慢病毒载体转染L23神经干细胞。Control shRNA组：阴性序列通过慢病毒转染体外培养的L23神经干细胞。Huwe1 shRNA组：Huwe1 shRNA通过慢病毒载体成功感染体外培养L23神经干细胞。分别收集三组体外培养的L23神经干细胞,应用western blot 方法分别检测三组L23神经干细胞Huwe1蛋白的表达。 结果 与control组、control shRNA组比, Huwe1shRNA组体外培养的L23神经干细胞huwe1蛋白表达条带显著减弱、变细（P < 005）。与control组Huwe1蛋白的相对表达量比,Huwe1shRNA组Huwe1蛋白的相对表达量是control组的25%（P < 005）。与control 组比,control shRNA组和Huwe1shRNA组在慢病毒载体转染L23神经干细胞,L23神经干细胞存活率无明显下降（P>005) 结论 Huwe1 shRNA通过慢病毒载体转染L23神经干细胞,可以靶向沉默L23神经干细胞Huwe1基因,却不影响L23神经干细胞的存活率。     

Effects of tamoxifen, fluphenazine and estradiol on ATP levels in preoptic area and hypothalamic slices from ovariectomized rats

Tamoxifen, the major adjuvant drug treatment for estrogen-dependent breast cancer, has been shown previously to affect both estrogen-dependent and calcium/calmodulin-dependent pathways. In the current study, we developed an in vitro slice system to study the effects of tamoxifen on ATP levels in hypothalamic (HTH) and preoptic areas (POA) of the rat brain. Baseline data showed that, following a 2-h incubation, HTH and POA slices had comparable ATP levels to hippocampal slices, a system used extensively by researchers examining the metabolic responsiveness of the hippocampal region (HPC) of the brain. HTH–POA slice ATP levels remained steady for 2, 4 and 6 h, but fell to 11% of initial levels by 12 h. Neurons from HTH–POA slices incubated for 4 h appeared healthy and demonstrated robust protein synthesis as measured autoradiographically by incorporation of [³H]leucine. We explored the effects of tamoxifen (TAM), fluphenazine (FLU) and estradiol (E₂) on ATP levels in HTH and POA slices. The effects of TAM were complex: a 4-h incubation with 10⁻⁶ M TAM led to decreased ATP levels in HTH (but not POA), and a 4-h incubation with 10⁻⁸ M led to increased ATP levels in POA (but not HTH); a 15-min exposure to 10⁻⁶ M TAM decreased ATP levels in POA (but not HTH) slices, while the exposure of slices to the lower concentration of TAM was without effect in either area. As with higher concentrations of TAM, 4-h incubation with 10⁻⁶ M FLU decreased ATP levels in HTH (but not POA), while incubation with E₂ did not affect slice ATP levels. These data are consistent with the hypothesis that both TAM and FLU alter ATP levels in HTH slices via calmodulin- or calcium-mediated processes.     

The accuracy of 1- and 3-mm slices in coronary calcium scoring using multi-slice CT in vitro and in vivo

The accuracy of coronary calcium scoring using 16-row MSCT comparing 1- and 3-mm slices was assessed. A thorax phantom with calcium cylinder inserts was scanned applying a non-enhanced retrospectively ECG-gated examination protocol: collimation 12×0.75 mm; 120 kV; 133 mAs_eff. Thirty-eight patients were examined using the same scan protocol. Image reconstruction was performed with an effective slice thickness of 3 and 1 mm. The volume score, calcium mass and Agatston score were determined. Image noise was measured in both studies. The volume score and calcium mass varied less than the Agatston score. The overall measured calcium mass compared to the actual calcium mass revealed a relative difference of +2.0% for 1-mm slices and −1.2% for 3-mm slices. Due to increased image noise in thinner slices in the patient study (26.1 HU), overall calcium scoring with a scoring threshold of 130 HU was not feasible. Interlesion comparison showed significantly higher scoring results for thinner slices (all P<0.001). A similar accuracy comparing calcium scoring results of 1- and 3-mm slices was shown in the phantom study; therefore, the potentially necessary increase of the patient's dose in order to achieve assessable 1-mm slices with an acceptable image-to-noise-ratio appears not to be justified. The study was supported by a “START” grant from the University Hospital of Aachen, Germany.     

四物汤传统饮片汤剂与配方颗粒汤剂有效成分比较

目的比较四物汤传统饮片汤剂与其配方颗粒汤剂有效成分的含有量差异。方法四物汤传统饮片汤剂与其配方颗粒汤剂溶液的分析采用依利特SinoChrom ODB-BP柱(4.6 mm×150 mm,5μm);流动相乙腈-0.1%磷酸溶液,梯度洗脱;体积流量0.8 mL/min;检测波长215 nm(没食子酸),230 nm(芍药苷),330 nm(绿原酸、咖啡酸、阿魏酸、毛蕊花糖苷);柱温30℃。结果 6种成分在各自范围内线性关系良好(r≥0.999 6),平均加样回收率96.81%～99.01%,RSD 0.81%～1.58%。结论该方法准确稳定,重复性好,可用于四物汤的质量控制。     

Paired pulse facilitation of GABAergic IPSCs in ventral horn neurons in neonatal rat spinal cord

Whole-cell patch-clamp recording of GABAergic inhibitory postsynaptic currents (IPSCs) were made in ventral horn neurons of neonatal rat lumbar spinal cord in slice. In contrast to the hippocampus where paired pulse depression is reported to be observed for GABAergic IPSCs, double pulse stimulation of GABAergic inputs resulted in enhancement in the amplitude of the second IPSC in the spinal ventral horn. The facilitation ratio was decreased during enhanced synaptic transmission by increasing Ca²⁺ concentration in the external recording solution. Baclofen and adenosine, which are reported to depress synaptic transmission by presynaptic mechanisms, depressed IPSCs and increased the facilitation ratio. A postsynaptic manipulation such as application of bicuculline or changing the driving force did not affect the facilitation ratio. These results suggest that paired pulse facilitation of GABAergic IPSCs observed in neonatal rat spinal ventral horn appears to be based upon a mechanism similar to that underlying frequency-dependent facilitation of excitatory synaptic transmission, and is sensitive to presynaptic changes in synaptic strength.     

Calculation of calcium dynamics from single wavelength fura-2 fluorescence recordings

We describe techniques for measurements of cytosolic calcium dynamics in single current- or voltage-clamped nerve cells. The calculations of calcium dynamics are based on continuous recordings of fura-2 fluorescence intensity at one excitation wavelength after an initial reference measurement at two excitation wavelengths. We show that such single wavelength recordings are not only sufficient for the calculation of Ca²⁺ concentrations, but also lead to a superior signal-to-noise ratio at a high temporal resolution. Moreover, this strategy diminishes requirements for the experimental setup, such as a device necessary to switch quickly between excitation filters. We have applied this approach on measurements of cytosolic free Ca²⁺ in single-electrode voltage-clamped CA3 pyramidal cells in hippocampal slice cultures.