Temperature Dependence of Pyrethroid Modification of Single Sodium Channels in Rat Hippocampal Neurons

Pyrethroid modulation of sodium channels is unique in the sense that it is highly dependent on temperature, the potency being augmented by lowering the temperature. To elucidate the mechanisms underlying the negative temperature dependence of pyrethroid action, single sodium channel currents were recorded from cultured rat hippocampal neurons using the inside-out configuration of patch-clamp technique, and the effects of the pyrethroid tetramethrin were compared at 22 and 12°C. Tetramethrin-modified sodium channels opened with short closures and/or transitions to subconductance levels at 22 and 12°C. The time constants of the burst length histograms for tetramethrin-modified channels upon depolarization to −60 mV were 7.69 and 14.46 msec at 22 and 12°C, respectively (Q₁₀= 0.53). Tetramethrin at 10 μm modified 17 and 23% of channels at 22 and 12°C, respectively, indicating that the sensitivity of the sodium channel of rat hippocampal neurons to tetramethrin was almost the same as that of tetrodotoxin-sensitive sodium channels of rat dorsal root ganglion neurons and rat cerebellar Purkinje neurons. The time constants for burst length in tetramethrin-modified sodium channels upon repolarization to −100 mV from −30 mV were 8.26 and 68.80 msec at 22 and 12°C (Q₁₀= 0.12), respectively. The prolongation of tetramethrin-modified whole-cell sodium tail currents upon repolarization at lower temperature was ascribed to a prolongation of opening of each channel. Simple state models were introduced to interpret behaviors of tetramethrin-modified sodium channels. The Q₁₀ values for transition rate constants upon repolarization were extremely large, indicating that temperature had a profound effect on tetramethrin-modified sodium channels. Received: 31 January 2000/Revised: 18 May 2000     

丙二醛破坏大鼠海马神经元胞质钙离子稳态的信号机制

目的研究丙二醛（MDA）对原代培养的海马神经元胞质中钙离子稳态的破坏作用及可能的信号机制。方法以Fur2／AM为荧光指示剂,采用荧光分光光度法定量测定原代培养海马神经元胞质游离钙浓度变化。结果随着MDA浓度的升高和作用时间的延长,导致胞质中游离钙水平显著升高,破坏其钙稳态。MDA所导致的海马神经元胞质游离钙水平升高包括两个过程：100μmol／L的MDA可使胞质[Ca2＋]i水平在0—10min内的早期渐进升高过程,经历中间大约5min的平台期后,接下来15—30min的晚期显著升高。以细胞膜电压依赖的Ca2＋通道抑制剂nimodipine抑制外钙内流后,可显著抑制晚期胞质[Ca2＋]i水平的升高,以PLC的抑制剂U73122作用后,则可抑制早期胞质[Ca2＋]i水平的升高。结论100μmol／L的MDA作用下,海马神经元胞质中早期钙离子水平的升高和晚期钙离子水平的升高可能分别由不同的信号机制所介导。     

芪丹通脉片对大鼠慢性脑缺血损伤大鼠学习记忆的影响

目的:研究芪丹通脉片对慢性脑缺血所致学习记忆障碍的治疗作用及其可能机制。方法:采用双侧颈动脉结扎方法复制慢性脑缺血模型。将健康雄性SD大鼠随机分为空白对照组、模型组、假手术组、芪丹通脉片低剂量组、芪丹通脉片中剂量组、芪丹通脉片高剂量组、阳性对照尼莫地平组,应用Morris水迷宫检测大鼠学习记忆能力,HE染色观察海马神经元形态学改变。结果:与对照组比较,模型组大鼠可见显著学习记忆障碍,并可见海马神经元呈现出典型的神经病理性改变,海马区神经细胞数量减少、固缩等改变。芪丹通脉片可显著减轻慢性脑缺血所致学习记忆能力,并减其轻海马神经元损伤,且有显著剂量依赖性。结论:本实验证实芪丹通脉片可显著减轻慢性脑缺血所致学习记忆障碍,其可能机制是通过减轻海马损伤来改善学习记忆能力。     

人（Homo sapiens）海马神经元microRNA调控网络的构建及其基本性质研究

目的：建立人海马神经元中的分子相互作用调控网络,研究miRNA在这个网络中是如何与其他信号通路相互作用并形成更复杂的生物网络,以及miRNA对网络中其靶点的调控如何影响生物网络的性质。方法：通过对已发表文献实验数据的挖掘分析,获得了哺乳动物海马神经元中主要信号通路的580个组分的一组相互作用数据,以及海马神经元中的miRNA表达谱。使用PITA,Miranda,TargetScan三个miRNA靶点预测软件计算出了这580个组分中的345个miRNA靶点。使用cytoscape对这些相互作用数据建立网络并对其性质进行计算分析。结果：建成了海马神经元中一个包含633个节点1653条边的miRNA调控网络,该网络中转录因子,adapter,酶更多的受到miRNA调控。结论：人海马神经元中,miRNA主要通过对转录因子,adapter和酶进行调控,与其他信号通路相互作用形成了一个更加复杂的网络,新形成的网络的集群系数,网络异质性,网络中心化程度,平均最短路径长度,平均邻点数都发生了变化。     

海马神经元信息传递的1／f涨落

采用元胞自动机建立了脑海马神经元信息传递的计算机仿真模型,发现海马神经元从个体到群体,在时间尺度上均呈现出１／ｆ涨落。分析了模型的动力学特性,认为产生１／ｆ涨落的机制在于脑海马神经元处于自组织临界状态,即在混沌的边缘上进化。     

人参皂苷Rg1对抑郁症大鼠抑郁行为和海马神经元损伤、PKA、PKC的影响

摘要 目的：探讨与分析人参皂苷Rg1对抑郁症大鼠抑郁行为和海马神经元损伤、蛋白激酶A（PKA）与蛋白激酶C（PKC）的影响。方法：抑郁症大鼠48只随机平分为三组-模型组、实验1组、实验2组,每组16只大鼠。实验1组、实验2组每天2次灌胃给药（1 mg/mL、4 mg/mL人参皂苷Rg1）,给药体积为10 mL;模型组以相同方式按体重给予双蒸水。观察与记录鼠抑郁行为和海马神经元损伤、PKA、PKC表达变化情况。结果：实验1组、实验2组治疗第7 d、第14 d的逃避潜伏期都显著低于模型组,实验2组与实验1组相比也显著缩短（P<0.05）。实验1组、实验2组治疗第7 d、第14 d的糖水偏好率高于模型组,实验2组与实验1组相比也显著升高（P<0.05）。实验1组、实验2组治疗第7 d、第14 d的血清5-羟色胺较模型组高,血清皮质酮含量较模型组低,实验2组与实验1组对比也有明显差异（P<0.05）。实验1组、实验2组治疗第7 d、第14 d的海马神经元组织的PKA、PKC蛋白相对表达水平显著低于模型组,实验2组与实验1组相比也显著缩短（P<0.05）。结论：人参皂苷Rg1在抑郁症大鼠的应用能改善抑郁行为,增加糖水偏好率,降低逃避潜伏期,还可提高大鼠的血清5-羟色胺含量,降低血清皮质酮含量,降低海马神经元组织的PKA、PKC蛋白表达水平。     

The Synthesis and Release of Acetylcholine by Depolarized Hippocampal Slices Is Increased by Increased Choline Available In Vitro Prior to Stimulation

The objective of these experiments was to determine whether preincubating hippocampal slices with choline provides precursor that can be used during a subsequent incubation to support or enhance the synthesis of acetylcholine (ACh). Slices were preincubated for 60 min with 0, 10, 25, or 50 microM choline, washed, resuspended, and then incubated for 10 min in choline-free buffer containing 4.74 (Krebs-Ringer bicarbonate, KRB) or 25 mM KCl. The tissue contents of ACh and choline were determined prior to and after the preincubation, as well as after the incubation; the amounts of ACh and choline released were measured, and ACh synthesis was calculated. Preincubation in the absence of choline increased the tissue content of ACh to 242% of original levels; preincubation with 10 microM choline did not lead to a further increase, but preincubation with 25 or 50 microM choline increased the ACh content to 272% of original levels, significantly greater than that of slices preincubated with either 0 or 10 microM choline. When tissues were subsequently incubated for 10 min with either KRB or 25 mM KCl, ACh release from slices preincubated with 50 microM choline was greater than from slices preincubated with 0, 10, or 25 microM choline. Incubation of slices with KRB did not alter the tissue content of ACh, but when tissues were incubated with 25 mM KCl, the ACh content of slices preincubated with 0 or 10 microM choline decreased significantly, whereas that of slices preincubated with 25 or 50 microM choline did not.(ABSTRACT TRUNCATED AT 250 WORDS)     

Axonal Transport of [35S]Methionine-Labeled Proteins in Two Intra-Brain Tracts of the Rat

[³⁵S]Methionine was stereotaxically injected into the dorsalateral geniculate body (DLGB) of adult male rats, and 1 h to 10 days post-injection the DLGB and projection site (striate cortex) were dissected out and solubilized in 1% sodium dodecyl sulfate. Samples were analyzed for acid-precipitable radioactivity, and radioactivity in different molecular weight classes was determined following discontinuous gel electrophoresis on both tube and slab gels. Acid-precipitable radioactivity in the DLGB peaked by 4 h and then declined over the time period studied. The molecular weight distribution pattern was complex and did not change appreciably with time. Radioactivity in the striate cortex arrived in at least three waves: rapidly transported proteins arrived between 2 and 4 h; a second wave of transport began to arrive at about 7 h post-injection and there was a slight rise in specific activity for 2 days; finally, at 3 days post-injection, there was a steep increase with the arrival of the bulk of the transported material. The electrophoretic distribution pattern of proteins arriving in the first wave included 40–50 identifiable bands ranging in molecular weight from 13,000 to 200,000. Of particular interest was a radioactive band of apparent molecular weight of 110,000, which was prominent at 4 h, but by 12 h showed very little labeling. The second wave of radioactivity contained primarily proteins of molecular weight classes already present, although there were quantitative differences. Several proteins in the molecular weight range of 43,000 to 78,000 were identifiable as characteristic of the third wave of transported material. Results from a study following injection of a hippocampus were similar: the electrophoretic distribution pattern of radioactive proteins extracted from the injected hippocampus resembled that of the DLGB, and also did not vary appreciably with time, while radioactive proteins in the contralateral hippocampus had an electrophoretic distribution pattern similar to that of the striate cortex and changed with time in a similar manner.     

Measurement of dendritic mRNA transport using ribosomal markers

mRNA is transported to the dendritic regions by forming RNA granules, an aggregate of mRNA, ribosomal proteins, rRNA, and RNA-binding proteins such as Staufen. In this study, the dendritic transport of RNA granules was measured using the individual antibodies to ribosome-specific markers such as ribosomal L4 or S6 protein, and Y10B, a monoclonal antibody specific to rRNA. All the markers showed significant immunoreactivity in the dendritic regions of the hippocampal neurons. In addition, a GFP-tagged Staufen, a marker protein of the RNA granules, was colocalized with the Y10B and S6 signals in the dendrites. The S6 signals were also colocalized with the Y10B signals in the dendrites. Consistent with previous studies, the depolarization induced by KCl stimulation increased the ribosomal level, revealed by the S6 or Y10B immunostaining in the distal dendrites. These results demonstrate the utility of ribosomal markers for detecting the RNA granules or mRNA transport in dendrites.