This paper demonstrate that cortical neurons in dissociated cultures exhibited synchronized spontaneous Ca2+ spikes.The synchronized spontaneous Ca2+ spikes are synaptically driven,as it is blocked by tetrodotoxin,and by the glutamate receptor antagonist CNQX/APV.The oscillatory activity is not influenced by GABAA receptor antagonist picrotoxin,suggesting that they entirely rely on glutamatergic neurotransmission.We have also found that these Ca2+ spikes are dependent on an influx of extracellular Ca2+ but are independent of mobilization of Ca2+ from intracellular Ca2+ stores.Ca2+ entry occurred primarily through L-type voltage-gated calcium channels,since nifedipine completely blocked these Ca2+ spikes.

Objective To observe the effects of chronic restraint stress on ultrastructure of neuron in the hippocampus of rats.Methods The experimental rats were restrained for 6 hours every day.After 21days,the ultrastructural changes of the pyramidal cells in rats'hippocampus CA 3 area were observed with transmission electromicroscopy.Results The organellae of the pyramidal cells in hippocampus CA3 area of the experimental rats decreased.The mitochondria swelled and the number of their crista decreased.The lipofuscin increased in cytoplasm.The nucleus membrane sank inside, and vacuolation degeneration was found in the nucleus and cytoplasm.Conclusion chronic restraint stress leads to the degeneration of hippocampus pyramidal cells.

对培养的细胞进行免疫细胞化学反应,可以在培养皿内直接进行,亦可经树脂包埋后,在半薄切片上进行.本实验应用上述二种方法,对培养的大鼠内耳血管纹的组织细胞进行了免疫细胞化学显示. 组织细胞处理:1)平皿培养和盖玻片上培养的大鼠内耳血管纹处组织细胞,经Bouin固定液固定4h,0.01Mol/L PBS浸洗,0.1%的Triton x-100处理20min;2)平皿培养的细胞经酶处理,离心收集,2%戊二醛固定液固定4h,经丙酮脱水与锇酸固定(-20℃)、树脂包埋、半薄切片.免疫细胞化学反应:上述处理的细胞或切片进行免疫细胞化学反应,其反应步骤如下:①3%H2O2 10min;②正常羊血清封闭,室温20min;③角蛋白抗血清1:1000;波形蛋白抗血清1:10 00,4℃,24h;④先后滴加生物素化羊抗兔IgG和ABC复合物,室温各孵育45 min;⑤用0.03%H2O2和0.05%DAB反应8～10min,终止反应 .反应步骤③～⑤过程中,每步骤后均用0.01Mol/L PBS,(pH7.3)清洗.切片经酒精脱水,二甲苯透明,树脂封片. 平皿培养和盖玻片上培养的大鼠内耳切片上可见来自血管纹上皮细胞的新增生的边缘细胞, 角蛋白免疫细胞化学反应呈阳性显示;也可见来自于螺旋韧带的结缔组织细胞,波形蛋白免疫细胞化学反应呈阳性显示.用以上二种方法处理的细胞进行免疫细胞化学反应的操作过程比较:1)培养皿内直接进行免疫细胞化学反应效果好,但需要抗体多;如果是塑料平皿,培养的细胞不宜用二甲苯透明、封片,细胞进行高倍照像时有一定的困难;2)盖玻片进行免疫细胞化学反应效果好,需要抗体少,标本能完成脱水、透明、封片,高倍照像无困难;但盖玻片进行培养时,培养的细胞可能有选择性.对于半薄切片的免疫细胞化学反应,虽然实验操作较为复杂,但只要能较好保存抗原性,免疫细胞化学反应一般没有困难.根据本实验的比较分析,我们认为细胞在充分脱水时的低温处理对抗原的保存有重要意义.

本实验应用PNAg和Con Ag细胞化学技术,对大鼠海马神经细胞膜上的相应受体进行凝集素细胞化学反应. 材料与方法:雄性SD大鼠4只,体重250～300g.动物经戊巴比妥钠腹腔注射麻醉后,进行心脏灌注固定(固定液为4%多聚甲醛+0.1M PB(pH7.3).动物静置于4℃,3hr后取出大脑,用上述固定液后固定3hr.取具有海马的脑组织,用梯度酒精脱水、透明、石蜡包埋、切片.PNAg和Con Ag的制备:1)枸橼酸还原法配制 7nm胶体金;2)胶体金分别与PNA和Con A稀释液按一定比例结合;3)超速离心, 4℃,36000/rpm 45分钟;4)收集PNAg和Con Ag结合物,保存备用. PNAg和Con Ag细胞化学反应:切片脱蜡至水后,分别用PNAg液或Con Ag 液混合孵育,37℃,2hr.对照组用D-半乳糖和α-甲基甘露糖作抑制对照试验.细胞化学反应后,切片进行银加强处理. 本实验结果显示,大鼠海马CA区神经细胞PNAg和Con Ag细胞化学明显呈阳性显示,用D-半乳糖和α-甲基甘露糖作抑制对照试验的对照组结果呈阴性显示. 存在于细胞膜上复合多糖末端的糖基常是唾液酸,当细胞表面唾液酸含量出现下降时,其下方的半乳糖胺或甘露糖糖基相对暴露增加.从而可引起细胞在诸如信号转导、分化、凋亡等生物学功能和行为方面产生重要的变化.由于凝集素Con A和PNA是半乳糖基和甘露醇糖基的重要配体,因此,本实验Con Ag和PNAg细胞化学在神经细胞的成功显示不仅在细胞生物学方面,而且在神经生物学研究方面均具有很好的应用前景.