应激对大鼠海马Glu-NMDA受体通路的影响及锌的保护机制

目的：探讨光-电应激对不同锌水平大鼠海马突触体谷氨酸摄取能力及海马N-甲基-D-门冬氨酸(NMDA)受体容量和亲和力的影响。方法：通过光-电刺激建立大鼠应激模型。观察实验动物旷场行为变化。以^3H-L-Glu作为放射配体进行海马NMDA受体结合反应,以放免法测定海马突触体谷氨酸摄取能力。结果：缺锌动物在旷场中活动较少,海马NMDA受体容量减少、海马突触体谷氨酸摄取能力显著下降;与相应非应激组比较,各应激组动物在旷场中停留时间延长,水平和垂直运动呈现减少趋势、海马NMDA受体容量均有增加趋势而海马突触体谷氨酸摄取能力有下降趋势,但以上各项指标仅缺锌应激组出现统计学差异。结论：光-电应激可导致大鼠在旷场中的行为异常,在缺锌情况下,实验动物出现更严重的异常反应。表明,海马NMDA受体容量及突触体谷氨酸摄取能力的变化参与了应激反应过程,推测其机制与海马Glu-NMDA受体通路的改变有关。     

川楝素在大鼠大脑皮层匀浆中的结合位点

从驱蛔中药楝属植物根皮提取的一种三萜化合物──川楝素是一个选择性地作用于突触前的神经肌肉传递阻遏剂。根据它作用的专一性和不可逆性推测,突触前末梢应存在着川楝素的结合位点。本工作利用３Ｈ－川楝素进行的结合实验表明,大鼠大脑皮层匀浆中存在川楝素的结合位点;３Ｈ－川楝素与结合位点复合物的平衡解离常数（ＫＤ）约为２．０×１０（－７）ｍｏｌ／Ｌ;皮层匀浆离心团粒（Ｐ２＇）的重悬液含川楝素结合位点约２．７×１０（－１２）ｍｏｌ／ｍｇ蛋白质;该结合位点的Ｈｉｌｌ系数为０．９７。研究还显示,Ｐ２＇重悬液经蔗糖密度梯度超离心得到的突触体组分是川楝素结合位点的富集亚细胞组分,其比结合活性是Ｐ２＇重悬液的２至３倍。     

A fluorescence polarization study of calcium and phase behaviour in synaptosomal lipids

Steady-state fluorescence polarization of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene reported temperature-dependent lipid order in l-α-dimyristoylphosphatidylcholine, egg phosphatidylcholine and synaptosomal membranes. No change in lipid order was detected after depolarization of synaptosomes by veratridine (150 μM) even in the presence of 2 mM CaCl₂. However, Ca²⁺ reduced the mobility of a second probe, dansylated dipalmitoylphosphatidylethanolamine, in dispersions of synaptosomal lipids. This effect, which was seen at a Ca²⁺/total phospholipid ratio as low as 0.1, may represent an interaction between the cation and negatively-charged phospholipids. It is suggested that Ca²⁺ promotes a phase separation in synaptosomal lipids which may be relevant to the process of neurotransmitter release.     

衰老性记忆障碍与脑突触体内游离[Ca~(2 )]_i的相关性

采用行为观察和生化检测相结合的方法 ,在过去工作的基础上 ,研究了 12月龄和 18月龄小鼠学习记忆能力的变化和 18月龄小鼠四个脑区 (海马、大脑皮层、四叠体和小脑 )突触体内 [Ca2 ]i 的水平 ,同时还比较了老年记忆保持良好组与记忆障碍组小鼠的脑钙水平。结果表明 ,随着年龄的增长 ,小鼠的学习记忆能力显著下降 ,上述脑区 (除大脑皮层外 )突触内 [Ca2 ]i 均明显升高 ,其中老年记忆障碍小鼠脑钙水平升高最为显著。提示 ,小鼠衰老性记忆障碍可能与其脑突触体内 [Ca2 ]i 的超载有关。     

Synaptosomes secrete and uptake functionally active microRNAs via exocytosis and endocytosis pathways

In this study, we first characterized synaptosome microRNA (miRNA) profiles using microarray and qRT‐PCR. MicroRNAs were detected in isolated synaptic vesicles, and Ago2 immunoprecipitation studies revealed an association between miRNAs and Ago2. Second, we found that miR‐29a, miR‐99a, and miR‐125a were significantly elevated in synaptosome supernatants after depolarization. MiRNA secretion by the synaptosome was Ca²⁺‐dependent and was inhibited by the exocytosis inhibitor, okadaic acid. Furthermore, application of nerve growth factor increased miRNA secretion without altering the spontaneous release of miRNAs. Conversely, kainic acid decreased miRNA secretion and enhanced the spontaneous release of miRNAs. These results indicate that synaptosomes could secrete miRNAs. Finally, synthesized miRNAs were taken up by synaptosomes, and the endocytosis inhibitor Dynasore blocked this process. After incubation with miR‐125a, additional miR‐125a was bound to Ago2 in the synaptosome, and expression of the miR‐125a target gene (PSD95 mRNA) was decreased; these findings suggest that the ingested miRNAs were assembled in the RNA‐induced silencing complex, resulting in the degradation of target mRNAs. To our knowledge, this is the first study that demonstrates the secretion of miRNAs by synaptosomes under physiological stimulation and demonstrates that secreted miRNAs might be functionally active after being taken up by the synaptic fraction via the endocytic pathway.     

Multiple roles of the vesicular-SNARE TI-VAMP in post-Golgi and endosomal trafficking

SNARE (Soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are the core machinery of membrane fusion. Vesicular SNAREs (v-SNAREs) interact with their target SNAREs (t-SNAREs) to form SNARE complexes which mediate membrane fusion. Here we review the basic properties and functions of the v-SNARE TI-VAMP/VAMP7 (Tetanus neurotoxin insensitive-vesicle associated membrane protein). TI-VAMP interacts with its t-SNARE partners, particularly plasmalemmal syntaxins, to mediate membrane fusion and with several regulatory proteins especially via its amino-terminal regulatory Longin domain. Partners include AP-3, Hrb/(Human immunodeficiency virus Rev binding) protein, and Varp (Vps9 domain and ankyrin repeats containing protein) and regulate TI-VAMP’s function and targeting. TI-VAMP is involved both in secretory and endocytic pathways which mediate neurite outgrowth and synaptic transmission, plasma membrane remodeling and lysosomal secretion.     

Maintenance of brain thyroid hormone level during peripheral hypothyroid condition in adult rat

Thyroid hormones are essential for normal functioning of adult mammalian brain. The present investigation deals with the understanding of the time course of thyroid hormone homeostasis in adult rat brain. Animals were rendered hypothyroid by PTU injections (2 mg/100 g bw) for 30 consecutive days. Serum and synaptosomal T3/T4 content, synaptosomal AChE and Na+-K+-ATPase activities were determined on alternate days. While serum T4 level initially increased on the second day compared to control, serum T3 declined in a triphasic pattern; the first phase lasting from the second day to the 6th day, the second phase ended on the 14th day and last phase continued till the 30th day. Cerebro-cortical synaptosomal T3 level increased on the 2nd day from the control, attained a peak on the 4th day, remained stable until the 18th day, and abruptly declined on the 20th day. Synaptosomal T4 content remained negligible or undetected throughout. Synaptosomal membrane Na+-K+-ATPase and AChE activity exhibited an inverse relationship during the experimental regime, being much more prominent on the 2nd, 18th and 20th day coinciding with the variations in brain T3 level. Thus, the study identifies the onset of central homeostasis between the first and second day, its continuation for about 16-18 days and its termination between the 18th and 20th day.     

Properties of an ATP-dependent Ca2+ pump and (Ca2+ + Mg2+)-ATPase in brain synaptosome membrane vesicles from the bertha armyworm,Mamestra configurata Wlk

Biochemical and kinetic properties under identical substrate and reaction conditions were obtained for an ATP-dependent Ca²⁺ pump and (Ca²⁺ + Mg²⁺)-ATPase in synaptosome membrane vesicles prepared from the brain of the moth, Mamestra configurata. Both the ATP-dependent Ca²⁺ pump and (Ca²⁺ + Mg²⁺)-ATPase had single, high-affinity binding sites for ATP (K_m = 14 and 116 μM, respectively), Ca²⁺_free (K_m = 0.13 nM and 0.072 nM, respectively), and Mg²⁺ (K_m = 1.1 mM and 0.07 mM, respectively). Both systems were relatively little affected by K⁺ and were insensitive to ouabain, an inhibitor of (Na⁺ + K⁺)-ATPase. The results indicate that the ATP-dependent Ca²⁺ pump and (Ca²⁺ + Mg²⁺)-ATPase are functionally coupled in synaptic membranes and constitute a mechanism for Ca²⁺ transport in the brain of M. configurata. Although moth brain (Ca²⁺ + Mg²⁺)-ATPase is maximally active at nanomolar concentrations of free calcium ion, the enzyme retains at least one-half of its maximal activity at micromolar calcium concentrations, indicating either that the enzyme has two binding sites for calcium (a high-affinity site at nanomolar Ca²⁺_free and a low-affinity site at micromolar Ca²⁺_free), or that there are two enzymes with high and low affinity for calcium, respectively. Calcium extrusion from brain neurones of M. configurata may operate in a two-stage, concentration-dependent process in which a first stage, low-affinity pump reduces intraneuronal calcium to a concentration at which a second stage, high-affinity pump becomes activated.