Developmental regulation of NMDA receptor-mediated synaptic currents at a central synapse.

The central nervous system has extraordinary plasticity in early life. This is thought to involve N-methyl-D-aspartate (NMDA) receptors which, along with the non-NMDA receptors, mediate fast excitatory synaptic transmission. Although NMDA receptors may be transiently enhanced early in life, it has not been possible to demonstrate directly a functional change in the NMDA receptor-mediated synaptic response because of the voltage-dependence of the NMDA conductance and the overlapping inhibitory synaptic conductances. Here I report that the duration of evoked NMDA-receptor-mediated excitatory postsynaptic currents (e.p.s.cs) in the superior colliculus is several times longer at early developmental stages compared to that measured in older animals. In contrast, the amplitude of NMDA-receptor-mediated miniature e.p.s.cs does not change during development. The kinetic response of excised membrane patches to a brief activation of NMDA receptors is similar to that of the NMDA e.p.s.c, which suggests that the time course of the NMDA e.p.s.c. in the superior colliculus reflects slow NMDA channel properties as in the hippocampus. Therefore, these data indicate that the molecular properties of NMDA receptors are developmentally regulated and thus may be controlling the ability of synapses to change in early life.     

Single and multiple vesicle fusion induce different rates of endocytosis at a central synapse

During synaptic transmission, neurotransmitter-laden vesicles fuse with the presynaptic membrane and discharge their contents into the synaptic cleft. After fusion, the vesicular membrane is retrieved by endocytosis for reuse. This recycling mechanism ensures a constant supply of releasable vesicles at the nerve terminal. The kinetics of endocytosis have been measured mostly after intense or non-physiological stimulation. Here we use capacitance measurements to resolve the fusion and retrieval of single and multiple vesicles following mild physiological stimulation at a mammalian central synapse. The time constant of endocytosis after single vesicle fusion was 56 ms; after a single action potential or trains at < or = 2 Hz it was about 115 ms, but increased gradually to tens of seconds as the frequency and the number of action potentials increased. These results indicate that an increase in the rate of exocytosis at the active zone induces a decrease in the rate of endocytosis. Existing models, including inhibition of endocytosis by Ca(2+), could not account for these results our results suggest that an accumulation of unretrieved vesicles at the plasma membrane slows endocytosis. These findings may resolve the debate about the dependence of endocytosis kinetics on the stimulation frequency, and suggest a potential role of regulation of endocytosis in short-term synaptic depression.     

Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C

Neurotransmitters are released at synapses by the Ca2(+)-regulated exocytosis of synaptic vesicles, which are specialized secretory organelles that store high concentrations of neurotransmitters. The rapid Ca2(+)-triggered fusion of synaptic vesicles is presumably mediated by specific proteins that must interact with Ca2+ and the phospholipid bilayer. We now report that the cytoplasmic domain of p65, a synaptic vesicle-specific protein that binds calmodulin contains an internally repeated sequence that is homologous to the regulatory C2-region of protein kinase C (PKC). The cytoplasmic domain of recombinant p65 binds acidic phospholipids with a specificity indicating an interaction of p65 with the hydrophobic core as well as the headgroups of the phospholipids. The binding specificity resembles PKC, except that p65 also binds calmodulin, placing the C2-regions in a context of potential Ca2(+)-regulation that is different from PKC. This is a novel homology between a cellular protein and the regulatory domain of protein kinase C. The structure and properties of p65 suggest that it may have a role in mediating membrane interactions during synaptic vesicle exocytosis.     

Possible role of acetylcholinesterase in regulation of postsynaptic receptor efficacy at a central inhibitory synapse of Aplysia

Most of the effects of acetylcholinesterase (AChE) on synaptic transmission are considered to be related to its acetylcholine (ACh) hydrolysing properties. This is clearly apparent from changes which occur in the characteristics of the miniature endplate potential and of the endplate potential at neuromuscular junctions when AChE is inhibited1-4 and during the development of enzymatic AChE activity at maturing synapses5. However, we report here that after inhibiting AChE in a cholinergic synapse in Aplysia, we found an increase not only in postsynaptic responses to presynaptic stimulation and to ionophoretic application of ACh on postsynaptic receptors, but also to ionophoretic application of carbachol. This could not be explained by the inhibition of the ACh hydrolysing function of the enzyme, as carbachol is not hydrolysed by AChE. A possible explanation of these observations is that inhibition of the enzyme affects a property of the ACh receptor (AChR) itself.     

Two modes of fusion pore opening revealed by cell-attached recordings at a synapse

Fusion of a vesicle with the cell membrane opens a pore that releases transmitter to the extracellular space. The pore can either dilate fully so that the vesicle collapses completely, or close rapidly to generate 'kiss-and-run' fusion. The size of the pore determines the release rate. At synapses, the size of the fusion pore is unclear, 'kiss-and-run' remains controversial, and the ability of 'kiss-and-run' fusion to generate rapid synaptic currents is questionable. Here, by recording fusion pore kinetics during single vesicle fusion, we found both full collapse and 'kiss-and-run' fusion at calyx-type synapses. For full collapse, the initial fusion pore conductance (G(p)) was usually >375 pS and increased rapidly at > or =299 pS ms(-1). 'Kiss-and-run' fusion was seen as a brief capacitance flicker (<2 s) with G(p) >288 pS for most flickers, but within 15-288 pS for the remaining flickers. Large G(p) (>288 pS) might discharge transmitter rapidly and thereby cause rapid synaptic currents, whereas small G(p) might generate slow and small synaptic currents. These results show that 'kiss-and-run' fusion occurs at synapses and that it can generate rapid postsynaptic currents, and suggest that various fusion pore sizes help to control the kinetics and amplitude of synaptic currents.     

Coupling of agonist binding to channel gating in the GABA(A) receptor

Neurotransmitters such as acetylcholine and GABA (gamma-aminobutyric acid) mediate rapid synaptic transmission by activating receptors belonging to the gene superfamily of ligand-gated ion channels (LGICs). These channels are pentameric proteins that function as signal transducers, converting chemical messages into electrical signals. Neurotransmitters activate LGICs by interacting with a ligand-binding site, triggering a conformational change in the protein that results in the opening of an ion channel. This process, which is known as 'gating', occurs rapidly and reversibly, but the molecular rearrangements involved are not well understood. Here we show that optimal gating in the GABA(A) receptor, a member of the LGIC superfamily, is dependent on electrostatic interactions between the negatively charged Asp 57 and Asp 149 residues in extracellular loops 2 and 7, and the positively charged Lys 279 residue in the transmembrane 2-3 linker region of the alpha1-subunit. During gating, Asp 149 and Lys 279 seem to move closer to one another, providing a potential mechanism for the coupling of ligand binding to opening of the ion channel.     

Histone H2AX-dependent GABA(A) receptor regulation of stem cell proliferation

Stem cell self-renewal implies proliferation under continued maintenance of multipotency. Small changes in numbers of stem cells may lead to large differences in differentiated cell numbers, resulting in significant physiological consequences. Proliferation is typically regulated in the G1 phase, which is associated with differentiation and cell cycle arrest. However, embryonic stem (ES) cells may lack a G1 checkpoint. Regulation of proliferation in the 'DNA damage' S/G2 cell cycle checkpoint pathway is known for its role in the maintenance of chromatin structural integrity. Here we show that autocrine/paracrine gamma-aminobutyric acid (GABA) signalling by means of GABA(A) receptors negatively controls ES cell and peripheral neural crest stem (NCS) cell proliferation, preimplantation embryonic growth and proliferation in the boundary-cap stem cell niche, resulting in an attenuation of neuronal progenies from this stem cell niche. Activation of GABA(A) receptors leads to hyperpolarization, increased cell volume and accumulation of stem cells in S phase, thereby causing a rapid decrease in cell proliferation. GABA(A) receptors signal through S-phase checkpoint kinases of the phosphatidylinositol-3-OH kinase-related kinase family and the histone variant H2AX. This signalling pathway critically regulates proliferation independently of differentiation, apoptosis and overt damage to DNA. These results indicate the presence of a fundamentally different mechanism of proliferation control in these stem cells, in comparison with most somatic cells, involving proteins in the DNA damage checkpoint pathway.     

Direct modulation of Aplysia S-K+ channels by a 12-lipoxygenase metabolite of arachidonic acid

Lipoxygenase metabolites of arachidonic acid have recently been shown to modulate the activity of ion channels in nerve and muscle cells. The mechanism of action of these metabolites is, however, unknown. In sensory neurons of Aplysia, the S-K- channel is under the dual modulatory control of 5-hydroxytryptamine (5-HT), which decreases the number of active S channels through cyclic AMP-dependent phosphorylation, and the neuropeptide FMRFamide, which increases the probability of S-channel opening through the 12-lipoxygenase metabolite 12-hydroperoxyeicosatetraenoic acid (12-HPETE). Here we report that the increase in the probability of S-channel opening with FMRFamide is mimicked by application of 12-HPETE to cell-free membrane patches that lack ATP and GTP. Thus, 12-HPETE can act directly to modulate S-channel activity, independently of protein phosphorylation or dephosphorylation, G-protein activation or cyclic nucleotides.     

运用交流阻抗技术直接监测DNA杂交

介绍了基于交流阻抗技术构建非标记型脱氧核糖核酸（DNA）杂交传感器的方法.以24个碱基长度的寡聚DNA作为实验对象,将5′端巯基化的单链寡聚DNA（SH-ssDNA）探针与巯基乙酸（RSH）同时自组装到金电极表面,形成杂交识别层,利用交流阻抗技术测量出杂交前后金电极表面电子传递电阻R_et的增量作为杂交信号.实验中对DNA探针的自组装时间、杂交温度、杂交时间和阻抗测量液等实验条件进行了观察和优化;通过选择自组装液中SH-ssDNA探针和RSH的浓度,减少DNA在金电极表面的非特异性吸附,同时保证金电极表面自组装的SH-ssDNA探针有合适的疏密度,提高了杂交效率.在各优化条件下,无需扩增杂交信号,此非标记型DNA杂交传感器的检测下限为3.0×10^-14mol/L;和完全互补序列相比,一个和三个碱基错配序列分别产生55.6%和1.3%的杂交信号.     

The C(2)B Ca(2+)-binding motif of synaptotagmin is required for synaptic transmission in vivo

Synaptotagmin is a synaptic vesicle protein that is postulated to be the Ca(2+) sensor for fast, evoked neurotransmitter release. Deleting the gene for synaptotagmin (syt(null)) strongly suppresses synaptic transmission in every species examined, showing that synaptotagmin is central in the synaptic vesicle cycle. The cytoplasmic region of synaptotagmin contains two C(2) domains, C(2)A and C(2)B. Five, highly conserved, acidic residues in both the C(2)A and C(2)B domains of synaptotagmin coordinate the binding of Ca(2+) ions, and biochemical studies have characterized several in vitro Ca(2+)-dependent interactions between synaptotagmin and other nerve terminal molecules. But there has been no direct evidence that any of the Ca(2+)-binding sites within synaptotagmin are required in vivo. Here we show that mutating two of the Ca(2+)-binding aspartate residues in the C(2)B domain (D(416,418)N in Drosophila) decreased evoked transmitter release by >95%, and decreased the apparent Ca(2+) affinity of evoked transmitter release. These studies show that the Ca(2+)-binding motif of the C(2)B domain of synaptotagmin is essential for synaptic transmission.     

B(H)上在恒等算子处可乘映射的特征

设A是代数,φ是A到自身的线性映射,如果对任意的5,T∈A且ST=Z,都有φ（ST）=φ（S）φ（T）成立,则称φ在Z处可乘．本文主要证明以下结果：设H是复数域上的无限维Hilbert空间,φ是B（H）到自身强算子拓扑连续的线性满射,若φ在恒等算子I处可乘,则φ是空间自同构．     

Β(H)上在恒等算子处可乘映射的特征

设A是代数,φ是A到自身的线性映射,如果对任意的S,T∈A且ST=Z,都有φ(ST)=φ(S)φ(T)成立,则称φ在Z处可乘.本文主要证明以下结果:设H是复数域上的无限维Hilbert空间,φ是Β(H)到自身强算子拓扑连续的线性满射,若φ在恒等算子I处可乘,则φ是空间自同构.     

B(H)上在某点处左可导映射的刻画

证明了如果为B(H)中一个非平凡投影,则从B(H)到自身的范数连续的在P处左可导映射恒为0.还证明了若δ是从B(H)到自身的范数连续的在0处左可导映射,则δ(A)=Aδ(I),对于任意的A∈B(H).     

溶液共沸法直接合成较高分子量的聚乳酸

采用溶液共沸法直接缩聚制备聚乳酸。探讨了复合催化剂、溶剂用量、反应时间及反应装置对聚乳酸相对分子质量的影响。结果表明:以乳酸质量分数w=0.01的复合催化剂(其中氯化亚锡与对甲苯磺酸质量比为1∶1)为催化剂,溶剂与乳酸的体积比为0.75∶1.00,在氮气氛保护下反应35 h左右,能得到重均分子量为6.6×104的聚乳酸。     

大脑前额叶扣带回皮层调制丘脑触觉信息的途径研究

大脑前额叶扣带回皮层是一个与感觉功能密切相关的脑区,它能够对丘脑触觉反应进行调制.实验在10只戊巴比妥钠（1%）麻醉的SD雄性大鼠上进行,采用辣根过氧化物酶（horseradish peroxidase,HRP）逆行标记的方法,对扣带皮层前部调制丘脑腹侧基底核（ventrobasal,VB）感觉信息的途径进行了研究.实验结果显示,VB核可以接受扣带皮层后部的投射,而且扣带皮层前部有纤维投射到扣带皮层后部.由此提示了前扣带皮层对丘脑腹侧基底核神经元触觉信息调制的可能神经环路是：扣带皮层前部-扣带皮层后部-丘脑腹侧基底核.     

后聚合直接法制备聚L-乳酸的研究

L-乳酸预聚物在SnCl2·2H2O和对甲苯磺酸双催化体系下,用直接法熔融后聚合合成聚乳酸,研究了后聚合反应时间、预聚物相对分子质量等因素对聚合产物的影响,采用核磁及粘度计法测定聚合物相对分子质量,通过红外、核磁、X-射线衍射等手段对聚合物进行了表征及测试,表明在适宜的工艺条件下,熔融后聚合直接法合成较高相对分子质量的聚L-乳酸是可行的.     

小型永磁直流直线电动机的设计

以电机内部磁场网络划分理论为基础，分析了典型直线电机磁通密度分布。以场的分布理论为指导，对一种小型永磁磁钢直流直线电机进行了设计计算。列出了这种水型永磁磁钢直流直线电机的详细计算公式，对设计结果进行了实际参数测试。