Presynaptic enhancement shown by whole-cell recordings of long-term potentiation in hippocampal slices

Long-term potentiation (LTP) of synaptic transmission in the hippocampus is a widely studied model system for understanding the cellular mechanisms of memory. In region CA1, LTP is triggered postsynaptically by Ca2(+)-dependent activation of protein kinases, but the locus of persistent modification remains controversial. Statistical analysis of synaptic variability has been proposed as a means of settling this debate, although a major obstacle has been the poor signal-to-noise ratio of conventional intracellular recordings. We have applied the whole-cell voltage clamp technique to study synaptic transmission in conventional hippocampal slices (compare refs 28-30). Here we report that robust LTP can be recorded with much improved signal resolution and biochemical access to the postsynaptic cell. Prolonged dialysis of the postsynaptic cell blocks the triggering of LTP, with no effect on expression of LTP. The improved signal resolution unmasks a large trial-to-trial variability, reflecting the probabilistic nature of transmitter release. Changes in the synaptic variability, and a decrease in the proportion of synaptic failures during LTP, suggest that transmitter release is significantly enhanced.     

Single cocaine exposure in vivo induces long-term potentiation in dopamine neurons

How do drugs of abuse modify neural circuitry and thereby lead to addictive behaviour? As for many forms of experience-dependent plasticity, modifications in glutamatergic synaptic transmission have been suggested to be particularly important. Evidence of such changes in response to in vivo administration of drugs of abuse is lacking, however. Here we show that a single in vivo exposure to cocaine induces long-term potentiation of AMPA (alpha-amino-3-hydroxy-5-methyl-isoxazole propionic acid)-receptor-mediated currents at excitatory synapses onto dopamine cells in the ventral tegmental area. Potentiation is still observed 5 but not 10 days after cocaine exposure and is blocked when an NMDA (N-methyl-d-aspartate) receptor antagonist is administered with cocaine. Furthermore, long-term potentiation at these synapses is occluded and long-term depression is enhanced by in vivo cocaine exposure. These results show that a prominent form of synaptic plasticity can be elicited by a single in vivo exposure to cocaine and therefore may be involved in the early stages of the development of drug addiction.     

Glutamate-induced long-term potentiation of the frequency of miniature synaptic currents in cultured hippocampal neurons.

Glutamate application at synapses between hippocampal neurons in culture produces long-term potentiation of the frequency of spontaneous miniature synaptic currents, together with long-term potentiation of evoked synaptic currents. The mini frequency potentiation is initiated postsynaptically and requires activity of NMDA receptors. Although the frequency of unitary quantal responses increases strongly, their amplitude remains little changed with potentiation. Tests of postsynaptic responsiveness rule out recruitment of latent glutamate receptor clusters. Thus, postsynaptic induction can lead to enhancement of presynaptic transmitter release. The sustained potentiation of mini frequency is expressed even in the absence of Ca2+ entry into presynaptic terminals.     

An essential role for postsynaptic calmodulin and protein kinase activity in long-term potentiation

The phenomenon of long-term potentiation (LTP), a long lasting increase in the strength of synaptic transmission which is due to brief, repetitive activation of excitatory afferent fibres, is one of the most striking examples of synaptic plasticity in the mammalian brain. In the CA1 region of the hippocampus, the induction of LTP requires activation of NMDA (N-methyl-D-aspartate) receptors by synaptically released glutamate with concomitant postsynaptic membrane depolarization. This relieves the voltage-dependent magnesium block of the NMDA-receptor ion channel, allowing calcium to flow into the dendritic spine. Although calcium has been shown to be a necessary trigger for LTP (refs 11, 12), little is known about the immediate biochemical processes that are activated by calcium and are responsible for LTP. The most attractive candidates have been calcium/calmodulin-dependent protein kinase II (CaM-KII) (refs 13-16), protein kinase C (refs 17-19), and the calcium-dependent protease, calpain. Extracellular application of protein kinase inhibitors to the hippocampal slice preparation blocks the induction of LTP (refs 21-23) but it is unclear whether this is due to a pre- and/or postsynaptic action. We have found that intracellular injection into CA1 pyramidal cells of the protein kinase inhibitor H-7, or of the calmodulin antagonist calmidazolium, blocks LTP. Furthermore, LTP is blocked by the injection of synthetic peptides that are potent calmodulin antagonists and inhibit CaM-KII auto- and substrate phosphorylation. These findings demonstrate that in the postsynaptic cell both activation of calmodulin and kinase activity are required for the generation of LTP, and focus further attention on the potential role of CaM-KII in LTP.     

The secreted form of the Alzheimer's amyloid precursor protein with the Kunitz domain is protease nexin-II

The A4 protein (or beta-protein) is a 42- or 43-amino-acid peptide present in the extracellular neuritic plaques in Alzheimer's disease and is derived from a membrane-bound amyloid protein precursor (APP). Three forms of APP have been described and are referred to as APP695, APP751 and APP770, reflecting the number of amino acids encoded for by their respective complementary DNAs. The two larger APPs contain a 57-amino-acid insert with striking homology to the Kunitz family of protease inhibitors. Here we report that the deduced amino-terminal sequence of APP is identical to the sequence of a cell-secreted protease inhibitor, protease nexin-II (PN-II). To confirm this finding, APP751 and APP695 cDNAs were over-expressed in the human 293 cell line, and the secreted N-terminal extracellular domains of these APPs were purified to near homogeneity from the tissue-culture medium. The relative molecular mass and high-affinity binding to dextran sulphate of secreted APP751 were consistent with that of PN-II. Functionally, secreted APP751 formed stable, non-covalent, inhibitory complexes with trypsin. Secreted APP695 did not form complexes with trypsin. We conclude that the secreted form of APP with the Kunitz protease inhibitor domain is PN-II.     

Structural basis of long-term potentiation in single dendritic spines

Dendritic spines of pyramidal neurons in the cerebral cortex undergo activity-dependent structural remodelling that has been proposed to be a cellular basis of learning and memory. How structural remodelling supports synaptic plasticity, such as long-term potentiation, and whether such plasticity is input-specific at the level of the individual spine has remained unknown. We investigated the structural basis of long-term potentiation using two-photon photolysis of caged glutamate at single spines of hippocampal CA1 pyramidal neurons. Here we show that repetitive quantum-like photorelease (uncaging) of glutamate induces a rapid and selective enlargement of stimulated spines that is transient in large mushroom spines but persistent in small spines. Spine enlargement is associated with an increase in AMPA-receptor-mediated currents at the stimulated synapse and is dependent on NMDA receptors, calmodulin and actin polymerization. Long-lasting spine enlargement also requires Ca2+/calmodulin-dependent protein kinase II. Our results thus indicate that spines individually follow Hebb's postulate for learning. They further suggest that small spines are preferential sites for long-term potentiation induction, whereas large spines might represent physical traces of long-term memory.     

Different voltage-dependent thresholds for inducing long-term depression and long-term potentiation in slices of rat visual cortex

In the hippocampus and neocortex, high-frequency (tetanic) stimulation of an afferent pathway leads to long-term potentiation (LTP) of synaptic transmission. In the hippocampus it has recently been shown that long-term depression (LTD) of excitatory transmission can also be induced by certain combinations of synaptic activation. In most hippocampal and all neocortical pathways studied so far, the induction of LTP requires the activation of N-methyl-D-aspartate (NMDA) receptor-gated conductances. Here we report that LTD can occur in neurons of slices of the rat visual cortex and that the same tetanic stimulation can induce either LTP or LTD depending on the level of depolarization of the postsynaptic neuron. By applying intracellular current injections or pharmacological disinhibition to modify the depolarizing response of the postsynaptic neuron to tetanic stimulation, we show that the mechanisms of induction of LTD and LTP are both postsynaptic. LTD is obtained if postsynaptic depolarization exceeds a critical level but remains below a threshold related to NMDA receptor-gated conductances, whereas LTP is induced if this second threshold is reached.     

Brain-state- and cell-type-specific firing of hippocampal interneurons in vivo

Neural-network oscillations at distinct frequencies have been implicated in the encoding, consolidation and retrieval of information in the hippocampus. Some GABA (gamma-aminobutyric acid)-containing interneurons fire phase-locked to theta oscillations (4-8 Hz) or to sharp-wave-associated ripple oscillations (120-200 Hz), which represent different behavioural states. Interneurons also entrain pyramidal cells in vitro. The large diversity of interneurons poses the question of whether they have specific roles in shaping distinct network activities in vivo. Here we report that three distinct interneuron types--basket, axo-axonic and oriens-lacunosum-moleculare cells--visualized and defined by synaptic connectivity as well as by neurochemical markers, contribute differentially to theta and ripple oscillations in anaesthetized rats. The firing patterns of individual cells of the same class are remarkably stereotyped and provide unique signatures for each class. We conclude that the diversity of interneurons, innervating distinct domains of pyramidal cells, emerged to coordinate the activity of pyramidal cells in a temporally distinct and brain-state-dependent manner.     

Novel form of long-term potentiation produced by a K+ channel blocker in the hippocampus.

Long-term potentiation (LTP) of synaptic transmission in the hippocampus is a widely studied model of memory processes. In the CA1 region, LTP is triggered by the entry of Ca2+ through N-methyl-D-aspartate (NMDA) receptor channels and maintained by the activation of Ca2(+)-sensitive intracellular messengers. We now report that in CA1, a transient block by tetraethylammonium of IC, IM and the delayed rectifier (IK) produces a Ca2(+)-dependent NMDA-independent form of LTP. Our results suggest that this new form of LTP (referred as to LTPK) is induced by a transient enhanced release of glutamate which generates a depolarization by way of the non-NMDA receptors and the consequent activation of voltage-dependent Ca2+ channels.     

植物病原丝状真菌分泌蛋白及CAZymes的研究进展

为了明确对植物病原丝状真菌分泌蛋白及CAZymes进一步研究的方向,笔者对丝状真菌分泌蛋白及CAZymes基本特征、分类情况以及预测方法、功能等方面进行综述。基于前人对于植物病原丝状真菌分泌蛋白以及CAZymes的报道,通过文献计量分析方法、SMART保守结构域和motif分析等方法,对丝状真菌分泌蛋白及CAZymes进行了综合分析,统计发现国内关于分泌蛋白的研究较多,对于CAZymes的研究报道较少,诸多分泌蛋白在病原菌侵染致病过程中发挥的功能如何尚未得到全面解析,有关CAZymes的研究也是如此,预测方法基本上是基于生物信息学分析,尚缺少有力的生物学试验验证证据。因此,今后研究需要进一步完善对于分泌蛋白及CAZymes预测的生物信息学分析工具或算法以及开展生物学试验验证; 通过比较植物病原菌与生防菌之间CAZymes数量及功能差异,明确不同菌之间的共性与特性; 此外,学术界应对丝状真菌中CAZymes的分类开展进一步细化研究,以及对比分析不同亚家族的分布情况,更好地解析CAZymes的功能。     

电针预处理对老龄大鼠长期术后认知功能障碍影响及相关机制

术后认知功能障碍(postoperative cognitive dysfunction, POCD)是老年人群常见的并发症。一种胆碱能依赖信号,α7-烟碱乙酰胆碱受体(α7-nAChR),已被认为在各种神经系统疾病中调节认知过程。为探讨电针预处理是否能改善老龄大鼠术后长期认知功能障碍以及其可能机制。通过将60只清洁级雄性SD(Sprague-Dawley)大鼠(20月龄)随机分为5组(样本数n=12):假手术组(S组)、模型组(M组)、电针组(EA组)、银环蛇毒+电针组(Y+EA组)、银环蛇毒+模型组(Y+M组)。在术后30 d后,分别对各组采用新物体识别实验评价认知功能,采用尼氏(Nissl)染色检测神经元数量,免疫荧光检测小胶质细胞活化、蛋白质印迹法(Western blot)检测海马IL-6、NF-κB和IL-1β的水平。实验结果表明：电针预处理增加了对新物体的探索,恢复了大鼠术后神经元的数量,降低了小胶质细胞激活,降低海马炎性因子蛋白表达,而α-BGT部分逆转了电针的作用。可见电针预处理改善了老龄大鼠长期术后认知功能障碍和海马炎症,其机制可能与激活α7nAChR介导的胆碱能抗...     

Degeneration in vitro of post-mitotic neurons overexpressing the Alzheimer amyloid protein precursor.

A pathological hallmark of Alzheimer's disease is the deposition of amyloid fibrils in the brain. The principal component of amyloid fibrils is beta/A4 amyloid protein, which can be generated by the aberrant processing of a large membrane-bound glycoprotein, the beta/A4 amyloid protein precursor (APP)3. To test whether overexpression of APP generates abnormally processed derivatives that affect the viability of neurons, we stably transfected full-length human APP complementary DNA into murine embryonal carcinoma P19 cells. These cells differentiate into post-mitotic neurons and astrocytes after exposure to retinoic acid. When differentiation of the APP cDNA-transfected P19 cells was induced, all neurons showed severe degenerative changes and disappeared within a few days. The degenerating neurons contained large amounts of APP derivatives that were truncated at the amino terminus and encompassed the entire beta/A4 domain. These results suggest that post-mitotic neurons are vulnerable to overexpressed APP, which undergoes aberrant processing to generate potentially amyloidogenic fragments.     

An in vivo recombinant RNA capable of autocatalytic synthesis by Q beta replicase

A variety of small RNAs ranging from tens to hundreds of nucleotides in length grow autocatalytically in a Q beta replicase (Q beta phage RNA-dependent RNA polymerase) reaction in the absence of added template, and similar RNAs are found in Q beta phage-infected Escherichia coli cells. Three such RNAs have been sequenced. One of them that is 221 nucleotides (nt) long ('MDV-1' RNA) has been found to be partially homologous to Q beta phage RNA 8, which might be considered as an indication of its origination from by-products of the Q beta RNA replication. To gain further insight into the origin and function of these RNAs, we have sequenced a new RNA, 120 nt long, isolated from the products of spontaneous synthesis by the nominally RNA-free Q beta replicase preparation. The minus strand of this RNA appeared to be a recombinant RNA, composed of the internal fragment of Q beta RNA (approximately 80 nt long) and the 33-nt-long 3'-terminal fragment of E. coli tRNA(1Asp). This seems to be the first strong indication of RNA recombination in bacterial cells. The various implications of this finding are discussed.     

Formation of amyloid-like fibrils in COS cells overexpressing part of the Alzheimer amyloid protein precursor

A pathological hallmark of Alzheimer's disease is the deposition of amyloid fibrils in the brain. The principal component of the amyloid fibril is beta/A4 protein, which is derived from a large membrane-bound glycoprotein, Alzheimer amyloid protein precursor (APP). Although the deposition of amyloid is thought to result from the aberrant processing of APP, the detailed molecular mechanisms of amyloidogenesis remain unclear. A C-terminal fragment of APP which spans the beta/A4 and cytoplasmic domains has a tendency to self-aggregate. In an attempt to establish a cultured-cell model for amyloid fibril formation, we have transfected COS-1 cells with complementary DNA encoding the C-terminal 100 residues of APP. In the perinuclear regions of a small population of DNA-transfected cells, we observed inclusion-like deposits which showed a strong immunohistochemical reaction towards an anti-C-terminal APP antibody or an anti-beta/A4 amyloid core-specific antibody. Electron microscope observations of the inclusion-carrying cells revealed an accumulation of amyloid-like fibrils of 8-22 nm diameter near and on the nuclear membrane. The fibrils showed a beaded or helical structure, and reacted positively with the anti-C-terminus antibody by immunoelectron microscopy. These results suggest that the formation of amyloid fibrils is an inherent characteristic of the C-terminal peptide of APP. The present system provides a suitable model for the molecular dissection of the process of brain amyloidogenesis.     

Role for a cortical input to hippocampal area CA1 in the consolidation of a long-term memory

A dialogue between the hippocampus and the neocortex is thought to underlie the formation, consolidation and retrieval of episodic memories, although the nature of this cortico-hippocampal communication is poorly understood. Using selective electrolytic lesions in rats, here we examined the role of the direct entorhinal projection (temporoammonic, TA) to the hippocampal area CA1 in short-term (24 hours) and long-term (four weeks) spatial memory in the Morris water maze. When short-term memory was examined, both sham- and TA-lesioned animals showed a significant preference for the target quadrant. When re-tested four weeks later, sham-lesioned animals exhibited long-term memory; in contrast, the TA-lesioned animals no longer showed target quadrant preference. Many long-lasting memories require a process called consolidation, which involves the exchange of information between the cortex and hippocampus. The disruption of long-term memory by the TA lesion could reflect a requirement for TA input during either the acquisition or consolidation of long-term memory. To distinguish between these possibilities, we trained animals, verified their spatial memory 24 hours later, and then subjected trained animals to TA lesions. TA-lesioned animals still exhibited a deficit in long-term memory, indicating a disruption of consolidation. Animals in which the TA lesion was delayed by three weeks, however, showed a significant preference for the target quadrant, indicating that the memory had already been adequately consolidated at the time of the delayed lesion. These results indicate that, after learning, ongoing cortical input conveyed by the TA path is required to consolidate long-term spatial memory.     

Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer's disease.

A locus segregating with familial Alzheimer's disease (AD) has been mapped to chromosome 21, close to the amyloid precursor protein (APP) gene. Recombinants between the APP gene and the AD locus have been reported which seemed to exclude it as the site of the mutation causing familial AD. But recent genetic analysis of a large number of AD families has demonstrated that the disease is heterogeneous. Families with late-onset AD do not show linkage to chromosome 21 markers. Some families with early-onset AD show linkage to chromosome 21 markers, but some do not. This has led to the suggestion that there is non-allelic genetic heterogeneity even within early onset familial AD. To avoid the problems that heterogeneity poses for genetic analysis, we have examined the cosegregation of AD and markers along the long arm of chromosome 21 in a single family with AD confirmed by autopsy. Here we demonstrate that in this kindred, which shows linkage to chromosome 21 markers, there is a point mutation in the APP gene. This mutation causes an amino-acid substitution (Val----Ile) close to the carboxy terminus of the beta-amyloid peptide. Screening other cases of familial AD revealed a second unrelated family in which this variant occurs. This suggests that some cases of AD could be caused by mutations in the APP gene.