Driving AMPA receptors into synapses by LTP and CaMKII: requirement for GluR1 and PDZ domain interaction

To elucidate mechanisms that control and execute activity-dependent synaptic plasticity, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPA-Rs) with an electrophysiological tag were expressed in rat hippocampal neurons. Long-term potentiation (LTP) or increased activity of the calcium/calmodulin-dependent protein kinase II (CaMKII) induced delivery of tagged AMPA-Rs into synapses. This effect was not diminished by mutating the CaMKII phosphorylation site on the GluR1 AMPA-R subunit, but was blocked by mutating a predicted PDZ domain interaction site. These results show that LTP and CaMKII activity drive AMPA-Rs to synapses by a mechanism that requires the association between GluR1 and a PDZ domain protein.     

Dynamic control of CaMKII translocation and localization in hippocampal neurons by NMDA receptor stimulation

Calcium-calmodulin-dependent protein kinase II (CaMKII) is thought to increase synaptic strength by phosphorylating postsynaptic density (PSD) ion channels and signaling proteins. It is shown that N-methyl-D-aspartate (NMDA) receptor stimulation reversibly translocates green fluorescent protein-tagged CaMKII from an F-actin-bound to a PSD-bound state. The translocation time was controlled by the ratio of expressed beta-CaMKII to alpha-CaMKII isoforms. Although F-actin dissociation into the cytosol required autophosphorylation of or calcium-calmodulin binding to beta-CaMKII, PSD translocation required binding of calcium-calmodulin to either the alpha- or beta-CaMKII subunits. Autophosphorylation of CaMKII indirectly prolongs its PSD localization by increasing the calmodulin-binding affinity.     

A pertussis toxin-sensitive G protein in hippocampal long-term potentiation

High-frequency (tetanic) stimulation of presynaptic nerve tracts in the hippocampal region of the brain can lead to long-term synaptic potentiation (LTP). Pertussis toxin prevented the development of tetanus-induced LTP in the stratum radiatum-CA1 synaptic system of rat hippocampal slices, indicating that a guanosine triphosphate-binding protein (G protein) may be required for the initiation of LTP. This G protein may be located at a site distinct from the postsynaptic neuron (that is, in presynaptic terminals or glial cells) since maximal activation of CA1 neuronal G proteins by intracellular injection of guanosine-5'-O-(3-thiotriphosphate), a nonhydrolyzable analog of guanosine 5'-triphosphate, did not occlude LTP.     

Postsynaptic calcium is sufficient for potentiation of hippocampal synaptic transmission

Brief repetitive activation of excitatory synapses in the hippocampus leads to an increase in synaptic strength that lasts for many hours. This long-term potentiation (LTP) of synaptic transmission is the most compelling cellular model in the vertebrate brain for learning and memory. The critical role of postsynaptic calcium in triggering LTP has been directly examined using three types of experiment. First, nitr-5, a photolabile nitrobenzhydrol tetracarboxylate calcium chelator, which releases calcium in response to ultraviolet light, was used. Photolysis of nitr-5 injected into hippocampal CA1 pyramidal cells resulted in a large enhancement of synaptic transmission. Second, in agreement with previous results, buffering intracellular calcium at low concentrations blocked LTP. Third, depolarization of the postsynaptic membrane so that calcium entry is suppressed prevented LTP. Taken together, these results demonstrate that an increase in postsynaptic calcium is necessary to induce LTP and sufficient to potentiate synaptic transmission.     

Trans-synaptic Eph receptor-ephrin signaling in hippocampal mossy fiber LTP

The site of induction of long-term potentiation (LTP) at mossy fiber-CA3 synapses in the hippocampus is unresolved, with data supporting both pre- and postsynaptic mechanisms. Here we report that mossy fiber LTP was reduced by perfusion of postsynaptic neurons with peptides and antibodies that interfere with binding of EphB receptor tyrosine kinases (EphRs) to the PDZ protein GRIP. Mossy fiber LTP was also reduced by extracellular application of soluble forms of B-ephrins, which are normally membrane-anchored presynaptic ligands for the EphB receptors. The application of soluble ligands for presynaptic ephrins increased basal excitatory transmission and occluded both tetanus and forskolin-induced synaptic potentiation. These findings suggest that PDZ interactions in the postsynaptic neuron and trans-synaptic interactions between postsynaptic EphB receptors and presynaptic B-ephrins are necessary for the induction of mossy fiber LTP.     

Long-term synaptic potentiation

Long-term synaptic potentiation (LTP) is a leading candidate for a synaptic mechanism of rapid learning in mammals. LTP is a persistent increase in synaptic efficacy that can be quickly induced. The biophysical process that controls one type of LTP is formally similar to a synaptic memory mechanism postulated decades ago by the psychologist Donald Hebb. A key aspect of the modification process involves the N-methyl-D-aspartate (NMDA) receptor-ionophore complex. This ionophore allows calcium influx only if the endogenous ligand glutamate binds to the NMDA receptor and if the voltage across the associated channel is also sufficiently depolarized to relieve a magnesium block. According to one popular hypothesis, the resulting increase in the intracellular calcium concentration activates protein kinases that enhance the postsynaptic conductance. Further biophysical and molecular understanding of the modification process should facilitate detailed explorations of the mnemonic functions of LTP.     

孕酮对绵羊输卵管上皮细胞β-防御素-1 mRNA表达的影响

旨在研究孕酮(P4)对绵羊输卵管上皮细胞内β-防御素-1( SBD-1) mRNA基因表达的影响及可能参与的信号通路,利用10~(-8) mol/L P4培养绵羊输卵管上皮细胞2 h、6 h、12 h、24 h、48 h,利用RT-qPCR技术检测 SBD-1 mRNA的表达变化,选取P4诱导 SBD-1 mRNA表达的最佳时间。分别添加P4核受体阻断剂RU-486、蛋白激酶C (PKC)阻断剂H-7、蛋白激酶A (PKA)阻断剂H-89干预绵羊输卵管上皮细胞1h,再使用P4处理细胞。结果显示,用10~(-8)mol/L P4诱导绵羊输卵管上皮细胞6 h, SBD-1 mRNA表达显著升高;添加RU-486和H-89显著抑制P4诱导的 SBD-1 mRNA的表达;添加H-7后 SBD-1 mRNA的表达量无显著性差异。以上结果表明,P4诱导的绵羊输卵管上皮细胞中 SBD-1 mRNA的表达通过孕酮核受体(PR)和PKA信号通路介导,与PKC信号通路激活无关。     

Requirement of AMPA receptor GluR2 phosphorylation for cerebellar long-term depression

Cerebellar long-term depression (LTD) is a model of synaptic memory that requires protein kinase C (PKC) activation and is expressed as a reduction in the number of postsynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors. LTD was absent in cultured cerebellar Purkinje cells from mutant mice lacking the AMPA receptor GluR2 subunit and could be rescued by transient transfection with the wild-type GluR2 subunit. Transfection with a point mutant that eliminated PKC phosphorylation of Ser880 in the carboxy-terminal PDZ ligand of GluR2 failed to restore LTD. In contrast, transfection with a point mutant that mimicked phosphorylation at Ser880 occluded subsequent LTD. Thus, PKC phosphorylation of GluR2 Ser880 is a critical event in the induction of cerebellar LTD.     

Postsynaptic Induction of BDNF-Mediated Long-Term Potentiation

Brain-derived neurotrophic factor (BDNF) and other neurotrophins are critically involved in long-term potentiation (LTP). Previous reports point to a presynaptic site of neurotrophin action. By imaging dentate granule cells in mouse hippocampal slices, we identified BDNF-evoked Ca2+ transients in dendrites and spines, but not at presynaptic sites. Pairing a weak burst of synaptic stimulation with a brief dendritic BDNF application caused an immediate and robust induction of LTP. LTP induction required activation of postsynaptic Ca2+ channels and N-methyl-d-aspartate receptors and was prevented by the blockage of postsynaptic Ca2+ transients. Thus, our results suggest that BDNF-mediated LTP is induced postsynaptically. Our finding that dendritic spines are the exclusive synaptic sites for rapid BDNF-evoked Ca2+ signaling supports this conclusion.     

Storage of spatial information by the maintenance mechanism of LTP

Analogous to learning and memory storage, long-term potentiation (LTP) is divided into induction and maintenance phases. Testing the hypothesis that the mechanism of LTP maintenance stores information requires reversing this mechanism in vivo and finding out whether long-term stored information is lost. This was not previously possible. Recently however, persistent phosphorylation by the atypical protein kinase C isoform, protein kinase Mzeta (PKMz), has been found to maintain late LTP in hippocampal slices. Here we show that a cell-permeable PKMz inhibitor, injected in the rat hippocampus, both reverses LTP maintenance in vivo and produces persistent loss of 1-day-old spatial information. Thus, the mechanism maintaining LTP sustains spatial memory.     

Recycling endosomes supply AMPA receptors for LTP

Long-term potentiation (LTP) of synaptic strength, the most established cellular model of information storage in the brain, is expressed by an increase in the number of postsynaptic AMPA receptors. However, the source of AMPA receptors mobilized during LTP is unknown. We report that AMPA receptors are transported from recycling endosomes to the plasma membrane for LTP. Stimuli that triggered LTP promoted not only AMPA receptor insertion but also generalized recycling of cargo and membrane from endocytic compartments. Thus, recycling endosomes supply AMPA receptors for LTP and provide a mechanistic link between synaptic potentiation and membrane remodeling during synapse modification.     

Transmission between pairs of hippocampal slice neurons: quantal levels, oscillations, and LTP

Long-term potentiation (LTP) of synaptic transmission after coincident pre- and postsynaptic activity is considered a cellular model of changes underlying learning and memory. In intact tissue, LTP has been observed only between populations of neurons, making analysis of mechanisms difficult. Transmission between individual pre- and postsynaptic hippocampal cells was studied, suggesting quantal amplitude distributions with little variability in quantal size. LTP between such pairs is manifested by large, persistent, and synapse-specific potentiation with a shift in amplitude distribution that suggests presynaptic changes. Oscillations in amplitude of transmission, apparently of presynaptic origin, are common and can be triggered by LTP.     

Protein synthesis and neurotrophin-dependent structural plasticity of single dendritic spines

Long-term potentiation (LTP) at glutamatergic synapses is considered to underlie learning and memory and is associated with the enlargement of dendritic spines. Because the consolidation of memory and LTP require protein synthesis, it is important to clarify how protein synthesis affects spine enlargement. In rat brain slices, the repetitive pairing of postsynaptic spikes and two-photon uncaging of glutamate at single spines (a spike-timing protocol) produced both immediate and gradual phases of spine enlargement in CA1 pyramidal neurons. The gradual enlargement was strongly dependent on protein synthesis and brain-derived neurotrophic factor (BDNF) action, often associated with spine twitching, and was induced specifically at the spines that were immediately enlarged by the synaptic stimulation. Thus, this spike-timing protocol is an efficient trigger for BDNF secretion and induces protein synthesis-dependent long-term enlargement at the level of single spines.     

Does BDNF have pre- or postsynaptic targets?

A type of synaptic plasticity in the brain called long-term potentiation (LTP) is thought to form the molecular basis of learning and memory. In a Perspective, Manabe discusses new findings (Kovalchuk et al.) showing brain-derived neurotropic factor modulates LTP by binding to TrkB receptors on the postsynaptic neuron.     

Pentobarbital: selective depression of excitatory postsynaptic potentials

The effects of pentobarbital (Nembutal) on synaptic transmission and postsynaptic potentials were studied by the use of several invertebrate preparations. Pentobarbital selectively and reversibly depressed both excitatory postsynaptic potentials and sodium-dependent postsynaptic responses to putative excitatory transmitters without affecting either inhibitory postsynaptic potentials or chloride- and potassium-dependent postsynaptic responses to putative transmitters. A selective depression of postsynaptic excitatory events was also observed with other central nervous system depressants (ethanol, chloroform, chloralose, diphenylhydantoin, and urethane). The results suggest that central and peripheral depression observed during general anesthesia is due to a selective depression of excitatory synaptic events.     

Comparison of two forms of long-term potentiation in single hippocampal neurons

In invertebrate nervous systems, some long-lasting increases in synaptic efficacy result from changes in the presynaptic cell. In the vertebrate nervous system, the best understood long-lasting change in synaptic strength is long-term potentiation (LTP) in the CA1 region of the hippocampus. Here the process is initiated postsynaptically, but the site of the persistent change is unresolved. Single CA3 hippocampal pyramidal cells receive excitatory inputs from associational-commissural fibers and from the mossy fibers of dentate granule cells and both pathways exhibit LTP. Although the induction of associational-commissural LTP requires in the postsynaptic cell N-methyl-D-aspartate (NMDA) receptor activation, membrane depolarization, and a rise in calcium, mossy fiber LTP does not. Paired-pulse facilitation, which is an index of increased transmitter release, is unaltered during associational-commissural LTP but is reduced during mossy fiber LTP. Thus, both the induction and the persistent change may be presynaptic in mossy fiber LTP but not in associational-commissural LTP.     

FSH和17β-雌二醇联合作用对仔猪睾丸 支持细胞Skp2表达的调节

 【目的】确定FSH和雌激素联合作用是否可通过ERK1/2级联调节培养条件下未成熟仔猪睾丸支持细胞中Skp2的表达。【方法】以培养的仔猪睾丸支持细胞为试验材料,通过添加各种信号通路的抑制剂,应用Western blotting和实时荧光定量PCR检测Skp2蛋白、mRNA的表达。【结果】FSH（50 ng·mL-1）和17β-雌二醇（10-9 mol·L-1）联合作用时以时间依赖的方式促进了Skp2蛋白和mRNA的表达（P＜0.05）,这一作用在30 min时到达高峰;FSH（50 ng·mL-1）、17β-雌二醇（10-9 mol·L-1）和forskolin单独作用均促进了Skp2蛋白和mRNA的表达（P＜0.05）,FSH（50 ng·mL-1）和17β-雌二醇（10-9 mol·L-1）联合作用对Skp2表达的影响与FSH或17β-雌二醇（10-9 mol·L-1）单独作用无显著差异（P≥0.05）,而环磷酸腺苷抑制剂（Rp-cAMP）、蛋白激酶A（PKA）抑制剂（H-89）、L-Ca2+离子通道抑制剂（verapamil）和ERK1/2抑制剂（U0126）单独作用时对Skp2蛋白和mRNA的表达与空白对照相比无显著影响（P＞0.05）,但都抑制了FSH（50 ng·mL-1）与17β-雌二醇（10-9 mol·L-1）联合作用对Skp2蛋白和mRNA表达的影响（P＜0.05）。H-89、verapamil单独作用对ERK1/2（细胞外信号调节的蛋白激酶1/2）活性没有影响,但降低了FSH（50 ng·mL-1）和17β-雌二醇（10-9 mol·L-1）联合作用对ERK1/2活性的影响。【结论】 FSH与17β-雌二醇联合作用激活了cAMP-PKA级联和Ca2+内流,而PKA和Ca2+内流又通过影响ERK1/2的活性进而影响Skp2的表达。     

Targeted protein degradation and synapse remodeling by an inducible protein kinase

Synaptic plasticity involves the reorganization of synapses at the protein and the morphological levels. Here, we report activity-dependent remodeling of synapses by serum-inducible kinase (SNK). SNK was induced in hippocampal neurons by synaptic activity and was targeted to dendritic spines. SNK bound to and phosphorylated spine-associated Rap guanosine triphosphatase activating protein (SPAR), a postsynaptic actin regulatory protein, leading to degradation of SPAR. Induction of SNK in hippocampal neurons eliminated SPAR protein, depleted postsynaptic density-95 and Bassoon clusters, and caused loss of mature dendritic spines. These results implicate SNK as a mediator of activity-dependent change in the molecular composition and morphology of synapses.     

Retrograde signaling by Syt 4 induces presynaptic release and synapse-specific growth

The molecular pathways involved in retrograde signal transduction at synapses and the function of retrograde communication are poorly understood. Here, we demonstrate that postsynaptic calcium 2+ ion (Ca2+) influx through glutamate receptors and subsequent postsynaptic vesicle fusion trigger a robust induction of presynaptic miniature release after high-frequency stimulation at Drosophila neuromuscular junctions. An isoform of the synaptotagmin family, synaptotagmin 4 (Syt 4), serves as a postsynaptic Ca2+ sensor to release retrograde signals that stimulate enhanced presynaptic function through activation of the cyclic adenosine monophosphate (cAMP)-cAMP-dependent protein kinase pathway. Postsynaptic Ca2+ influx also stimulates local synaptic differentiation and growth through Syt 4-mediated retrograde signals in a synapse-specific manner.     

Regulation of chloride channels by protein kinase C in normal and cystic fibrosis airway epithelia

Apical membrane chloride channels control chloride secretion by airway epithelial cells. Defective regulation of these channels is a prominent characteristic of cystic fibrosis. In normal intact cells, activation of protein kinase C (PKC) by phorbol ester either stimulated or inhibited chloride secretion, depending on the physiological status of the cell. In cell-free membrane patches, PKC also had a dual effect: at a high calcium concentration, PKC inactivated chloride channels; at a low calcium concentration, PKC activated chloride channels. In cystic fibrosis cells, PKC-dependent channel inactivation was normal, but activation was defective. Thus it appears that PKC phosphorylates and regulates two different sites on the channel or on an associated membrane protein, one of which is defective in cystic fibrosis.