Persistent Posttetanic Depression at Cerebellar Parallel Fiber to Purkinje Cell Synapses

Plasticity at the cerebellar parallel fiber to Purkinje cell synapse may underlie information processing and motor learning. In vivo, parallel fibers appear to fire in short high frequency bursts likely to activate sparsely distributed synapses over the Purkinje cell dendritic tree. Here, we report that short parallel fiber tetanic stimulation evokes a ∼7–15% depression which develops over 2 min and lasts for at least 20 min. In contrast to the concomitantly evoked short-term endocannabinoid-mediated depression, this persistent posttetanic depression (PTD) does not exhibit a dependency on the spatial pattern of synapse activation and is not caused by any detectable change in presynaptic calcium signaling. This persistent PTD is however associated with increased paired-pulse facilitation and coefficient of variation of synaptic responses, suggesting that its expression is presynaptic. The chelation of postsynaptic calcium prevents its induction, suggesting that post- to presynaptic (retrograde) signaling is required. We rule out endocannabinoid signaling since the inhibition of type 1 cannabinoid receptors, monoacylglycerol lipase or vanilloid receptor 1, or incubation with anandamide had no detectable effect. The persistent PTD is maximal in pre-adolescent mice, abolished by adrenergic and dopaminergic receptors block, but unaffected by adrenergic and dopaminergic agonists. Our data unveils a novel form of plasticity at parallel fiber synapses: a persistent PTD induced by physiologically relevant input patterns, age-dependent, and strongly modulated by the monoaminergic system. We further provide evidence supporting that the plasticity mechanism involves retrograde signaling and presynaptic diacylglycerol.     

The CNS Synapse Revisited: Gaps,Adhesive Welds,and Borders

Although processes leading up to the point of synapse formation are fairly well understood, the precise sequence of events in which the membranes of two separate cells “lock in” to form a mature synaptic junctional complex is poorly understood. A careful study of the molecules operating at the synapse indicates that their roles are more multifarious than once imagined. In this review we posit that the synapse is a functional organelle with poorly defined boundaries and a complex biochemistry. The role of adhesion molecules, including the integration of their signaling and adhesive properties in the context of synaptic activity is discussed. Special issue dedicated to Anthony Campagnoni     

Microcircuit Rules Governing Impact of Single Interneurons on Purkinje Cell Output In Vivo

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Muscle Fiber Types in Crabs: Studies on Single Identified Muscle Fibers

SYNOPSIS. Based on electrophysiological and histochemical data,four types of muscle fibers (types I, II, III and IV) can beidentified in the closer of the crab Eriphia. Although characteristicsused for typing vary among the fibers of a particular type,the combination of several parameters permits an assignment.Of particular significance for typing is the myosin ATPase activityand its stability after preincubation at different pH levels.The fiber types defined for the closer muscle can also be foundin the other leg muscles of riphia. Single, electrophysiologically identified fibers of each typewere quantitatively analyzed for several key enzymes of oxidativeand glycolytic energy metabolism (GAPDH, LDH, CS, IDH, HAD).Despite the variations found, different metabolic types canbe defined. The typing derived from biochemical studies correlateswell with that obtained electrophysiologically and histochemically.The variability of the biochemical properties, however, seemsto be considerably larger. The type I fibers can be regarded as slow oxidative, the typeII and III fibers as fast oxidative glycolytic, and the typeIV fibers as fast glycolytic.     

Measurement of Elastic Modulus of Collagen Type I Single Fiber

Collagen fibers are the main components of the extra cellular matrix and the primary contributors to the mechanical properties of tissues. Here we report a novel approach to measure the longitudinal component of the elastic moduli of biological fibers under conditions close to those found in vivo and apply it to type I collagen from rat tail tendon. This approach combines optical tweezers, atomic force microscopy, and exploits Euler-Bernoulli elasticity theory for data analysis. This approach also avoids drying for measurements or visualization, since samples are freshly extracted. Importantly, strains are kept below 0.5%, which appear consistent with the linear elastic regime. We find, surprisingly, that the longitudinal elastic modulus of type I collagen cannot be represented by a single quantity but rather is a distribution that is broader than the uncertainty of our experimental technique. The longitudinal component of the single-fiber elastic modulus is between 100 MPa and 360 MPa for samples extracted from different rats and/or different parts of a single tail. Variations are also observed in the fibril-bundle / fibril diameter with an average of 325±40 nm. Since bending forces depend on the diameter to the fourth power, this variation in diameter is important for estimating the range of elastic moduli. The remaining variations in the modulus may be due to differences in composition of the fibril-bundles, or the extent of the proteoglycans constituting fibril-bundles, or that some single fibrils may be of fibril-bundle size.     

Cell lineage dependent and independent control of Purkinje cell number in the mammalian CNS: further quantitative studies of lurcher chimeric mice

Recent quantitative studies of lurcher chimeric mice have shown that the adult population of cerebellar Purkinje cells can properly be described as a small number of developmental clones of cells. The clones are not seen as patches of contiguous neurons; rather, the cells of any one clone distribute throughout the half-cerebellum that contains them, intermingling extensively with the Purkinje cells of other linkages. Lurcher----wild-type chimeras were analyzed using the cell autonomous Purkinje-cell-lethal mutant, lurcher (+/Lc), as a cell marker. Cell counts from these chimeras revealed that the number of surviving Purkinje cells was always an integral multiple of a unit clone size. These numerical quanta are the evidence for the existence of Purkinje cell developmental clones. When two different inbred strains of mouse were compared (C3H/HeJ and C57BL/6), the resulting clonal analysis showed that the unit clone size (i.e., the number of Purkinje cells in one quantum) is an autonomous property of the lineage and hence, presumably, intrinsic to the progenitor cell that founded it. The current study uses the lurcher chimeric mouse system to examine the cell lineage relationships among the Purkinje cells of a third inbred strain of mouse, AKR/J. The data both support and extend our previous studies. Quantitative analysis reveals that the Purkinje cells of this strain also exist in clones, and the size of these clones is also strain-specific. The number of cells in a single clone (7850), however, is different from either C3H/HeJ (10,200) or C57BL/6 (9200). The fact that this value is so highly polymorphic among the inbred strains of mouse makes it likely that, rather than being a function of different alleles at a single genetic locus, clone size may well represent a multifactorial (but still cell-autonomous) property of developing Purkinje cells. Additional results from a single chimeric animal suggest strongly that clone number (i.e., the number of progenitors selected to found the population) is not strain-specific but results instead from cell:cell interactions during early nervous system formation.     

A One-Step Immunostaining Method to Visualize Rodent Muscle Fiber Type within a Single Specimen

In this study, we present a quadruple immunostaining method for rapid muscle fiber typing of mice and rats using antibodies specific to the adult myosin heavy chain (MyHC) isoforms MyHC1, 2A, 2X, and 2B, which are common marker proteins of distinct muscle fiber types. We developed rat monoclonal antibodies specific to each MyHC isoform and conjugated these four antibodies to fluorophores with distinct excitation and emission wavelengths. By mixing the four types of conjugated antibodies, MyHC1, 2A, 2X, and 2B could be distinguished within a single specimen allowing for facile delineation of skeletal muscle fiber types. Furthermore, we could observe hybrid fibers expressing MyHC2X and MyHC2B together in single longitudinal muscle sections from mice and rats, that was not attained in previous techniques. This staining method is expected to be applied to study muscle fiber type transition in response to environmental factors, and to ultimately develop techniques to regulate animal muscle fiber types.     

Chemically Mediated Transmission at a Giant Fiber Synapse in the Central Nervous System of a Vertebrate

The hatchetfish, Gasteropelecus, possesses large pectoral fin adductor muscles whose simultaneous contraction enables the fish to dart upwards at the approach of a predator. These muscles can be excited by either Mauthner fiber. In the medulla, each Mauthner fiber forms axo-axonic synapses on four "giant fibers," two on each side of the midline. Each pair of giant fibers innervates ipsilateral motoneurons controlling the pectoral fin adductor muscles. Mauthner fibers and giant fibers can be penetrated simultaneously by microelectrodes close to the synapses between them. Electrophysiological evidence indicates that transmission from Mauthner to giant fiber is chemically mediated. Under some conditions miniature postsynaptic potentials (PSP's) are observed, suggesting quantal release of transmitter. However, relatively high frequency stimulation reduces PSP amplitude below that of the miniature potentials, but causes no complete failures of PSP's. Thus quantum size is reduced or postsynaptic membrane is desensitized. Ramp currents in Mauthner fibers that rise too slowly to initiate spikes can evoke responses in giant fibers that appear to be asynchronous PSP's. Probably both spikes and ramp currents act on the same secretory mechanism. A single Mauthner fiber spike is followed by prolonged depression of transmission; also PSP amplitude is little affected by current pulses that markedly alter presynaptic spike height. These findings suggest that even a small spike releases most of an immediately available store of transmitter. If so, the probability of release by a single spike is high for any quantum of transmitter within this store.     

The Wnt/Planar Cell Polarity Pathway Component Vangl2 Induces Synapse Formation through Direct Control of N-Cadherin

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The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents ³. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells ¹¹ are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period ⁴. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.     

Proteomic analysis of Giardia: Studies from the pre- and post-genomic era

The proteome of Giardia duodenalis has been under study for the last 25 years and has lead to the discovery of valuable information on the biology and variation of the parasite. Proteomic techniques, mainly SDS-PAGE and 2D-PAGE, have been used to investigate protein variation, cellular structure and host parasite interactions. This has allowed for the identification of assemblage and host specific proteins, structural proteins, proteins released by trophozoites upon exposure to host cell monolayers and immunoreactive proteins. These data are important in understanding the pathogenesis of G. duodenalis infections, as well as highlighting potential drug and vaccine targets. There is, however, a large amount of future work needed to fully understand the proteome of this parasite.     

The Fine Structure of the Rod-Bipolar Cell Synapse in the Retina of the Albino Rat

The fine structure of the rod-bipolar synapse is described and illustrated. Each rod spherule possesses a large, single, oval or elongate mitochondrion approximately 0.5 x 2.0 microns. Surrounding the mitochondrion are elements of agranular endoplasmic reticulum. The bipolar dendrite projects into the lower pole of the spherule and usually terminates in two lobes separated by a cleft. The plasma membranes appear dense and thicker in the region of the synapse. In the rod spherule cytoplasm, contiguous with the plasma membrane is a dense, slightly concave arciform structure, the rod arciform density, extending from the base of the bipolar bifid process through the cleft to an equivalent point on the opposite side. Also within the spherule, and external (towards the sclera) to the rod arciform density, is a parallel, dense, thin lamella, the rod synaptic lamella. This is approximately 25 mµ in thickness and 400 mµ in width at its widest extent. This halfmoon-shaped plate straddles the cleft between the two lobes of the bipolar process. The lamella appears to consist of short regular rodlets or cylinders 5 to 7 mµ in diameter, oriented with their long axes perpendicular to the plane of the lamella. Minute cytoplasmic vesicles found in the cytoplasm of both the rod spherule and the bipolar terminal are most abundant near the rod synaptic lamella.     

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer''s attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.     

Human Mycobacterium tuberculosis CD8 T Cell Antigens/Epitopes Identified by a Proteomic Peptide Library

Identification of CD8⁺ T cell antigens/epitopes expressed by human pathogens with large genomes is especially challenging, yet necessary for vaccine development. Immunity to tuberculosis, a leading cause of mortality worldwide, requires CD8⁺ T cell immunity, yet the repertoire of CD8 antigens/epitopes remains undefined. We used integrated computational and proteomic approaches to screen 10% of the Mycobacterium tuberculosis (Mtb) proteome for CD8 Mtb antigens. We designed a weighting schema based upon a Multiple Attribute Decision Making:framework to select 10% of the Mtb proteome with a high probability of containing CD8⁺ T cell epitopes. We created a synthetic peptide library consisting of 15-mers overlapping by 11 aa. Using the interferon-γ ELISPOT assay and Mtb-infected dendritic cells as antigen presenting cells, we screened Mtb-specific CD8⁺ T cell clones restricted by classical MHC class I molecules (MHC class Ia molecules), that were isolated from Mtb-infected humans, against this library. Three novel CD8 antigens were unambiguously identified: the EsxJ family (Rv1038c, Rv1197, Rv3620c, Rv2347c, Rv1792), PE9 (Rv1088), and PE_PGRS42 (Rv2487c). The epitopes are B5701-restricted EsxJ_24–34, B3905-restricted PE9_53–67, and B3514-restricted PE_PGRS42_48–56, respectively. The utility of peptide libraries in identifying unknown epitopes recognized by classically restricted CD8⁺ T cells was confirmed, which can be applied to other intracellular pathogens with large size genomes. In addition, we identified three novel Mtb epitopes/antigens that may be evaluated for inclusion in vaccines and/or diagnostics for tuberculosis.     

Heat Shock Protein Beta-1 Modifies Anterior to Posterior Purkinje Cell Vulnerability in a Mouse Model of Niemann-Pick Type C Disease

Selective neuronal vulnerability is characteristic of most degenerative disorders of the CNS, yet mechanisms underlying this phenomenon remain poorly characterized. Many forms of cerebellar degeneration exhibit an anterior-to-posterior gradient of Purkinje cell loss including Niemann-Pick type C1 (NPC) disease, a lysosomal storage disorder characterized by progressive neurological deficits that often begin in childhood. Here, we sought to identify candidate genes underlying vulnerability of Purkinje cells in anterior cerebellar lobules using data freely available in the Allen Brain Atlas. This approach led to the identification of 16 candidate neuroprotective or susceptibility genes. We demonstrate that one candidate gene, heat shock protein beta-1 (HSPB1), promoted neuronal survival in cellular models of NPC disease through a mechanism that involved inhibition of apoptosis. Additionally, we show that over-expression of wild type HSPB1 or a phosphomimetic mutant in NPC mice slowed the progression of motor impairment and diminished cerebellar Purkinje cell loss. We confirmed the modulatory effect of Hspb1 on Purkinje cell degeneration in vivo, as knockdown by Hspb1 shRNA significantly enhanced neuron loss. These results suggest that strategies to promote HSPB1 activity may slow the rate of cerebellar degeneration in NPC disease and highlight the use of bioinformatics tools to uncover pathways leading to neuronal protection in neurodegenerative disorders.     

Proteomic Studies of Saliva: A Proposal for a Standardized Handling of Clinical Samples

Background

In recent years, differential analysis of proteins from human saliva, i.e., proteomic analysis, has received much attention mainly due to its unstressful sampling and its great potential for biomarker research. It is widely considered that saliva is a highly stable medium for proteins thanks to a large amount of antiprotease agents, even at ambient and physiological temperatures.

Objective

To find the best protocol for the handling of samples, we have investigated the stability of saliva proteins stored at different temperatures (from ?80 to 20°C) by one- and two-dimensional electrophoresis.

Results

At 20°C, no major changes were observed on protein one-dimensional profiles following 1 day of storage; however, between 7 days and 30 days, the native alpha-amylase band decreased slightly to give several bands with molecular weight between 35 and 25 kDa. The same phenomenon appeared after 30 days of storage at 4°C. Two-dimensional analysis of salivary maps revealed degradation from day 7 of several protein groups for samples stored at 20°C.

Conclusion

All these findings have to be carefully considered when saliva is collected for clinical proteomic analysis. We can conclude that, to maintain the optimum stability of saliva proteins, saliva samples should be collected on ice followed by the addition of protease inhibitor cocktail, centrifuged to remove insoluble material, and stored at ?20 or ?80°C.     

Pten^＋/＋MEFs与Pten^-/-MEFs细胞系的差异表达蛋白质组分析

目的：研究PTEN缺失细胞的蛋白表达规律。方法：用双向电泳技术比较Pten^＋/＋MEFs与Pten^-/-EFs细胞的蛋白表达差异;用基质辅助激光解吸／电离飞行时间质谱（MALDI-TOF-MS）对差异蛋白进行质谱分析;用肽质量指纹图谱检索数据库对差异蛋白进行鉴定;用Northern印迹和Western印迹验证蛋白的差异表达。结果：与Pten^＋/＋MEFs细胞相比。Pten^-/-MEFs细胞有明显的差异性蛋白表达谱,Pten^-/-MEFs细胞中表达下降的蛋白有铜锌超氧化物歧化酶、过氧化物氧化还原酶5和6等,表达增加的蛋白有低分子量蛋白酪氨酸磷酸酶和丝切蛋白（cofflin）1。结论：PTEN的缺失引起细胞内多种蛋白表达改变,这些表达改变的蛋白可能与PTEN缺失后细胞癌变相关。     

槲皮素抑制中枢神经突触前兴奋性依赖的胞吞动力学（英文）

目的 槲皮素是一种广泛分布于药用植物中的黄酮类化合物，传统被认为具有神经保护作用。本研究利用位于大鼠脑干花萼状突触的突触前神经末梢进行膜片钳记录，研究槲皮素调控突触传递和可塑性的突触前机制。方法 利用全细胞膜片钳结合膜电容记录，在突触后记录微小兴奋性突触后电流（m EPSC），在突触前神经末梢记录钙內流和神经囊泡的释放、回收以及可立即释放库（RRP）的恢复动力学。并且利用纤维刺激在轴突给予5～200 Hz的刺激，诱发突触后EPSC，记录突触后短时程抑制（STD）。结果 100μmol/L槲皮素不影响突触后m EPSC的振幅、频率以及AMPA受体的动力学特征。在突触前神经末梢，槲皮素不改变钙内流或囊泡的释放，但显著抑制胞吐后网格蛋白依赖的慢速胞吞。抑制胞吞会导致突触前囊泡动员的减慢，降低RRP的补充速率，并且增强高频刺激下的短时程可塑性STD。结论 本研究为槲皮素调控中枢神经突触传递提供全新的突触前神经机制，槲皮素有助于抑制中枢神经过度兴奋，进而发挥神经保护作用。     

Single Cell Analysis of a Bacterial Sender-Receiver System

Monitoring gene expression dynamics on the single cell level provides important information on cellular heterogeneity and stochasticity, and potentially allows for more accurate quantitation of gene expression processes. We here study bacterial senders and receivers genetically engineered with components of the quorum sensing system derived from Aliivibrio fischeri on the single cell level using microfluidics-based bacterial chemostats and fluorescence video microscopy. We track large numbers of bacteria over extended periods of time, which allows us to determine bacterial lineages and filter out subpopulations within a heterogeneous population. We quantitatively determine the dynamic gene expression response of receiver bacteria to varying amounts of the quorum sensing inducer N-3-oxo-C6-homoserine lactone (AHL). From this we construct AHL response curves and characterize gene expression dynamics of whole bacterial populations by investigating the statistical distribution of gene expression activity over time. The bacteria are found to display heterogeneous induction behavior within the population. We therefore also characterize gene expression in a homogeneous bacterial subpopulation by focusing on single cell trajectories derived only from bacteria with similar induction behavior. The response at the single cell level is found to be more cooperative than that obtained for the heterogeneous total population. For the analysis of systems containing both AHL senders and receiver cells, we utilize the receiver cells as ‘bacterial sensors’ for AHL. Based on a simple gene expression model and the response curves obtained in receiver-only experiments, the effective AHL concentration established by the senders and their ‘sending power’ is determined.