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.     

Type-1 metabotropic glutamate receptor in cerebellar Purkinje cells: a key molecule responsible for long-term depression, endocannabinoid signalling and synapse elimination

The cerebellum is a brain structure involved in the coordination, control and learning of movements, and elucidation of its function is an important issue. Japanese scholars have made seminal contributions in this field of neuroscience. Electrophysiological studies of the cerebellum have a long history in Japan since the pioneering works by Ito and Sasaki. Elucidation of the basic circuit diagram of the cerebellum in the 1960s was followed by the construction of cerebellar network theories and finding of their neural correlates in the 1970s. A theoretically predicted synaptic plasticity, long-term depression (LTD) at parallel fibre to Purkinje cell synapse, was demonstrated experimentally in 1982 by Ito and co-workers. Since then, Japanese neuroscientists from various disciplines participated in this field and have made major contributions to elucidate molecular mechanisms underlying LTD. An important pathway for LTD induction is type-1 metabotropic glutamate receptor (mGluR1) and its downstream signal transduction in Purkinje cells. Sugiyama and co-workers demonstrated the presence of mGluRs and Nakanishi and his pupils identified the molecular structures and functions of the mGluR family. Moreover, the authors contributed to the discovery and elucidation of several novel functions of mGluR1 in cerebellar Purkinje cells. mGluR1 turned out to be crucial for the release of endocannabinoid from Purkinje cells and the resultant retrograde suppression of transmitter release. It was also found that mGluR1 and its downstream signal transduction in Purkinje cells are indispensable for the elimination of redundant synapses during post-natal cerebellar development. This article overviews the seminal works by Japanese neuroscientists, focusing on mGluR1 signalling in cerebellar Purkinje cells.     

A Biophysical Model of Synaptic Delay Learning and Temporal Pattern Recognition in a Cerebellar Purkinje Cell

It has been suggested that information in the brain is encoded in temporal spike patterns which are decoded by a combination of time delays and coincidence detection. Here, we show how a multi-compartmental model of a cerebellar Purkinje cell can learn to recognise temporal parallel fibre activity patterns by adapting latencies of calcium responses after activation of metabotropic glutamate receptors (mGluRs). In each compartment of our model, the mGluR signalling cascade is represented by a set of differential equations that reflect the underlying biochemistry. Phosphorylation of the mGluRs changes the concentration of receptors which are available for activation by glutamate and thereby adjusts the time delay between mGluR stimulation and voltage response. The adaptation of a synaptic delay as opposed to a weight represents a novel non-Hebbian learning mechanism that can also implement the adaptive timing of the classically conditioned eye-blink response.     

Highly Sensitive Immunoassay for Rat Brain-Type Creatine Kinase: Determination in Isolated Purkinje Cells

Ultrasensitive enzyme immunoassay method for the measurement of rat brain-type creatine kinase BB (CK-BB) was developed by use of purified antibodies specific to the B subunit of creatine kinase. The antibody immunoglobulin G was purified with immunoaffinity chromatography of the antiserum raised in rabbits by injecting the purified rat CK-BB. The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The assay was specific to the B subunit of CK (CK-B), showing about 10% cross-reactivity with CK-MB, but it did not cross-react with CK-MM and neuron-specific gamma gamma enolase. The minimum detection limit of the assay was 0.1 pg or 1 amol CK-BB, being sufficiently sensitive for the measurement of CK-B contents in the isolated Purkinje cell bodies at the level of single cells. The average content of CK-B in a single Purkinje cell was 1.64 pg. The CK-B concentration in rat cerebellum (about 22 micrograms/mg protein) was about twofold higher than that (about 13 micrograms/mg protein) in the cerebrum. High levels (greater than 5 micrograms/mg protein) of CK-B were also found in the peripheral tissues such as gastrointestinal tract and urinary bladder, all of which are composed of smooth muscle. Immunohistochemical localization of CK-B antigens in the CNS revealed that the antigens is distributed not only in the neurons but also in the glial cells.     

Ghostbursting: A Novel Neuronal Burst Mechanism

Pyramidal cells in the electrosensory lateral line lobe (ELL) of weakly electric fish have been observed to produce high-frequency burst discharge with constant depolarizing current (Turner et al., 1994). We present a two-compartment model of an ELL pyramidal cell that produces burst discharges similar to those seen in experiments. The burst mechanism involves a slowly changing interaction between the somatic and dendritic action potentials. Burst termination occurs when the trajectory of the system is reinjected in phase space near the ghost of a saddle-node bifurcation of fixed points. The burst trajectory reinjection is studied using quasi-static bifurcation theory, that shows a period doubling transition in the fast subsystem as the cause of burst termination. As the applied depolarization is increased, the model exhibits first resting, then tonic firing, and finally chaotic bursting behavior, in contrast with many other burst models. The transition between tonic firing and burst firing is due to a saddle-node bifurcation of limit cycles. Analysis of this bifurcation shows that the route to chaos in these neurons is type I intermittency, and we present experimental analysis of ELL pyramidal cell burst trains that support this model prediction. By varying parameters in a way that changes the positions of both saddle-node bifurcations in parameter space, we produce a wide gallery of burst patterns, which span a significant range of burst time scales.     

A Mathematical Model of the Cerebellar-Olivary System I: Self-Regulating Equilibrium of Climbing Fiber Activity

We use a mathematical model to investigate how climbing fiber-dependent plasticity at granule cell to Purkinje cell (grPkj) synapses in the cerebellar cortex is influenced by the synaptic organization of the cerebellar-olivary system. Based on empirical studies, grPkj synapses are assumed to decrease in strength when active during a climbing fiber input (LTD) and increase in strength when active without a climbing fiber input (LTP). Results suggest that the inhibition of climbing fibers by cerebellar output combines with LTD/P to self-regulate spontaneous climbing fiber activity to an equilibrium level at which LTP and LTD balance and the expected net change in grPkj synaptic weights is zero. The synaptic weight vector is asymptotically confined to an equilibrium hyperplane defining the set of all possible combinations of synaptic weights consistent with climbing fiber equilibrium. Results also suggest restrictions on LTP/D at grPkj synapses required to produce synaptic weights that do not drift spontaneously.     

The p75 Neurotrophin Receptor Mediates Neuronal Apoptosis and Is Essential for Naturally Occurring Sympathetic Neuron Death

Abstract. To determine whether the p75 neurotrophin receptor (p75NTR) plays a role in naturally occurring neuronal death, we examined neonatal sympathetic neurons that express both the TrkA tyrosine kinase receptor and p75NTR. When sympathetic neuron survival is maintained with low quantities of NGF or KCl, the neurotrophin brain-derived neurotrophic factor (BDNF), which does not activate Trk receptors on sympathetic neurons, causes neuronal apoptosis and increased phosphorylation of c-jun. Function-blocking antibody studies indicate that this apoptosis is due to BDNF-mediated activation of p75NTR. To determine the physiological relevance of these culture findings, we examined sympathetic neurons in BDNF−/− and p75NTR−/− mice. In BDNF−/− mice, sympathetic neuron number is increased relative to BDNF+/+ littermates, and in p75NTR−/− mice, the normal period of sympathetic neuron death does not occur, with neuronal attrition occurring later in life. This deficit in apoptosis is intrinsic to sympathetic neurons, since cultured p75NTR−/− neurons die more slowly than do their wild-type counterparts. Together, these data indicate that p75NTR can signal to mediate apoptosis, and that this mechanism is essential for naturally occurring sympathetic neuron death.     

A Semi-Markovian Model for Cell Survival after Irradiation

To study the behaviour of a living cell exposed to radiations we investigate a stochastic model, employing for its analysis the theory of semi-Markov and Markov renewal processes. Four states of the cell namely, normal state, damaged state 1, damaged state 2 and altered state are defined and various characteristics of interest pertaining to the cell behaviour are studied.     

猪瘟病毒野毒株持续感染细胞模型的稳定性研究

以本实验室建立的CSFV39-PKl5持续感染细胞模型为实验材料,综合运用免疫荧光、RT-PCR、流式细胞仪,对其稳定性进行研究。实验结果均表明,该细胞模型有着良好的稳定性。即使在连续传至128代的CSFV39-PKl5传代细胞中,CSFV仍持续存在：呈免疫荧光抗体反应阳性和RT-PCR检测阳性。同时,该细胞与正常的PK-15细胞相比,细胞周期无显著差异。通过同段序列的同源性比较,发现CSFV39与CSFV石门株的同源性最高,达99．02％。     

Neuronal cell cultures: A tool for investigations in developmental neurobiology

The aim of this review is to describe environmental requirements for survival of neuronal cells in culture, and secondly to survey the complex interplay between hormones, neurotrophic factors, transport- and extracellular matrix- proteins, which characterize the developmental program of differentiating neurons. An overall reconsideration of the literature in this vast field is above the limits of the present paper; since progress and refinement in the techniques of neuronal cell cultures have paralleled the advancement in Developmental Neurobiology, we will run instead through the main steps which form the conceptual framework of neuronal cell cultures.     

Organotypic Hippocampal Slice Cultures: A Model System to Study Basic Cellular and Molecular Mechanisms of Neuronal Cell Death,Neuroprotection, and Synaptic Plasticity

The hippocampus has become one of the most extensively studied areas of the mammalian brain, and its proper function is of utmost importance, particularly for learning and memory. The hippocampus is the most susceptible brain region for damage, and its impaired function has been documented in many human brain diseases, e.g. hypoxia, ischemia, and epilepsy regardless of the age of the affected patients. In addition to experimental in vivo models of these disorders, the investigation of basic anatomical, physiological, and molecular aspects requires an adequate experimental in vitro model, which should meet the requirements for well-preserved representation of various cell types, and functional information processing properties in the hippocampus. In this review, the characteristics of organotypic hippocampal slice cultures (OHCs) together with the main differences between the in vivo and in vitro preparations are first briefly outlined. Thereafter, the use of OHCs in studies focusing on neuron cell death and synaptic plasticity is discussed. Special issue dedicated to Dr. Simo S. Oja     

Molecular Dynamics Simulations for 1:1 Solvent Primitive Model Electrolyte Solutions

Abstract

Molecular dynamics (MD) simulations at constant temperature have been carried out for systems of 1:1 solvent primitive model (SPM) electrolyte solutions. Equilibrium thermodynamics, mean cluster size, self-diffusion coefficients, and collision frequencies were computed to examine the electrostatic effects on the structural and dynamical properties. Coherent ionic cluster motion was deduced from a cluster analysis and from the dependence of the velocity and force autocorrelation functions (FACFs). The resulting MD data for the collision frequencies and self-diffusivities of both ions and hard-spheres were shown to be in good agreement with the theoretical predictions.     

Question 7: Construction of a Semi-Synthetic Minimal Cell: A Model for Early Living Cells

Using a Synthetic Biology approach we are building a semi-synthetic minimal cell. This represents an exercise to shape a minimal-cell model system recalling the simplicity of early living cells in early evolution. We have recently introduced into liposome compartments a minimal set of enzymes named “Puresystem” (PS) synthesizing EGFP proteins. To establish reproduction of the shell compartment with a minimal set of genes we have cloned the genes for the Fatty Acid Synthase (FAS) type I enzymes. These FAS genes introduced into liposomes, translated into FAS enzymes by PS and in the presence of precursors produce fatty acids. The resulting release of fatty acid molecules within liposome vesicles should promote vesicle growth and reproduction. The core reproduction of a minimal cell corresponding to the replication of the minimal genome will require a few genes for the DNA replication and the PS, and a minimum set of genes for the synthesis of t-RNAs. In future the reconstruction of a minimal ribosome will bring the number of genes for ribosomal proteins from 54 of an existing minimal genome down to 30–20 genes. A Synthetic Biology approach could bring the number of essential genes for a minimal cell down to 100 or less. International School of Complexity–4th Course: Basic Questions on the Origins of Life; “Ettore Majorana” Foundation and Centre for Scientific Culture, Erice, Italy, 1–6 October 2006.     

人乳腺癌MDA-MB-231细胞裸鼠移植瘤模型的建立及生物学特性研究

目的-建立人乳腺癌MDA-MB-231细胞株裸小鼠模型,研究其生物学特性,观察MDA-MB-231乳腺癌细胞在移植前后的形态学变化。方法-将人乳腺癌细胞MDA-MB-231接种于裸鼠腋窝处皮下,每3天测量肿瘤大小,第30天处死小鼠。肿瘤组织及相关脏器送病理切片。皮下肿瘤组织细胞及细胞株培养HE染色。结果-肿瘤生长较快,成功率为72%,病理检查符合人乳腺癌细胞特征。肿瘤组织细胞及培养细胞形态学未见显著差异。结论-人乳腺癌细胞株MDA-MB-231裸小鼠模型建立方法较简便,细胞形态无明显差异,且保持了人乳腺癌的生物学特性。     

Overview of Mathematical Approaches Used to Model Bacterial Chemotaxis I: The Single Cell

Mathematical modeling of bacterial chemotaxis systems has been influential and insightful in helping to understand experimental observations. We provide here a comprehensive overview of the range of mathematical approaches used for modeling, within a single bacterium, chemotactic processes caused by changes to external gradients in its environment. Specific areas of the bacterial system which have been studied and modeled are discussed in detail, including the modeling of adaptation in response to attractant gradients, the intracellular phosphorylation cascade, membrane receptor clustering, and spatial modeling of intracellular protein signal transduction. The importance of producing robust models that address adaptation, gain, and sensitivity are also discussed. This review highlights that while mathematical modeling has aided in understanding bacterial chemotaxis on the individual cell scale and guiding experimental design, no single model succeeds in robustly describing all of the basic elements of the cell. We conclude by discussing the importance of this and the future of modeling in this area.     

The Human Platelet as a Model for the Glutamatergic Neuron: Platelet Uptake of L-Glutamate

Abstract: L-Glutamate uptake into human platelets revealed two components: a high-affinity system ( K_mH = 3.1 μM), which was sodium-dependent, and a low-affinity site ( K_mL = 88 μM) displaying temperature rather than sodium dependency. These kinetic properties were similar to those found in crude synaptosomal preparations and brain slices. However, V_max values were far higher in brain(V_maxH= 325 ± 96, V_maxH= 3759 ± 1116 pmol/mg wet weight per min) than in platelets (V_maxH, = 14 ± 6, V_maxL= 313 ± 63 pmol/mg platelet protein per 10 min), indicating a denser population in brain than in platelets of qualitatively similar sites. Pharmacological analysis substantiated the resemblance of nerve endings and platelets: the specific uptake inhibitors threo-3-hydroxy-DL-aspartate and DL-aspartate-β-hydroxamate as well as D-and L-glutamate and L-aspartate showed similar—though not identical—IC₅₀ values in both preparations; a spectrum of compounds devoid of inhibitory effects in synaptosomes also did not interfere with glutamate uptake in platelets. Uptake parameters were studied in a population of human volunteers to determine the variability of platelet glutamate uptake. Whole blood could be stored up to 6 h after venipuncture without any appreciable change in experimental values. Percentage of variation between 0.09 and 0.28 for three repetitive (weekly) assays in single subjects indicated that glutamate uptake measurements in human platelets are sufficiently suited for future clinical studies.     

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines¹ support our understanding of invasion of the uterine wall² and remodeling of uterine spiral arteries^3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts^5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation^7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies^10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu¹³.We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues^7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models^10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.