Generation, description and storage of dendritic morphology data

It is generally assumed that the variability of neuronal morphology has an important effect on both the connectivity and the activity of the nervous system, but this effect has not been thoroughly investigated. Neuroanatomical archives represent a crucial tool to explore structure–function relationships in the brain. We are developing computational tools to describe, generate, store and render large sets of three–dimensional neuronal structures in a format that is compact, quantitative, accurate and readily accessible to the neuroscientist. Single–cell neuroanatomy can be characterized quantitatively at several levels. In computer–aided neuronal tracing files, a dendritic tree is described as a series of cylinders, each represented by diameter, spatial coordinates and the connectivity to other cylinders in the tree. This ‘Cartesian’ description constitutes a completely accurate mapping of dendritic morphology but it bears little intuitive information for the neuroscientist. In contrast, a classical neuroanatomical analysis characterizes neuronal dendrites on the basis of the statistical distributions of morphological parameters, e.g. maximum branching order or bifurcation asymmetry. This description is intuitively more accessible, but it only yields information on the collective anatomy of a group of dendrites, i.e. it is not complete enough to provide a precise ‘blueprint’ of the original data. We are adopting a third, intermediate level of description, which consists of the algorithmic generation of neuronal structures within a certain morphological class based on a set of ‘fundamental’, measured parameters. This description is as intuitive as a classical neuroanatomical analysis (parameters have an intuitive interpretation), and as complete as a Cartesian file (the algorithms generate and display complete neurons). The advantages of the algorithmic description of neuronal structure are immense. If an algorithm can measure the values of a handful of parameters from an experimental database and generate virtual neurons whose anatomy is statistically indistinguishable from that of their real counterparts, a great deal of data compression and amplification can be achieved. Data compression results from the quantitative and complete description of thousands of neurons with a handful of statistical distributions of parameters. Data amplification is possible because, from a set of experimental neurons, many more virtual analogues can be generated. This approach could allow one, in principle, to create and store a neuroanatomical database containing data for an entire human brain in a personal computer. We are using two programs, L–NEURON and ARBORVITAE, to investigate systematically the potential of several different algorithms for the generation of virtual neurons. Using these programs, we have generated anatomically plausible virtual neurons for several morphological classes, including guinea pig cerebellar Purkinje cells and cat spinal cord motor neurons. These virtual neurons are stored in an online electronic archive of dendritic morphology. This process highlights the potential and the limitations of the ‘computational neuroanatomy’ strategy for neuroscience databases.

Effects of dendritic morphology on CA3 pyramidal cell electrophysiology: a simulation study

We investigated the effect of morphological differences on neuronal firing behavior within the hippocampal CA3 pyramidal cell family by using three-dimensional reconstructions of dendritic morphology in computational simulations of electrophysiology. In this paper, we report for the first time that differences in dendritic structure within the same morphological class can have a dramatic influence on the firing rate and firing mode (spiking versus bursting and type of bursting). Our method consisted of converting morphological measurements from three-dimensional neuroanatomical data of CA3 pyramidal cells into a computational simulator format. In the simulation, active channels were distributed evenly across the cells so that the electrophysiological differences observed in the neurons would only be due to morphological differences. We found that differences in the size of the dendritic tree of CA3 pyramidal cells had a significant qualitative and quantitative effect on the electrophysiological response. Cells with larger dendritic trees: (1) had a lower burst rate, but a higher spike rate within a burst, (2) had higher thresholds for transitions from quiescent to bursting and from bursting to regular spiking and (3) tended to burst with a plateau. Dendritic tree size alone did not account for all the differences in electrophysiological responses. Differences in apical branching, such as the distribution of branch points and terminations per branch order, appear to effect the duration of a burst. These results highlight the importance of considering the contribution of morphology in electrophysiological and simulation studies.

An epigenetic signature of developmental potential in neural stem cells and early neurons.

A cardinal property of neural stem cells (NSCs) is their ability to adopt multiple fates upon differentiation. The epigenome is widely seen as a read-out of cellular potential and a manifestation of this can be seen in embryonic stem cells (ESCs), where promoters of many lineage-specific regulators are marked by a bivalent epigenetic signature comprising trimethylation of both lysine 4 and lysine 27 of histone H3 (H3K4me3 and H3K27me3, respectively). Bivalency has subsequently emerged as a powerful epigenetic indicator of stem cell potential. Here, we have interrogated the epigenome during differentiation of ESC-derived NSCs to immature GABAergic interneurons. We show that developmental transitions are accompanied by loss of bivalency at many promoters in line with their increasing developmental restriction from pluripotent ESC through multipotent NSC to committed GABAergic interneuron. At the NSC stage, the promoters of genes encoding many transcriptional regulators required for differentiation of multiple neuronal subtypes and neural crest appear to be bivalent, consistent with the broad developmental potential of NSCs. Upon differentiation to GABAergic neurons, all non-GABAergic promoters resolve to H3K27me3 monovalency, whereas GABAergic promoters resolve to H3K4me3 monovalency or retain bivalency. Importantly, many of these epigenetic changes occur before any corresponding changes in gene expression. Intriguingly, another group of gene promoters gain bivalency as NSCs differentiate toward neurons, the majority of which are associated with functions connected with maturation and establishment and maintenance of connectivity. These data show that bivalency provides a dynamic epigenetic signature of developmental potential in both NSCs and in early neurons. Stem Cells 2013;31:1868-1880.

Morphometry and morphology of nucleus of the Sertoli and interstitial cells of the tambaqui Colossoma macropomum (Cuvier, 1881) (Pisces: Characidae) during the reproductive cycle

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Aspiration of equine oocytes from immature follicles after treatment with equine pituitary extract (EPE) alone or in combination with hCG

This study examined the effect of treating mares with equine pituitary extract (EPE) alone or in combination with hCG on the recovery rate of immature follicles by transvaginal follicular aspiration (ovum pick-up; OPU). Ten normally cycling crossbred mares aged 3-15 years and weighing 350-400 kg were subjected to each of three treatments in a random sequence with each exposure to a new treatment separated by a rest cycle during which a spontaneous ovulation occurred. The treatments were (1) superovulated with 25 mg EPE and treated with 2500 IU hCG, (2) superovulation with 25 mg EPE, and (3) control (no exogenous treatment). Treatments 7 days after spontaneous ovulation; and all the follicles > 10 mm were aspirated 24 h after the largest follicle achieved a diameter of 27-30 mm for control group, and most follicles reached 22-27 mm for the EPE alone treatment. To the group EPE+hCG, when the follicles reached 22-27 mm, hCG was administered, 24 h before OPU. Superovulation increased the number of follicles available for aspiration. The total number of follicles available for aspiration was 61 in the EPE/hCG group. 63 in the EPE group and 42 in the control. The proportion of follicles aspirated varied from 63.5% to 73.8%. Oocyte recovery rate ranged from 15.0% to 16.7% and the proportion of mares that yielded at least one oocyte was 70% (7/10) in the EPE/hCG, 60% (6/10) in the EPE alone and 50% (5/10) in control group. The EPE/hCG treatment had a higher proportion of follicles with expanded granulose cells (64.4%) than the control (3.3%: p < 0.05) and the EPE treatment (25.0%). The intervals from spontaneous ovulation to aspiration were similar for all treatments (11-12 days). However, superovulatory treatment significantly increased the aspiration to ovulation interval from 15 +/- 4 days for control to 27 +/- 15 days for EPE (p < 0.05) and to 23 +/- 13 days for EPE/hCG treatment with commensurate increases in the time between spontaneous ovulations. (c) 2008 Elsevier B.V. All rights reserved.

Collection Rates and Morphology of Equine Oocytes Obtained from Immature Follicles after Treatment with Equine Pituitary Extract (EPE) and Human Chorionic Gonadotropin

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Tissue Reaction to a Triantibiotic Paste Used for Endodontic Tissue Self-regeneration of Nonvital Immature Permanent Teeth

Introduction: The endodontic regenerative procedure (ERP), which is an alternative to calcium hydroxide induced apexification, involves the use of a triple antibiotic paste (TAP) as a dressing material. The aim of this study was to evaluate the response of rat subcutaneous tissue to implanted polyethylene tubes that were filled with TAP or calcium hydroxide. Methods: Thirty rats received 2 individual implants of polyethylene tubes filled with TAP or calcium hydroxide paste (CHP) and another empty tube as a control. Thirty additional rats received 2 individual implants consisting of polyethylene tubes filled with dressing material carriers (macrogol and propylene glycol) and a sham procedure. After 7, 15, 30, 60, and 90 days, 12 animals were euthanized, and the tubes and surrounding tissue were removed and processed for histology by using glycol methacrylate and stained with hematoxylin and eosin. The histological score ranged from 0 to 3 depending on the content of inflammatory cells; the fibrous capsule was considered thin or thick, and necrosis and calcification were recorded as present or absent. The results were analyzed using the Kruskal-Wallis test. Results: Both dressing materials induced moderate reactions at 7 and 15 days. These reactions were similar to the control (P>.05) and reduced in intensity (to mild) from day 30 onward (P>.05). The carriers did not interfere with the reaction of the dressing materials. Conclusions: TAP and CHP were biocompatible over the different experimental periods examined. (J Endod 2012;38:91-94)

Relationship between Mast Cells and the Colitis with Relapse Induced by Trinitrobenzesulphonic Acid in Wistar Rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Differentiation of Leydig Cells in the Mongolian Gerbil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Selective Ablation of the Androgen Receptor in Mouse Sertoli Cells Affects Sertoli Cell Maturation, Barrier Formation and Cytoskeletal Development

The observation that mice with a selective ablation of the androgen receptor (AR) in Sertoli cells (SC) (SCARKO mice) display a complete block in meiosis supports the contention that SC play a pivotal role in the control of germ cell development by androgens. To delineate the physiological and molecular mechanism responsible for this control, we compared tubular development in pubertal SCARKO mice and littermate controls. Particular attention was paid to differences in SC maturation, SC barrier formation and cytoskeletal organization and to the molecular mediators potentially involved. Functional analysis of SC barrier development by hypertonic perfusion and lanthanum permeation techniques and immunohistochemical analysis of junction formation showed that SCARKO mice still attempt to produce a barrier separating basal and adluminal compartment but that barrier formation is delayed and defective. Defective barrier formation was accompanied by disturbances in SC nuclear maturation (immature shape, absence of prominent, tripartite nucleoli) and SC polarization (aberrant positioning of SC nuclei and cytoskeletal elements such as vimentin). Quantitative RT-PCR was used to study the transcript levels of genes potentially related to the described phenomena between day 8 and 35. Differences in the expression of SC genes known to play a role in junction formation could be shown from day 8 for Cldn11, from day 15 for Cldn3 and Espn, from day 20 for Cdh2 and Jam3 and from day 35 for ZO-1. Marked differences were also noted in the transcript levels of several genes that are also related to cell adhesion and cytoskeletal dynamics but that have not yet been studied in SC (Actn3, Ank3, Anxa9, Scin, Emb, Mpzl2). It is concluded that absence of a functional AR in SC impedes the remodeling of testicular tubules expected at the onset of spermatogenesis and interferes with the creation of the specific environment needed for germ cell development.

Role of natural killer cells in antitumor resistance

Natural killer cells constitute a population of lymphocytes able to non-specifically destroy virus-infected and some kinds of tumor cells. Since this lytic activity was shown by non-immunized animals the phenomenon is denominated natural killer (NK) activity and contrasts with specific cytotoxicity performed by cytolytic T lymphocytes (CTLs) because it does not depends on MHC-restricted peptides recognition. In fact, the main feature of most functional receptors of NK cells (NKRs) is their ability to be inhibited by different kinds of class I MHC antigens. In the middle of the 1950's, Burnet & Thomas forged the concept of tumor immunosurveillance and NK cells can be considered one of the main figures in this phenomenon both for effector and regulatory functions. In the present review the early studies on the biology of NK cells were revisited and both their antitumor activity and dependence on the activation by cytokines are discussed.

Relationships between dendritic morphology, spatial distribution and firing patterns in rat layer 1 neurons

The cortical layer 1 contains mainly small interneurons, which have traditionally been classified according to their axonal morphology. The dendritic morphology of these cells, however, has received little attention and remains ill defined. Very little is known about how the dendritic morphology and spatial distribution of these cells may relate to functional neuronal properties. We used biocytin labeling and whole cell patch clamp recordings, associated with digital reconstruction and quantitative morphological analysis, to assess correlations between dendritic morphology, spatial distribution and membrane properties of rat layer 1 neurons. A total of 106 cells were recorded, labeled and subjected to morphological analysis. Based on the quantitative patterns of their dendritic arbor, cells were divided into four major morphotypes: horizontal, radial, ascendant, and descendant cells. Descendant cells exhibited a highly distinct spatial distribution in relation to other morphotypes, suggesting that they may have a distinct function in these cortical circuits. A significant difference was also found in the distribution of firing patterns between each morphotype and between the neuronal populations of each sublayer. Passive membrane properties were, however, statistically homogeneous among all subgroups. We speculate that the differences observed in active membrane properties might be related to differences in the synaptic input of specific types of afferent fibers and to differences in the computational roles of each morphotype in layer 1 circuits. Our findings provide new insights into dendritic morphology and neuronal spatial distribution in layer 1 circuits, indicating that variations in these properties may be correlated with distinct physiological functions.

Histopathological Condition of the Remaining Tissues after Endodontic Infection of Rat Immature Teeth

Introduction: Recently, case reports have shown that immature teeth diagnosed with necrotic pulp and periapical periodontitis can be repaired through a regenerative endodontic procedure. True regeneration depends on the presence of stem cells in the remaining vital tissues. The aim of this study was to evaluate the histologic condition of the pulp tissue, root apical papilla, and periapical tissues after inducing endodontic infection in immature rat teeth for different periods. Methods: This study evaluated 18 first upper rat molars (36 roots). Periapical lesions were induced and were confirmed radiographically, and the animals were divided into 3 groups according to the days of pulp exposure for endodontic infection induction: 30, 60, and 90 days. Histologic analysis was performed in 5 different areas (ie, cervical, middle, and apical root canal thirds; the apical papilla; and the periapex surrounding the apical papilla). Results: At 30 days, one third of the specimens still showed vital but intensely inflamed pulp tissue in the apical third and vital apical papilla with varying degrees of inflammation. After 60 days, the results were similar with respect to the apical pulp tissue and apical papilla. Completely necrotic pulp tissue in the space canal and vital apical papilla were observed in about 67% of the cases after 90 days. Conclusions: Vital pulp tissue was observed in the apical third until 60 days and in the vital apical papilla until 90 days of infection in a rat model.

Gene Expression in Bovine Oocytes and Cumulus Cells After Meiotic Inhibition with the Cyclin-Dependent Kinase Inhibitor Butyrolactone I

Contents The aim of this study was to determine the effect of temporary inhibition of meiosis using the cyclin-dependent kinase inhibitor butyrolactone I (BLI) on gene expression in bovine oocytes and cumulus cells. Immature bovine cumulusoocyte complexes (COCs) were assigned to groups: (i) Control COCs collected immediately after recovery from the ovary or (ii) after in vitro maturation (IVM) for 24 h, (iii) Inhibited COCs collected 24 h after incubation with 100 mu m BLI or (iv) after meiotic inhibition for 24 h followed by IVM for a further 22 h. For mRNA relative abundance analysis, pools of 10 denuded oocytes and respective cumulus cells were collected. Transcripts related to cell cycle regulation and oocyte competence were evaluated in oocytes and cumulus cells by quantitative real-time PCR (qPCR). Most of the examined transcripts were downregulated (p < 0.05) after IVM in control and inhibited oocytes (19 of 35). Nine transcripts remained stable (p > 0.05) after IVM in control oocytes; only INHBA did not show this pattern in inhibited oocytes. Seven genes were upregulated after IVM in control oocytes (p < 0.05), and only PLAT, RBP1 and INHBB were not upregulated in inhibited oocytes after IVM. In cumulus cells, six genes were upregulated (p < 0.05) after IVM and eight were downregulated (p < 0.05). Cells from inhibited oocytes showed the same pattern of expression regarding maturation profile, but were affected by the temporary meiosis inhibition of the oocyte when the same maturation stages were compared between inhibited and control groups. In conclusion, changes in transcript abundance in oocytes and cumulus cells during maturation in vitro were mostly mirrored after meiotic inhibition followed by maturation.