A method for reconstructing patterns of somatosensory cerebral cortical activity

An interactive graphics package was developed in order to acquire, display, and manipulate images of cerebral cortical autoradiographic data. The primary purpose for development of the system was to reconstruct accurate 2-dimensional maps of the functional activity within the somatosensory cerebral cortex. A Datacube Q-bus graphics module (QVG/QAF-123) was interfaced with the Micro PDP-11/23 to accept a standard RS170 video input signal, and autoradiographs of serial sections (each 20 microns thick) of a cerebral cortex were digitized individually to 768 X 512 X 8 bit resolution. Input look-up tables were used to standardize the autoradiographic data. Boundaries of the somatosensory cortex were entered (with a Summagraphics MM 1201 digitizer), and the image data was stored on disk file (a method of data compression was devised). A method for segmenting the image data for many (sequential) sections was developed that provided arrays from which the maps were generated. Thresholding, histogram equalization, edge detection and edge enhancement, and filters in both the spatial and frequency domains were employed to process the images of the maps. Plots of optical density values along any axis of the maps and gray level histograms of any map region could also be generated. Maps made by the described method are much higher in resolution than those produced by traditional (manual) methods, and permit analysis of the reconstructions in both the frequency and spatial domains.     

Synaptic circuitry identified by intracellular labeling with horseradish peroxidase

During the past two decades new techniques have been developed to directly test the dogma that neuronal structure is correlated with neuronal function. In the earliest experiments, Procion yellow was injected into neurons after they had been characterized physiologically; these neurons were then viewed through the light microscope. Recent advances in the method generally employ horseradish peroxidase as the dye which is injected since it diffuses quite readily throughout the injected neuron and produces a stable reaction product for both light and electron microscopic studies. This review explores the utility of examining synaptic circuitry after physiologically recording from axons or neurons and then injecting horseradish peroxidase into them. As a model system, we studied the cat lateral geniculate nucleus and investigated, at the electron microscopic level, the synaptic contribution to this nucleus from retinogeniculate axons, from interneurons, and from the axon collaterals of neurons that project to visual cortex.     

Analysis of neuronal networks: A review of techniques for labeling axonal projections

In order to analyze connections between neurons in the vetebrate central nervous system, methods have been developed to label a given population of axons of known origin so that they can be differentiated from other, non-labeled structures. Three such methods are reviewed here: experimentally induced orthograde (Wallerian) degeneration, axon transport of radioactive proteins demonstrated by autoradiography, and axon transport of macromolecules that can be reacted histochemically to yield a visible reaction product. Each of the methods has particular strengths and weaknesses. Degeneration methods may differentiate between different functional classes of axons which have different fiber diameters. However, degeneration distorts the morphology of axon terminals, making them more difficult to interpret, and degenerating terminals may be removed rapidly by phagocytosis. Autoradiography of radioactive terminals preserves normal fine structure, but the necessary exposure times extend the method by weeks or months, and care must be exercised to distinguish labeled axons from other structures exhibiting background or transneuronal radioactivity. Histochemical methods, such as those used to demonstrate horseradish peroxidase conjugated to wheat germ lectin (WGA-HRP), are sensitive and rapid, but the injection site must be carefully characterized, and the presence of transneuronal label may make interpretation of the results difficult. Experimental methods of axonal labeling have been invaluable in studying neuronal networks. Each of the methods described here may be of particular value, given the nature of the system to be analyzed.     

Degeneration and regeneration of ganglion cell axons

The retino-tectal system has been used to study developmental aspects of axon growth, synapse formation and the establishment of a precise topographic order as well as degeneration and regeneration of adult retinal ganglion cell (RGC) axons after axonal lesion. This paper reviews some novel findings that provide new insights into the mechanisms of developmental RGC axon growth, pathfinding, and target formation. It also focuses on the cellular and molecular cascades that underlie RGC degeneration following an axonal lesion and on some therapeutic strategies to enhance survival of axotomized RGCs in vivo. In addition, this review deals with problems related to the induction of regeneration after axonal lesion in the adult CNS using the retino-tectal system as model. Different therapeutic approaches to promote RGC regeneration and requirements for specific target formation of regenerating RGCs in vitro and in vivo are discussed.     

Ultrastructural studies of microtubules and microtubule organizing centers of the vertebrate olfactory neuron.

The olfactory neuron is specialized along its length into highly determined morphological regions. These regions include the dendritic cilia, dendritic vesicle, dendritic shaft proper, perikaryon, axon, zone of transition where the axon widens as it approaches its termination, and the axon terminal. Except for the zone of transition and the terminal, characteristic populations of microtubules occur in these compartments. In the olfactory vesicle, three discrete microtubule organizing centers (MTOCs) nucleate microtubules: the basal body, the lateral foot associated with the body, and dense masses of nearby material. Little is known about MTOCs elsewhere in the neuron, although the polarity of the axonal microtubules indicate that they originate at or near the perikaryon. An attempt is made to summarize what is known of the origin, structure, distribution, and function of microtubules in vertebrate olfactory neurons, which are useful model systems in which to study microtubules. Information about olfactory neuron microtubules may be applicable to neurons in general (e.g., the discovery that axons contain microtubules of uniform polarity was first made in the olfactory neuron) or to microtubules in other eukaryotic cells.     

Electron microscopy and morphometric analyses of microtubules in two differently sized types of axons in the protocerebral tract of a crustacean

Despite several reports on the morphology and functions associated with the morphometry of the vertebrate axoplasm cytoskeleton, the subject has not been thoroughly explored in invertebrates. In vertebrates, among many other functions, microtubules (MTs) serve as scaffolding for axon assembly, and neurofilaments (NFs) as the elements that determine the axon caliber. Intermediate filaments have never been described by electron microscopy in arthropods, although NF proteins have been revealed in the MT side-arms of the axoplasm of certain species, such as the crab Ucides cordatus. Thus, it is not known which elements of the cytoskeleton of invertebrates are responsible for determination of the axon caliber. We studied, by electron microscopy and morphometric analyses, the MT and axon area variability in differently sized axons of the protocerebral tract of the crab Ucides cordatus. Our results revealed differences in the distance between MTs, in MT density and number, and in the areas of differently sized axons. The number of MTs increases with the axon area, but this relationship is not directly proportional. Therefore, MT density is greater in smaller axons than in medium axons, similar to the morphometry of the vertebrate axon MT. The distance between MTs is, however, directly related to the axonal area. On the basis of the results shown here, and on previous reports by us and others, we suggest that MTs may be involved in the determination of the axon caliber, possibly due to the presence of NF proteins found in the side-arms.     

Quantitative aspects of synapses on Golgi-impregnated neurons.

With the classical Golgi techniques, numerous types of neurons can be distinguished in the cerebral cortex, each with a specific dendritic geometry and pattern of axonal ramifications. In the present review we describe two techniques which allow quantification of synapses on identified neurons: (1) Golgi-rapid impregnation-gold toning-electron microscopy, and (2) Golgi-Kopsch impregnation-gold toning-electron microscopy in combination with staining of the tissue with ethanolic phosphotungstic acid (E-PTA). Both techniques were applied on neurons in the visual cortex of young and adult rabbits. By means of rotating and tilting specimens in the electron microscope, the nondistinctive ultrastructure of obliquely sectioned synapses can be circumvented, leading to precise estimates of asymmetrical vs. symmetrical synapses without complete reconstruction of the neuron.     

Use of lipophilic dyes in studies of axonal pathfinding in vivo

Fluorescent lipophilic dyes are an ideal tool to study axonal pathfinding. Because these dyes do not require active axonal transport for their spreading, they can be used in fixed tissue. Here, we describe the method we have used to study the molecular mechanisms of commissural axon pathfinding in the embryonic chicken spinal cord in vivo. Based on in vitro studies, different families of molecules had been suggested to play a role in the guidance of developing axons. In order to test their function in vivo, we used the commissural neurons that are located at the dorsolateral border of the chicken spinal cord as a model system [Stoeckli and Landmesser (1995) Neuron 14:1165-1179]. Axonin-1, NgCAM, and NrCAM, three members of the immunoglobulin (Ig) superfamily of cell adhesion molecules (CAMs), were shown to be important for the correct growth pattern of commissural axons. We studied the effect of perturbations of specific CAM/CAM interactions by injection of function-blocking antibodies into the central canal of the spinal cord in ovo. After 2 days, the embryos were sacrificed and fluorescent tracers, such as Fast-DiI, were used to visualize commissural axons, and thus, to analyze their response to these perturbations in two different types of fixed preparations: transverse vibratome sections and whole-mount preparations of the spinal cord. Both pathfinding errors and defasciculation of axons were observed as a result of the perturbation of CAM/CAM interactions.     

An application of numerical taxonomy to the study of the cerebral cortex of Natrix maura (L)

The separate cellular regions of the reptilian cerebral cortex were studied using Numerical Taxonomy. Three parameters were employed: a) The area occupied by each region at the several levels studied. b) The total area of the cell nuclei present in each level. c) The average are of these nuclei. Numerical Taxonomy resolves the problem by means of a dendrogram which represents the normalised distances, which indicate levels of similarity on absciassas. The elements studies are on the ordinate axis. The dendrogram shows the different levels of similarity existing between each one of the chosen populations. Depending upon the degree of similarity one may deduce the similarities or differences existing between these populations, and also the characteristics of each cellular population throughout the length of its presence in the cerebral cortex and the variations between the regions. These results, in the first place, relate to the four cortical regions: medialis cortex, dorsomedialis cortex, dorsalis cortex, and lateralis cortex, and in the second place, to each one of the regions within the entire telencephalic cortex.     

Improvement in cytoarchitectonic mapping by combining electrodynamic modeling with local orientation in high‐resolution images of the cerebral cortex

To detect changes of cortical cytoarchitectonics, digital images of cortical laminations are analyzed. Cortical regions are transformed into a rectangular grid for subsequent evaluations. Transformations are realized by stepwise scanning using perpendicular testlines. 3D cytoarchitectonic data of the human brain at a histological resolution are not available and 2D sections deliver partial information only. The problem is to find an optimal scanning‐technique that introduces a minimum of distortions and noise by the transformation of the curvilinear cortex to a rectangular presentation. In the past this was solved by constructing testlines dependent on the outlined cortical contours only. An advanced approach was to model the contours as electrically charged surfaces and to use the resulting field lines as testlines. However, local information of cell distributions were not considered. Hence a novel hybrid approach was developed which is able to construct significantly better testlines in cortical images with mixtures of columnar rich (local orientation rich) and orientation poor parts of strongly curved and large regions of the cerebral cortex. The novel hybrid approach is based on the computer vision methods such as the structure tensor and constrained anisotropic diffusion. In addition, the introduction of projective transformations yields a significant improvement of cortical fingerprints, thereby offering the possibility for detecting weakly pronounced regions of cytoarchitectonic transitions. The statistical evaluation of the novel hybrid approach confirms robustness. This technique can be generalized and applied to different types of cerebral cortex with any kind and amount of local orientation information. Microsc. Res. Tech. 2010. © 2010 Wiley‐Liss, Inc.     

Three-dimensional microscopy data exploration by interactive volume visualization

This paper presents a new volume visualization approach for three-dimensional (3-D) interactive microscopy data exploration. Because of their unique image characteristics, 3-D microscopy data are often not able to be visualized effectively by conventional volume visualization techniques. In our approach, microscopy visualization is carried out in an interactive data exploration environment, based on a combination of interactive volume rendering techniques and image-based transfer function design methods. Interactive volume rendering is achieved by using two-dimensional (2-D) texture mapping in a Shear-Warp volume rendering algorithm. Image processing techniques are employed and integrated into the rendering pipeline for the definition and searching of appropriate transfer functions that best reflect the user's visualization intentions. These techniques have been implemented successfully in a prototype visualization system on low-end and middle-range SGI desktop workstations. Since only 2-D texture mapping is required, the system can also be easily ported to PC platforms.     

Tolerance design of robot parameters using Taguchi method

A robotic arm must be able to manipulate objects with high accuracy and repeatability. As with every physical system, there are number of noise factors cause uncertainty in the performance. A probabilistic approach has been used to model, the otherwise difficult to model, noise effects. This paper presents the approach utilised in selection of tolerance specification of robot kinematic and dynamic parameters using experimental design technique for reduction of performance variations. The concept of inner and outer orthogonal arrays proposed by Taguchi is employed to identify the significant parameters and select the optimal tolerance range. The performance measure, i.e. signal-to-noise ratio is utilised to validate by Monte Carlo simulations and to complement above study individual parameter tolerance sensitivity are investigated. To provide insight to investigation, parameter sensitivity maps are plotted. The tolerance specification selection methodology of a manipulator is illustrated by taking 2-DOF RR planar manipulator with payload.     

Three‐dimensional microscopy data exploration by interactive volume visualization

This paper presents a new volume visualization approach for three‐dimensional (3‐D) interactive microscopy data exploration. Because of their unique image characteristics, 3‐D microscopy data are often not able to be visualized effectively by conventional volume visualization techniques. In our approach, microscopy visualization is carried out in an interactive data exploration environment, based on a combination of interactive volume rendering techniques and image‐based transfer function design methods. Interactive volume rendering is achieved by using two‐dimensional (2‐D) texture mapping in a Shear‐Warp volume rendering algorithm. Image processing techniques are employed and integrated into the rendering pipeline for the definition and searching of appropriate transfer functions that best reflect the user's visualization intentions. These techniques have been implemented successfully in a prototype visualization system on low‐end and middle‐range SGI desktop workstations. Since only 2‐D texture mapping is required, the system can also be easily ported to PC platforms.     

Strategies for studying microtubule transport in the neuron

Microtubules are prominent cytoskeletal elements within the neuron. They are essential for the differentiation, growth, and maintenance of axons and dendrites. The microtubules within each type of process have a distinct pattern of organization, and these distinct patterns result in many of the morphological and structural features that distinguish axons and dendrites from one another. There are a number of challenges that must be met in order for the neuron to establish the microtubule arrays of axons and dendrites. One attractive model invokes the active transport of microtubules from the cell body of the neuron into and down these processes. In support of this model, specific motor proteins have now been identified within neurons that have the necessary properties to transport microtubules into developing axons and dendrites with the appropriate orientation for each type of process. An important goal is to develop microscopic methods that permit the visualization of microtubule transport within different regions of the neuron. To date, achieving this goal has met with mixed success, probably as a result of the geometry of the neuron and the inherent complexity of the neuronal microtubule arrays. While some approaches have failed to reveal microtubule transport, other more recent approaches have proven successful. These approaches provide strong visual support for a model based on microtubule transport, and provide hope that future approaches can provide even clearer demonstrations of this transport.     

Towards correlative imaging of plant cortical microtubule arrays: combining ultrastructure with real-time microtubule dynamics

There are a variety of microscope technologies available to image plant cortical microtubule arrays. These can be applied specifically to investigate direct questions relating to array function, ultrastructure or dynamics. Immunocytochemistry combined with confocal laser scanning microscopy provides low resolution "snapshots" of cortical microtubule arrays at the time of fixation whereas live cell imaging of fluorescent fusion proteins highlights the dynamic characteristics of the arrays. High-resolution scanning electron microscopy provides surface detail about the individual microtubules that form cortical microtubule arrays and can also resolve cellulose microfibrils that form the innermost layer of the cell wall. Transmission electron microscopy of the arrays in cross section can be used to examine links between microtubules and the plasma membrane and, combined with electron tomography, has the potential to provide a complete picture of how individual microtubules are spatially organized within the cortical cytoplasm. Combining these high-resolution imaging techniques with the expression of fluorescent cytoskeletal fusion proteins in live cells using correlative microscopy procedures will usher in an radical change in our understanding of the molecular dynamics that underpin the organization and function of the cytoskeleton.     

Immunohistochemistry of GluR1 subunits of AMPA receptors of rat cerebellar nerve cells

The localization of GluR1 subunits of ionotropic glutamate receptors in the glial cells and inhibitory neurons of cerebellar cortex and their association with the climbing and parallel fibers, and basket cell axons were studied. Samples of P14 and P21 rat cerebellar cortex were exposed to a specific antibody against GluR1 subunit(s) of AMPA receptors and were examined with confocal laser scanning microscopy. GluR1 strong immunoreactivity was confined to Purkinje cell and the molecular layer. Weak GluR1 immunoreactivity was observed surrounding some Golgi cells in the granule cell layer. Intense GluR1 immunoreactivity was localized around Purkinje, basket, and stellate cells. Purkinje cells expressed strong GluR1 immunoreactivity surrounding the cell body, primary dendritic trunk and secondary and tertiary spiny dendritic branches. Marked immunofluorescent staining was also detected in the Bergmann glial fibers at the level of middle and outer third molecular layer. Positive immunofluorescence staining was also observed surrounding basket and stellate cells, and in the capillary wall. These findings suggest the specific localization of GluR1 subunits of AMPA receptors in Bergmann glial cells, inhibitory cerebellar neurons, and the associated excitatory glutamatergic circuits formed by climbing and parallel fibers, and by the inhibitory basket cell axons     

基于过渡面的同速区间规划与质量预估研究

针对过渡曲面加工表面质量往往比普通曲面更难控制的特殊性,开展与过渡面适应的速度规划、加工轨迹的局部优化等研究来改善过渡面的表面质量,并通过运动参数可视化来预测过渡面的加工质量。在一定变化范围内,使空间曲率、挠率具有连续性的连续程序段属于一个区间,以建立过渡面的刀具轨迹“同速区间”。在同速区间内建立弧长参数化、拟合递推式Akima样条曲线,实现刀具轨迹的局部优化。通过数控模拟软件采集运动参数(速度、加速度及电流等),生成的插补文件导入自主开发的运动参数可视化软件,形成可视化图以此来预估加工表面质量。分别对可乐瓶底与变曲率曲面的过渡面进行同速区间规划与轨迹优化,得到了优化前后的可视化图。进一步,验证预估表面质量的有效性,通过对变曲率过渡面和五指山零件实际加工,验证了加工表面质量提高与否与可视化图好坏的一致性。因此建立过渡面同速区间及运动参数可视化,以实现过渡面的质量预估及提升效果。     

Ceramic wear maps

Ceramic wear maps have been developed to elucidate the complex interactions of the operating parameters, environments, and wear mechanisms. This paper summarizes these interactions for four ceramics, alumina, yttria-doped zirconia, silicon carbide and silicon nitride. Wear maps of these ceramics are systematically constructed using measured data under dry sliding, water, and paraffin lubricated conditions. For each material, different wear level regions acid wear transition zones are identified as a function of operating conditions and lubrication conditions. Wear mechanism studies performed within each wear region give rise to the wear mechanism maps. These maps facilitate material comparison and selection. The knowledge of wear, wear transitions, and wear mechanisms for a material pair enables realistic wear model development. One outcome of this approach is the recognition that a single wear model for a material pair cannot cover all operating conditions and environments.

As wear maps are constructed today, they are material pair specific. Within a material pair, there are microstructural dependence and surface properties influence. These parameters can change substantially for a given chemical composition of the material. How to incorporate these factors into the wear map research remains an issue. The search for a universal parameter such as the “asperity temperature” in Ashby's wear map continues in spite of mounting evidence that this may not be practical or feasible. But the hope remains that some parameters can be identified to normalize a large number of materials, operating conditions, and environments for tribological applications. Systematic wear maps are the first steps in this direction.     

Cryofixation rapidly preserves cytoskeletal arrays of leaf epidermal cells revealing microtubule co-alignments between neighbouring cells and adjacent actin and microtubule bundles in the cortex

Accurate preservation of microtubule and actin microfilament arrays is crucial for investigating their roles in plant cell development. Aldehyde fixatives such as paraformaldehyde or glutaraldehyde preserve cortical microtubule arrays but, unless actin microfilaments are stabilized with drugs such as m-maleimidobenzoyl N-hydroxysuccinimide ester (MBS), ethylene glycol bis[sulfosuccinimidylsuccinate] (sulfo-EGS) or phalloidin, their arrays are often poorly preserved. Cryofixation, used primarily for electron microscopy, preserves actin microfilaments well but is used rarely to fix plant cells for optical microscopy. We developed a novel whole-mount cryofixation method to preserve microtubule and microfilament arrays within Tradescantia virginiana leaf epidermal cells for investigation using confocal microscopy. Cortical microtubule arrays were often oriented in different directions on the internal and external faces of the epidermal cells. A number of arrays were aligned in several directions, parallel to microtubules of neighbouring cells. Actin microfilaments were particularly well preserved possibly due to the speed with which they were immobilized. No transverse cortical microfilament arrays were observed. On occasion, we observed co-aligned microfilament and microtubule bundles lying adjacent to the plasma membrane and positioned side by side suggesting a potential direct interaction between the cytoskeletal filaments at these locations. Cryofixation is therefore a valuable tool to investigate the interactions between cytoskeletal arrays in plant cells using confocal microscopy.     

Basic techniques for long distance axon tracing in the spinal cord

The regeneration of axons after a spinal cord injury or disease is attracting a significant amount of interest among researchers. Being able to assess these axons in terms of morphology, length and origin is essential to our understanding of the regeneration process. Recently, two specific axon tracers have gained much recognition; biotinylated dextran amine (BDA) 10 kDa as an anterograde tracer and cholera toxin‐B as a retrograde tracer. However, there are still several complexities when using these tracers, including the volume that should be administered and the best administration site so that a significant amount of axons are labeled in the area of interest. In this article, we describe some simple procedures for injecting the tracers and detecting them. We also quantified the number of axons at different locations of the spinal cord. Our results show axons labeled from motor cortex injections traveled down to the lumbosacral spinal cord in 2 weeks, while BDA injections into the lateral vestibular nucleus and reticular formation took 3 weeks to label axons in the lumbosacral spinal cord. Moreover, this protocol outlines some basic procedures that could be used in any laboratory and gives insight into the number of axons labeled and how procedures could be tailored to meet specific researcher's needs. Microsc. Res. Tech. 76:1240–1249, 2013. © 2013 Wiley Periodicals, Inc.