The structure of the first auditory cortex (A I) in the cat. I.--Light microscopic observations on its organization

The cyto- and mieloarchitecture of the first auditory cortex (A I) was studied in the cat. The cortical layers II, III and IV are very densely populated by relatively uniform, round or stellate cells with 20 to 30 micro perikaryal diameter. The separation between these three layers, which is not possible in Nissl stained sections, becomes visible in 1 to 3 micro thick sections of plastic embedded material. nerve cells in layer II are randomly disposed, whilst they form in laver III loose rounded cellular groups, and in layer IV vertical cylinders which have 50 to 60 micro in outside diameter and a cell poor centre. These cylinders are best visible in 100 micro thick Nissl preparations, cut parallel to the pial surface. The cylinders may extend into layer V, which is comparatively cell poor. The VIth layer contains numerous round, stellate or fusiform cells with 20 to 30 micro in diameter. The IIIrd and Vth layers have few pyramidal perikarya which are small. Large or giant pyramidal cells are not found in A I. The overall thickness of the cortex in the convexity of A I is 2,000 micro, measured in sections of plastic blocks. The thickness of the 6 layers is 200 to 250 micro for layer I; 300 micro for layer II; 300 micro for layer III; 300 to 400, for layer IV; 350 micro for layer V; and 400 micro for layer VI. In preparations stained for myelin sheats A I is characterized by the presence of a very dense plexus of fibres running in all directions in the IVth, Vth anti VIth layers. These plexus obscurs the radiations of Meynert, giving a characteristic appearance to A I, since these radiations are prominent in the neighbouring cortical areas. In preliminary studies of Golgi rapid preparations of A I the cell types commonly present in others cortical areas were found. Pyramidal cells have small perikarya, and very long (600 micro) horizontal basal dendrites. Modified pyramidal cells (star pyramids) are the main cellular element in layer II and constitute one of the main sources of efferent fibres of A I. Several types of stellate cells were found, including a particular cell type, found very often in the IVth layer, with a very long horizontal axon. The specific thalamic afferents were identified as fibres with 5 or 8 micro in diameter, which run obliquely and sinuously through the VIth and Vth layers of A I. These fibres give off many branches with 1 to 2 micro in diameter, which pass to the IVth layer where they give off very thin sinuous branches, ending in small terminal knobs. The ramification of one of these fibres may spread horizontally over 800 micros, at the level of the IVth layer.     

The Influence of Electric Field and Confinement on Cell Motility

The ability of cells to sense and respond to endogenous electric fields is important in processes such as wound healing, development, and nerve regeneration. In cell culture, many epithelial and endothelial cell types respond to an electric field of magnitude similar to endogenous electric fields by moving preferentially either parallel or antiparallel to the field vector, a process known as galvanotaxis. Here we report on the influence of dc electric field and confinement on the motility of fibroblast cells using a chip-based platform. From analysis of cell paths we show that the influence of electric field on motility is much more complex than simply imposing a directional bias towards the cathode or anode. The cell velocity, directedness, as well as the parallel and perpendicular components of the segments along the cell path are dependent on the magnitude of the electric field. Forces in the directions perpendicular and parallel to the electric field are in competition with one another in a voltage-dependent manner, which ultimately govern the trajectories of the cells in the presence of an electric field. To further investigate the effects of cell reorientation in the presence of a field, cells are confined within microchannels to physically prohibit the alignment seen in 2D environment. Interestingly, we found that confinement results in an increase in cell velocity both in the absence and presence of an electric field compared to migration in 2D.     

van der Waals forces between cylinders: Arrays of thin cylinders and three body forces

We study the van der Waals energy of interaction of an array of parallel dielectric cylinders immersed in a dielectric medium. We consider only “thin” cylinders which have radius small compared to the separation of the cylinders. The energy is calculated as a sum of two body plus three body interactions. The case of hexagonally close packed cylinders is studied in more detail. Some biophysical applications are discussed and in particular the van der Waals energy of the myosin lattice in striated muscle is examined.     

Characterization and reassembly of a regular array in the cell wall of Clostridium difficile GAI 4131

The cell wall of Clostridium difficile GAI 4131 was revealed by electron microscopy to have an outer layer composed of a nearly square array and contained the two major proteins with molecular weights of 38 kDa and 42 kDa. The properties and reassembly of the two major proteins into the regular array were investigated. When the isolated cell walls were treated with hydrophobic bond-disrupting agents or a chelating agent specific for Ca2+, the two major proteins were effectively removed and the regularly arranged outer layer disappeared. The amino acid composition of the two major proteins differed from each other. The two major proteins also gave different peptide maps from each other upon proteolysis with Staphylococcus aureus V8 protease. The major proteins solubilized from the isolated cell walls with 8 M urea or 4 M guanidine hydrochloride could be reassembled into open-ended cylinders possessing the native regular pattern by dialysis against neutral buffer containing 5 mM CaCl2. The reassembled cylinders purified by centrifugation on a Percoll density gradient were composed of almost equal amounts of the 38 kDa and 42 kDa proteins and freed from the other proteins. These results suggest that the regular array in the outer cell wall layer is constructed from the two major cell wall proteins and requires Ca2+ for its assembly.     

A fiber matrix model for interstitial fluid flow and permeability in ligaments and tendons

Collagen fibrils in ligaments and tendons are highly organized into parallel arrays which influence interstitial fluid transport. Finite element (FE) models were developed analogous to the fibrillar arrays in ligaments and tendons to investigate interstitial fluid flow and tissue permeability as a function of interfibrillar spacing and fluid properties. Collagen fibrils were assumed to be a periodic square array of impermeable cylinders. A two-dimensional FE model was used to study transverse fluid flow and a three-dimensional model was used to study flow parallel to the collagen fibrils. Parametric FE analysis provided data to formulate empirical expressions for permeability (kappa) as a function of porosity (phi). Results show that longitudinal permeability (kappa = 1.1.10(-15)phi 2.5[1 - phi]-0.333) can be up to 50 times higher than transverse permeability (kappa = 1.2.10(-15)phi 0.5[phi - phi min]2.5) in a compact array. Maximum fluid shear stresses occur at the narrowest zones of adjacent fibrils (1.21 Pa or 12.1 dyn/cm2 at 10 microns/s of average transverse influx). If interstitial fluid is highly non-Newtonian, the permeability should be considered as flow (shear)-dependent. The computational results suggest that tissue permeability in ligaments and tendons is highly anisotropic, porosity-dependent, and can be estimated by analytic expressions.     

Modeling the electromobility of ions in a target tissue

Electroporation is a clinical and laboratory technique for the delivery of molecules to cells. This method imposes electric fields onto cells or tissues through the use of electrodes and a set of electrical parameters to ultimately incorporate molecules into the cells. Clinical applications may include using directional fields to bring therapeutics to the target tissues before triggering an electroporation event. The choice of applicator may also have a significant influence on this molecular flow. Modeling ionic flow in tissues will yield insight into selecting the appropriate parameters or electroporation signature for a desired target application. In this paper, the motion of tissue injected ions was modeled for two common electroporation applicator configurations-the parallel plate, and the four needle electrodes. This electric field induced fluid flow model predicts that the parallel plate applicator ultimately directs the movement of an ionic therapeutic in a forward manner with side motion due only to obstruction, while the four-needle applicator directs anisotropic flow within the field ultimately forcing the therapeutic into a mound at the fringes of the induced electric field.     

Effect of the endothelial surface layer on transmission of fluid shear stress to endothelial cells

Responses of vascular endothelial cells to mechanical shear stresses resulting from blood flow are involved in regulation of blood flow, in structural adaptation of vessels, and in vascular disease. Interior surfaces of blood vessels are lined with a layer of bound or adsorbed macromolecules, known as the endothelial surface layer (ESL). In vivo investigations have shown that this layer has a width of order 1 microm, that it substantially impedes plasma flow, and that it excludes flowing red blood cells. Here, the effect of the ESL on transmission of shear stress to endothelial cells is examined using a theoretical model. The layer is assumed to consist of a matrix of molecular chains extending from the surface, held in tension by a slight increase in colloid osmotic pressure relative to that in free-flowing plasma. It is shown that, under physiological conditions, shear stress is transmitted to the endothelial surface almost entirely by the matrix, and fluid shear stresses on endothelial cell membranes are very small. Rapid fluctuations in shear stress are strongly attenuated by the layer. The ESL may therefore play an important role in sensing of shear stress by endothelial cells.     

Dynamic microtubule-dependent interactions position homotypic neurones in regular monolayered arrays during retinal development

In the vertebrate retina cell layers support serial processing, while monolayered arrays of homotypic neurones tile each layer to allow parallel processing. How neurones form layers and arrays is still largely unknown. We show that monolayered retinal arrays are dynamic structures based on dendritic interactions between the array cells. The analysis of three developing retinal arrays shows that these become regular as a net of dendritic processes links neighbouring array cells. Molecular or pharmacological perturbations of microtubules within dendrites lead to a stereotyped and reversible disruption of array organization: array cells lose their regular spacing and the arrangement in a monolayer. This leads to a micro-mechanical explanation of how monolayers of regularly spaced 'like-cells' are formed.     

Using a Spread Sheet Program to Model the Interaction of Low-Frequency Electric Fields with Inhomogeneous,Irregularly Shaped Objects

This paper describes the use of an ordinary business spread sheet program to calculate, using a two-stage finite difference method, the electric fields and current densities produced inside irregularly shaped models of the upper arm and forearm. The limb interiors are inhomogeneous, being represented as realistic cross-sections of bone and muscle.

A spread sheet forms a two-dimensional array. Each cell of the sheet can correspond to a physical element of space. Equations entered into the cells then represent the relationships among the potentials of the corresponding spatial elements.

The model is validated for a two-layer, lossy dielectric cylinder. Good agreement is obtained between the numerical and analytical solutions in this case except near the boundaries of the outer layer. The electric field within the limb depends on the shape and orientation of the limb relative to the applied field. The flow of current around the less conductive bones in the forearm can be observed.     

Biomechanics of microscopic appendages: functional shifts caused by changes in speed

Many diverse animals use arrays of hair-like structures to perform important jobs such as feeding, gas exchange, smelling, and swimming. Since these functions involve hair interactions with the surrounding water or air, analysis of the fluid dynamics of diverse hair-bearing appendages reveals how the morphology of an array of hairs affects it performance. Mathematical and physical models of flow between cylinders have shown that arrays of large, rapidly moving cylinders are leaky sieves, whereas little fluid moves through a row of small, slow rods. The purpose of the present study was to test this prediction for realistic appendage morphologies and to elucidate whether the design of a hairy leg can affect the range of speeds in which this transition in function occurs. We studied flow through hairy food-capturing appendages (second maxillae) of calanoid copepods, abundant planktonic crustaceans whose feeding on unicellular algae forms an important link in many marine food webs. Using dynamically scaled physical models, we found that hairy appendages undergo a transition between paddle- and sieve-like function at a critical range of sizes and speeds. The coarser the mesh of hairs on second maxillae, the smaller the size and speed at which this functional shift occurs. Thus, a simple increase in size (ontogenetic or evolutionary) or speed can generate a novel function (a paddle can become a filter), but the morphology of a hairy appendage determines the size and speed range at which leakiness to fluid flow can be affected by behavior or growth.     

Propagated disturbances of transverse potential gradient in intracellular fibrils as the source of motive forces for longitudinal transport in cells

Summary It is proposed as a working hypothesis that conformational changes propagated like waves along intracellular fibrils (tubules, microtubules, microfilaments) have an electric component,i.e., there are waves of disturbance of electric potential in the fibrils. The paper considers the unavoidable consequences of the wave. The latter is accompanied by local electric field in the boundary layer of cytoplasmic fluid. Both positively and negatively charged particles may be attracted to the fibril in certain regions of the field and, being attracted, the particle may be under the action of longitudinal component of electric force. When the force is strong enough to move the particle with wave velocity, the particle will travel smoothly along the fibril, otherwise the movement will be saltatory or of agitation type. Net electroosmotic flow in one direction in the boundary layer of fluid may be expected when the waves are propagated in series. Turbulent motion of the fluid caused by the waves may provide the basis for activated diffusion. Asymmetry of the wave may account for polar transport of this sort. The electric field transmitted along the fibril across a sieve pore in phloem may facilitate electroosmotically the flow through the pore. Quantitative requirements of the hypothesis that electric field generated by the waves may account for different aspects of longitudinal transport in cells are apparently met.     

Mathematical model of nitric oxide convection and diffusion in a renal medullary vas rectum

In this study, the generation, convection, diffusion, and consumption of nitric oxide (NO) in and around a single renal medullary descending or ascending vas rectum in rat were modeled using CFD. The vascular lumen (with a core RBC-rich layer and a parietal layer), the endothelium, the pericytes and the interstitium were represented as concentric cylinders. We accounted for the generation of NO by vascular endothelial cells, and that by the epithelial cells of medullary thick ascending limbs (mTALs) and inner medullary collecting ducts (IMCDs), the latter via interstitial boundary conditions. Luminal velocity profiles were obtained by modeling blood flow dynamics. Our results suggest that convection (i.e., blood flow per se) does not significantly affect NO concentrations along the cortico–medullary axis, because the latter are mostly determined by the rate of NO production and that of NO consumption by hemoglobin. However, the shear stress-mediated effects of blood flow on NO generation rates, and therefore NO concentrations, were predicted to be important. Finally, we found that unless epithelial NO generation rates (per unit tubular surface area) are at least 10 times lower than endothelium NO generation rates, NO production by mTALs and IMCDs affects vascular NO concentrations, with possible consequences for medullary blood flow distribution.     

Spatially and temporally controlled gene transfer by electroporation into adherent cells on plasmid DNA-loaded electrodes

Functional characterization of human genes is one of the most challenging tasks in current genomics. Owing to a large number of newly discovered genes, high-throughput methodologies are greatly needed to express in parallel each gene in living cells. To develop a method that allows efficient transfection of plasmids into adherent cells in spatial- and temporal-specific manners, we studied electric pulse-triggered gene transfer using a plasmid-loaded electrode. A plasmid was loaded on a gold electrode surface having an adsorbed layer of poly(ethyleneimine), and cells were then plated directly onto this modified surface. The plasmid was detached from the electrode by applying a short electric pulse and introduced into the cells cultured on the electrode, resulting in efficient gene expression, even in primary cultured cells. The location of transfected cells could be restricted within a small area on a micropatterned electrode, showing the versatility of the method for spatially controlled transfection. Plasmid transfection could also be performed in a temporally controlled manner without a marked loss of the efficiency when an electric pulse was applied within 3 days after cell plating. The method described here will provide an efficient means to transfer multiple genes, in parallel, into cultured mammalian cells for high-throughput reverse genetics research.     

Staining kinetics in single cells. Part I. Influence of convective diffusion on the staining rate

The staining kinetics of single cells have been investigated using a perfusion cuvette in combination with a computer controlled microscope spectrometer. The physicochemical hydrodynamics of staining are characterized. Using a steady-state laminar flow parallel to the cell surface a hydrodynamic and a diffusional boundary layer are observed which are determined by the flow rate. The thickness of the diffusional boundary layer revealed by experimental data is in agreement with theoretically calculated values. At certain well-defined hydrodynamic conditions convective diffusion has no further effect on the staining rate.     

Electric fields induced by low frequency magnetic fields in inhomogeneous biological structures that are surrounded by an electric insulator

Electric fields induced by low-frequency magnetic fields into inhomogeneous structures, which have electric conductivities and dielectric permittivities of typical biological substances, are evaluated. Closed-form approximate and numerical solutions are obtained for nonconcentric cylinders with different electric properties (such as bone embedded in muscle), which are surrounded by a good electrical insulator (such as air). It is shown that even a single inhomogeneity in an otherwise homogenous cylinder, which is exposed to a uniform, axially directed magnetic field, can lead to substantial deviations from the direction and distribution of the induced electric field that would exist in the homogenous cylinder. Thus the induced field is not everywhere circumferential, nor does it magnitude at all angular positions increase linearly with the radial distance. Radially and circumferentially directed field components depend on size, electrical properties, and eccentricity of the inhomogeneities. Equations as well as graphical presentations are given that describe the induced fields when the enclosed inhomogeneities consist either of eccentrically located single cylinders or pairs of coaxial cylinders with different electrical conductivities or dielectric permittivities.     

Electrofusion of fibroblasts on the porous membrane

Electric fusion of cells is usually performed in two steps: the first is the creation of tight intercellular contact, the second is an application of electric pulses which induce membrane fusion proper. In the present work a new technique of cell electrofusion on the porous film is described. It consists of preliminary cultivation of cell monolayer on the porous film (protein-coated cellophane). Then cells of the same or any other type are added from above to form a second cell layer upon the first one. The pulses of the electric field are applied normally to the plane of the double cell layer to induce cell fusion. After pulse application a picture of mass polynucleation was observed. At the same time we did not obtain fusion of L cells by means of dielectrophoretic electrofusion technique. This difference in efficiency could be explained by the formation of broad zones of membrane contact between the cells adherent to the film, while during intensive dielectrophoresis only the point contacts were revealed. The high-conducting medium for electric treatment providing an efficient fusion on the film and high cell viability was composed. Neither cytochalasin B nor colcemid affected cell fusion noticeably; however the sodium azide (added with 2-deoxyglucose) inhibited fusion completely. The short hypotonic shock after electric treatment enhanced the rate of polycaryon formation.     

Stability of elliptical cylinders in two-dimensional channel flow

The flow around rigid cylinders of elliptical cross section placed transverse to Poiseuille flow between parallel plates was simulated to investigate issues related to the tumbling of red blood cells and other particles of moderate aspect ratio in the similar flow in a Field Flow Fractionation (FFF) channel. The torque and transverse force on the cylinder were calculated with the cylinder freely translating, but prevented from rotating, in the flow. The aspect ratios (long axis to short axis) of the elliptical cylinders were 2, 3, 4, and 5. The cylinder was placed transversely at locations of y0/H = 0.1, 0.2, 0.3, and 0.4, where y0 is the distance from the bottom of the channel and H is the height of the channel, and the orientation of the cylinder was varied from 0 to 10 deg with respect to the axis of the channel for a channel Reynolds number of 20. The results showed that equilibrium orientations (indicated by a zero net torque on the cylinder) were possible for high-aspect-ratio cylinders at transverse locations y0/H < 0.2. Otherwise, the net torque on the cylinder was positive, indicating that the cylinder would rotate. For cylinders with a stable orientation, however, a transverse lift forced existed up to about y0/H = 0.25. Thus, a cylinder of neutral or low buoyancy might be lifted with a stable orientation from an initial position near the wall until it reached y0/H < 0.2, whereupon it would begin to tumble or oscillate. The dependence of lift and torque on cylinder orientation suggested that neutral or low-buoyancy cylinders may oscillate in both transverse location and angular velocity. Cylinders more dense than the carrier fluid could be in equilibrium both in terms of orientation and transverse location if their sedimentation force matched their lift force for a location y0/H < 0.2.     

Determination of the induced ELF electric field distribution in a two layer in vitro system simulating biological cells in nutrient solution

In-vitro studies of biological effects of electromagnetic fields are often conducted with cultured cells either in suspension or grown in a monolayer. In the former case, the exposed medium can be assumed to be homogeneous; however, eventually the cells settle to the bottom of the container forming a two layer system with different dielectric and conductive properties. In the present work the effect of this separation on the electric field distribution is calculated and experimentally measured at selected positions for a commonly used exposure configuration. The settled cell suspension is modeled by a well-defined two layer system placed in a rectangular container with the base of the container parallel to the direction of the magnetic field. Theoretical calculations based on numerical techniques are done for various two layer systems with different conductivities in each layer. The agreement between the theoretical calculations and the experimental measurements is within ± 1.5 mV/m, or 10% of the maximum induced field when the conductivity of the lower layer is ten times that of the upper layer. This result is well within experimental error. When the thickness of one of the layers is small compared to the thickness of the other layer, it is found that the electric field distribution is essentially that of the homogeneous case. The latter situation corresponds to a typical cell exposure condition. © 1993 Wiley-Liss, Inc.     

Relative motion inAmoeba proteus in respect to the adhesion sites

Summary The whole ectoplasmic layer of polytactic and heterotactic forms ofA. proteus behaves as self-contractile structure. Depending on the configuration of cell body and on the cell-to-substrate attachment conditions it continuously retracts from each distal cell projection toward its centre and/or from each free body end toward the actual adhesion sites. As in the monotactic forms, it leads to the withdrawal of the tail region behind the retraction center and may result in the fountain movement in front of it. In the long unattached pseudopodia of heterotactic forms the ectoplasm is retracted in the fountain form, with the velocity linearly increasing from the basis of pseudopodium up to its tip. In polytactic cells the fountain is often absent, if the advancing fronts immediately adhere to the substrate. When they develop in unattached condition, or are experimentally obliged to detach, the ectoplasmic cylinders of frontal pseudopodia are retracted backwards. On the substrates which do not offer firm points of support the cell periphery moves back as a whole,i.e., the principal ectoplasmic cylinder retracts together with the cylinders of lateral pseudopodia, and the direction and speed of movement in any spot is the resultant of forces produced by all other segments. The retraction of ectoplasmic gel layer is independent of the endoplasmic flow in such extent that a pseudopodium may be withdrawn as a whole in spite of the endoplasm streaming directed forwards in its interior. On the cell surface the particles attached by adhesion (glass rods) strictly follow the movements of the internal ectoplasmic structures, whereas the unattached particles flow forward in the direction of endoplasm streaming.Study supported by Research Project II. 1 of the Polish Academy of Science.     

Fibrillar Array in the Cell Wall of a Gliding Filamentous Cyanobacterium

The cell walls of a number of filamentous, gliding cyanobacteria of the genus Oscillatoria were examined by transmission electron microscopy of ultrathin sections, of freeze-etched replicas, and of whole cells crushed between glass slides and negatively stained. All three techniques revealed the presence of a highly ordered array of parallel fibrils, seen in transverse sections to be situated between the peptidoglycan and the outer membrane. Approximately 200 individual fibrils, each 25 to 30 nm in width, form a parallel, helical array that completely surrounds each cyanobacterial filament, running at an angle of 25 to 30° to its long axis. This highly regular arrangement of the fibrillar layer may imply some underlying symmetry responsible for its organization. A possible source of such symmetry would be the peptidoglycan, and some form of interaction between this layer and the fibrils might provide the necessary scaffolding for the fibrillar array. In crushed, negatively stained samples of fresh cells, individual fibrils were seen outside the filament, released from the cell wall. These released fibrils were of the same width as those observed in situ but were in short lengths, mostly of 100 to 200 nm, and were invariably bent, sometimes even into U shapes, implying great flexibility. Negative staining of released fibrils showed no evidence that they were hollow tubes but did give some indication of a substructure, implying that they were composed of many subunits. The function of this fibrillar array is unknown, although its position in the cell wall, as well as the correspondence between the angle of the fibrils with respect to the long axis of the filament and the rotation of the filament during gliding, may imply an involvement in gliding motility.