“Clock-scan” protocol for image analysis

2006 ◽  
Vol 291 (5) ◽  
pp. C869-C879 ◽  
Author(s):  
Maxim Dobretsov ◽  
Dmitry Romanovsky
Keyword(s):  
Image Analysis ◽  
Spatial Resolution ◽  
Neuronal Cell ◽  
Region Of Interest ◽  
Dorsal Root Ganglion Neurons ◽  
Protein Markers ◽  
Pixel Intensity ◽  
Good Spatial Resolution ◽  
Ganglion Neurons ◽  
Scan Protocol

Comparative analysis of extra- and intracellular distributions of protein markers in immunohistochemical and immunofluorescent studies relies on techniques of image analysis. Line or region of interest pixel intensity scans are methods routinely used. However, although having good spatial resolution, linear pixel intensity scans fail to produce integral image of the cellular distribution of the label. On the other hand, the regions of interest scans have good integrative capacity but low spatial resolution. In this work, we describe a “clock-scan” protocol that, when applied to convex objects (such as neuronal cell bodies and the majority of cells in culture), combines advantages and circumnavigates limitations of the above-mentioned techniques. The protocol 1) collects multiple radial pixel intensity profiles scanned from the cell center to the periphery, 2) scales these profiles according to the cell radius measured in the direction of the scan, and finally, 3) averages these individual profiles into one integral radial pixel intensity profile. Because of scaling, the mean pixel intensity profiles produced by the clock-scan protocol depend on neither the cell size nor, within reasonable limits, the cell shape. This allows direct comparison or, if required, averaging or subtraction of profiles of different cells. We have successfully tested the clock-scan protocol in experiments with immunostained dorsal root ganglion neurons. In addition, the protocol seems to be equally applicable for studies in a variety of other preparations.

scholarly journals Permeation and block of TRPV1 channels by the cationic lidocaine derivative QX-314

2013 ◽  
Vol 109 (7) ◽  
pp. 1704-1712 ◽  
Author(s):  
Michelino Puopolo ◽  
Alexander M. Binshtok ◽  
Gui-Lan Yao ◽  
Seog Bae Oh ◽  
Clifford J. Woolf ◽  
...  
Keyword(s):  
Transient Receptor Potential ◽  
Neuronal Cell ◽  
Reversal Potential ◽  
Dorsal Root Ganglion Neurons ◽  
C6 Glioma Cells ◽  
Transient Receptor Potential Vanilloid ◽  
Trpv1 Channels ◽  
Inward Currents ◽  
Pore Dilation ◽  
Ganglion Neurons

QX-314 ( N-ethyl-lidocaine) is a cationic lidocaine derivative that blocks voltage-dependent sodium channels when applied internally to axons or neuronal cell bodies. Coapplication of external QX-314 with the transient receptor potential vanilloid 1 protein (TRPV1) agonist capsaicin produces long-lasting sodium channel inhibition in TRPV1-expressing neurons, suggestive of QX-314 entry into the neurons. We asked whether QX-314 entry occurs directly through TRPV1 channels or through a different pathway (e.g., pannexin channels) activated downstream of TRPV1 and whether QX-314 entry requires the phenomenon of “pore dilation” previously reported for TRPV1. With external solutions containing 10 or 20 mM QX-314 as the only cation, inward currents were activated by stimulation of both heterologously expressed and native TRPV1 channels in rat dorsal root ganglion neurons. QX-314-mediated inward current did not require pore dilation, as it activated within several seconds and in parallel with Cs-mediated outward current, with a reversal potential consistent with PQX-314/ PCs = 0.12. QX-314-mediated current was no different when TRPV1 channels were expressed in C6 glioma cells, which lack expression of pannexin channels. Rapid addition of QX-314 to physiological external solutions produced instant partial inhibition of inward currents carried by sodium ions, suggesting that QX-314 is a permeant blocker. Maintained coapplication of QX-314 with capsaicin produced slowly developing reduction of outward currents carried by internal Cs, consistent with intracellular accumulation of QX-314 to concentrations of 50–100 μM. We conclude that QX-314 is directly permeant in the “standard” pore formed by TRPV1 channels and does not require either pore dilation or activation of additional downstream channels for entry.

scholarly journals Antibodies to the L1 adhesion molecule inhibit Schwann cell ensheathment of neurons in vitro.

1989 ◽  
Vol 109 (6) ◽  
pp. 3095-3103 ◽  
Author(s):  
B Seilheimer ◽  
E Persohn ◽  
M Schachner
Keyword(s):  
Cell Differentiation ◽  
Schwann Cell ◽  
Schwann Cells ◽  
Adhesion Molecule ◽  
Neuronal Cell ◽  
Dorsal Root Ganglion Neurons ◽  
Neural Adhesion Molecule ◽  
Ganglion Neurons ◽  
Schwann Cell Differentiation

To investigate whether neural adhesion molecules are involved in neuron-induced Schwann cell differentiation, cocultures of pure dorsal root ganglion neurons, and Schwann cells were maintained in the presence of antibodies to evaluate possible perturbing effects. Several parameters characteristic of differentiating Schwann cells were studied, such as transition of spindle-shaped to flattened, i.e., more epithelioid morphology, association with neuronal cell bodies, ensheathment of neurites, production of basal lamina and collagen fibrils, and expression of the myelin associated glycoprotein (MAG). A complete ablation of Schwann cell differentiation in all features studied was seen with antibodies to the neural adhesion molecule L1. Antibodies to N-CAM did not reduce the association of Schwann cells with neurites but abolished the interdigitation of Schwann cell processes into neurite bundles, while leaving the other parameters studied unaffected. Fab fragments of antibodies to J1, MAG, and mouse liver membranes did not interfere with the manifestation of any of these parameters. None of the antibodies changed incorporation of [3H]thymidine into Schwann cells.

scholarly journals Neuronal cell lines as model dorsal root ganglion neurons

2016 ◽  
Vol 12 ◽  
pp. 174480691664611 ◽  
Author(s):  
Kathleen Yin ◽  
Gregory J Baillie ◽  
Irina Vetter
Keyword(s):  
Dorsal Root Ganglion ◽  
Cell Lines ◽  
Dorsal Root ◽  
Neuronal Cell ◽  
Dorsal Root Ganglion Neurons ◽  
Neuronal Cell Lines ◽  
Ganglion Neurons

scholarly journals Improved Spatial Resolution of Electroencephalogram Using Tripolar Concentric Ring Electrode Sensors

2020 ◽  
Vol 2020 ◽  
pp. 1-9 ◽  
Author(s):  
Xiang Liu ◽  
Oleksandr Makeyev ◽  
Walter Besio
Keyword(s):  
Spatial Resolution ◽  
Signal To Noise Ratio ◽  
Human Subjects ◽  
Region Of Interest ◽  
Ring Electrode ◽  
Brain Diseases ◽  
Concentric Ring ◽  
Healthy Human ◽  
Good Spatial Resolution ◽  
Visual Evoked

The electroencephalogram (EEG) is broadly used for research of brain activities and diagnosis of brain diseases and disorders. Although EEG provides good temporal resolution of millisecond or less, it does not provide good spatial resolution. There are two main reasons for the poor spatial resolution: the blurring effects of the head volume conductor and poor signal-to-noise ratio. We have developed a tripolar concentric ring electrode (TCRE) Laplacian sensor and now report on computer simulations comparing spatial resolution between conventional EEG disc electrode sensors and TCRE Laplacian sensors. We also performed visual evoked stimulus experiments and acquired visual evoked potentials (VEPs) from healthy human subjects. From the simulations, we found that TCRE Laplacian sensors can provide approximately a tenfold improvement in spatial resolution and pass signals from specific volumes. Placing TCRE sensors near the brain region of interest will allow passage of the wanted signals and rejection of distant interference signals. We were also able to detect VEPs on the scalp surface and show that TCREs separated VEP sources better than conventional disc electrodes.

scholarly journals Increased Na+ and K+ currents in small mouse dorsal root ganglion neurons after ganglion compression

2011 ◽  
Vol 106 (1) ◽  
pp. 211-218 ◽  
Author(s):  
Ni Fan ◽  
Parul Sikand ◽  
David F. Donnelly ◽  
Chao Ma ◽  
Robert H. LaMotte
Keyword(s):  
Dorsal Root Ganglion ◽  
Dorsal Root ◽  
Neuronal Response ◽  
Small Diameter ◽  
Neuronal Cell ◽  
Radicular Pain ◽  
Dorsal Root Ganglion Neurons ◽  
Delayed Rectifier ◽  
Ganglion Neurons

We investigated the effects of chronic compression (CCD) of the L3 and L4 dorsal root ganglion (DRG) on pain behavior in the mouse and on the electrophysiological properties of the small-diameter neuronal cell bodies in the intact ganglion. CCD is a model of human radicular pain produced by intraforaminal stenosis and other disorders affecting the DRG, spinal nerve, or root. On days 1, 3, 5, and 7 after the onset of compression, there was a significant decrease from preoperative values in the threshold mechanical force required to elicit a withdrawal of the foot ipsilateral to the CCD (tactile allodynia). Whole cell patch-clamp recordings were obtained, in vitro, from small-sized somata and, for the first time, in the intact DRG. Under current clamp, CCD neurons exhibited a significantly lower rheobase compared with controls. A few CCD but no control neurons exhibited spontaneous action potentials. CCD neurons showed an increase in the density of TTX-resistant and TTX-sensitive Na+ current. CCD neurons also exhibited an enhanced density of voltage-dependent K+ current, due to an increase in delayed rectifier K+ current, without a change in the transient or “A” current. We conclude that CCD in the mouse produces a model of radicular pain, as we have previously demonstrated in the rat. While the role of enhanced K+ current remains to be elucidated, we speculate that it represents a compensatory neuronal response to reduce ectopic or aberrant levels of neuronal activity produced by the injury.

scholarly journals Restriction of 480/270-kD Ankyrin G to Axon Proximal Segments Requires Multiple Ankyrin G-specific Domains

1998 ◽  
Vol 142 (6) ◽  
pp. 1571-1581 ◽  
Author(s):  
Xu Zhang ◽  
Vann Bennett
Keyword(s):  
Plasma Membrane ◽  
Dorsal Root Ganglion ◽  
Dorsal Root ◽  
Membrane Binding ◽  
Neuronal Cell ◽  
Lateral Diffusion ◽  
Dorsal Root Ganglion Neurons ◽  
Rich Domain ◽  
Ankyrin G ◽  
Ganglion Neurons

AnkyrinG (−/−) neurons fail to concentrate voltage-sensitive sodium channels and neurofascin at their axon proximal segments, suggesting that ankyrinG is a key component of a structural pathway involved in assembly of specialized membrane domains at axon proximal segments and possibly nodes of Ranvier (Zhou, D., S. Lambert, D.L. Malen, S. Carpenter, L. Boland, and V. Bennett, manuscript submitted for publication). This paper addresses the mechanism for restriction of 270-kD ankyrinG to axon proximal segments by evaluation of localization of GFP-tagged ankyrinG constructs transfected into cultured dorsal root ganglion neurons, as well as measurements of fluorescence recovery after photobleaching of neurofascin– GFP-tagged ankyrinG complexes in nonneuronal cells. A conclusion is that multiple ankyrinG-specific domains, in addition to the conserved membrane-binding domain, contribute to restriction of ankyrinG to the axonal plasma membrane in dorsal root ganglion neurons. The ankyrinG-specific spectrin-binding and tail domains are capable of binding directly to sites on the plasma membrane of neuronal cell bodies and axon proximal segments, and presumably have yet to be identified docking sites. The serine-rich domain, which is present only in 480- and 270-kD ankyrinG polypeptides, contributes to restriction of ankyrinG to axon proximal segments as well as limiting lateral diffusion of ankyrinG–neurofascin complexes. The membrane-binding, spectrin-binding, and tail domains of ankyrinG also contribute to limiting the lateral mobility of ankyrinG–neurofascin complexes. AnkyrinG thus functions as an integrated mechanism involving cooperation among multiple domains heretofore regarded as modular units. This complex behavior explains ability of ankyrinB and ankyrinG to sort to distinct sites in neurons and the fact that these ankyrins do not compensate for each other in ankyrin gene knockouts in mice.

scholarly journals A few axonal proteins distinguish ventral spinal cord neurons from dorsal root ganglion neurons.

1984 ◽  
Vol 98 (1) ◽  
pp. 364-368 ◽  
Author(s):  
P Sonderegger ◽  
M C Fishman ◽  
M Bokoum ◽  
H C Bauer ◽  
E A Neale ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Dorsal Root Ganglia ◽  
Dorsal Root ◽  
Synapse Formation ◽  
Neuronal Cell ◽  
Dorsal Root Ganglion Neurons ◽  
Neuronal Diversity ◽  
Spinal Cord Neurons ◽  
Ganglion Neurons ◽  
Ventral Spinal Cord

A series of proteins putatively involved in the generation of axonal diversity was identified. Neurons from ventral spinal cord and dorsal root ganglia were grown in a compartmented cell-culture system which offers separate access to cell somas and axons. The proteins synthesized in the neuronal cell somas and subsequently transported into the axons were selectively analyzed by 2-dimensional gel electrophoresis. The patterns of axonal proteins were substantially less complex than those derived from the proteins of neuronal cell bodies. The structural and functional similarity of axons from different neurons was reflected in a high degree of similarity of the gel pattern of the axonal proteins from sensory ganglia and spinal cord neurons. Each axonal type, however, had several proteins that were markedly less abundant or absent in the other. These neuron-population enriched proteins may be involved in the implementation of neuronal diversity. One of the proteins enriched in dorsal root ganglia axons had previously been found to be expressed with decreased abundance when dorsal root ganglia axons were co-cultured with ventral spinal cord cells under conditions in which synapse formation occurs (P. Sonderegger, M. C. Fishman, M. Bokoum, H. C. Bauer, and P.G. Nelson, 1983, Science [Wash. DC], 221:1294-1297). This protein may be a candidate for a role in growth cone functions, specific for neuronal subsets, such as pathfinding and selective axon fasciculation or the initiation of specific synapses. The methodology presented is thus capable of demonstrating patterns of protein synthesis that distinguish different neuronal subsets. The accessibility of these proteins for structural and functional studies may contribute to the elucidation of neuron-specific functions at the molecular level.

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