Use of a Custom Macro in Analysis of Rat Sciatic Nerve Sections

Introduction: Technology is a powerful analytic tool. This abstract outlines the use of a custom macro to analyze rat sciatic nerve histology sections as part of a larger study to evaluate functional and histologic outcomes after segmental nerve injury. The problem facing analysis of nerve sections is that the microscopic features of a nerve, such as the individual axon, cannot be viewed on the same scale as the entire nerve bundle. The goal of this analysis was to extrapolate the average total values of axon count, axon density, fiber diameter, and myelin thickness on a nerve section image taken at 400x magnification to the total area captured at 40x magnification. Methods: Images of nerve sections stained with toluidine blue were captured using a microscope with a mounted camera and image acquisition software (Nikon Eclipse E800; Nikon DS-Ri1; NIS Elements BR 3.10 SP3 Hotfix5). Multiple images at different light intensities were used to determine a contrast and saturation that would grant the best accuracy. From these images, a custom macro using Image-Pro Premier (Version 9.1.5262.28) was developed to compute the desired measurements (Media Cybernetics, Rockville MD). Results: At 400x magnification, circular shape recognition was used to determine each axon myelin sheath pair in the selected area. Area analyzed was within the “circle of good definition”. After an axon myelin pair was recognized, multiple diameter measurements were taken, and these values were the key to calculations of axon area, fiber diameter, and myelin thickness. Two images taken at 400x magnification for each sample were used to calculate an average for each measurement. An image of the entire nerve slice at 40x was taken, and this calculated area was used extrapolate measurements to the entire nerve section. Discussion:The utility of this software and macro is exciting due to its reproducibility, accuracy, and efficiency. Not only does it take human processing and potential error out of the equation, but it solves the scale discrepancy when trying to analyze microscopic parameters on a larger scale. This procedure is versatile and can be implemented in future histologic analysis of nerve sections

A Run-Length Encoding Approach for Path Analysis of C. elegans

The nematode Caenorhabditis elegans explores the environment using a combination of different movement patterns, which include straight movement, reversal, and turns. We propose to quantify C. elegans movement behavior using a computer vision approach based on run-length encoding of step-length data. In this approach, the path of C. elegans is encoded as a string of characters, where each character represents a path segment of a specific type of movement. With these encoded string data, we perform k-means cluster analysis to distinguish movement behaviors resulting from different genotypes and food availability. We found that shallow and sharp turns are the most critical factors in distinguishing the differences among the movement behaviors. To validate our approach, we examined the movement behavior of tph-1 mutants that lack an enzyme responsible for serotonin biosynthesis. A k-means cluster analysis with the path string-encoded data showed that tph-1 movement behavior on food is similar to that of wild-type animals off food. We suggest that this run-length encoding approach is applicable to trajectory data in animal or human mobility data

The Dystrophin Complex Controls BK Channel Localization and Muscle Activity in Caenorhabditis elegans

Genetic defects in the dystrophin-associated protein complex (DAPC) are responsible for a variety of pathological conditions including muscular dystrophy, cardiomyopathy, and vasospasm. Conserved DAPC components from humans to Caenorhabditis elegans suggest a similar molecular function. C. elegans DAPC mutants exhibit a unique locomotory deficit resulting from prolonged muscle excitation and contraction. Here we show that the C. elegans DAPC is essential for proper localization of SLO-1, the large conductance, voltage-, and calcium-dependent potassium (BK) channel, which conducts a major outward rectifying current in muscle under the normal physiological condition. Through analysis of mutants with the same phenotype as the DAPC mutants, we identified the novel islo-1 gene that encodes a protein with two predicted transmembrane domains. We demonstrate that ISLO-1 acts as a novel adapter molecule that links the DAPC to SLO-1 in muscle. We show that a defect in either the DAPC or ISLO-1 disrupts normal SLO-1 localization in muscle. Consistent with observations that SLO-1 requires a high calcium concentration for full activation, we find that SLO-1 is localized near L-type calcium channels in muscle, thereby providing a mechanism coupling calcium influx with the outward rectifying current. Our results indicate that the DAPC modulates muscle excitability by localizing the SLO-1 channel to calcium-rich regions of C. elegans muscle

Presynaptic BK channel localization is dependent on the hierarchical organization of alpha-catulin and dystrobrevin and fine-tuned by CaV2 calcium channels

BACKGROUND: Large conductance, calcium-activated BK channels regulate many important physiological processes, including smooth muscle excitation, hormone release and synaptic transmission. The biological roles of these channels hinge on their unique ability to respond synergistically to both voltage and cytosolic calcium elevations. Because calcium influx is meticulously regulated both spatially and temporally, the localization of BK channels near calcium channels is critical for their proper function. However, the mechanism underlying BK channel localization near calcium channels is not fully understood. RESULTS: We show here that in C. elegans the localization of SLO-1/BK channels to presynaptic terminals, where UNC-2/CaV2 calcium channels regulate neurotransmitter release, is controlled by the hierarchical organization of CTN-1/alpha-catulin and DYB-1/dystrobrevin, two proteins that interact with cortical cytoskeletal proteins. CTN-1 organizes a macromolecular SLO-1 channel complex at presynaptic terminals by direct physical interaction. DYB-1 contributes to the maintenance or stabilization of the complex at presynaptic terminals by interacting with CTN-1. We also show that SLO-1 channels are functionally coupled with UNC-2 calcium channels, and that normal localization of SLO-1 to presynaptic terminals requires UNC-2. In the absence of UNC-2, SLO-1 clusters lose the localization specificity, thus accumulating inside and outside of presynaptic terminals. Moreover, CTN-1 is also similarly localized in unc-2 mutants, consistent with the direct interaction between CTN-1 and SLO-1. However, localization of UNC-2 at the presynaptic terminals is not dependent on either CTN-1 or SLO-1. Taken together, our data strongly suggest that the absence of UNC-2 indirectly influences SLO-1 localization via the reorganization of cytoskeletal proteins. CONCLUSION: CTN-1 and DYB-1, which interact with cortical cytoskeletal proteins, are required for the presynaptic punctate localization of SLO-1 in a hierarchical manner. In addition, UNC-2 calcium channels indirectly control the fidelity of SLO-1 puncta localization at presynaptic terminals. We suggest that the absence of UNC-2 leads to the reorganization of the cytoskeletal structure that includes CTN-1, which in turn influences SLO-1 puncta localization

An Alpha-Catulin Homologue Controls Neuromuscular Function through Localization of the Dystrophin Complex and BK Channels in Caenorhabditis elegans

The large conductance, voltage- and calcium-dependent potassium (BK) channel serves as a major negative feedback regulator of calcium-mediated physiological processes and has been implicated in muscle dysfunction and neurological disorders. In addition to membrane depolarization, activation of the BK channel requires a rise in cytosolic calcium. Localization of the BK channel near calcium channels is therefore critical for its function. In a genetic screen designed to isolate novel regulators of the Caenorhabditis elegans BK channel, SLO-1, we identified ctn-1, which encodes an α-catulin homologue with homology to the cytoskeletal proteins α-catenin and vinculin. ctn-1 mutants resemble slo-1 loss-of-function mutants, as well as mutants with a compromised dystrophin complex. We determined that CTN-1 uses two distinct mechanisms to localize SLO-1 in muscles and neurons. In muscles, CTN-1 utilizes the dystrophin complex to localize SLO-1 channels near L-type calcium channels. In neurons, CTN-1 is involved in localizing SLO-1 to a specific domain independent of the dystrophin complex. Our results demonstrate that CTN-1 ensures the localization of SLO-1 within calcium nanodomains, thereby playing a crucial role in muscles and neurons

Testing the Effect of Family Characteristics on Locational Choice for Residence

This paper examines the importance of family characteristics in choosing location for residence, especially school districts. In testing this fact, I employ two data set the PSID and the Census of Government. Finance Statistics. The main finding is that families with a lower total child-care time available choose school districts with a higher expenditure to compensate for their time. If we recognize that parental child-care time is important in occurring intergenerational wealth transfer through the cognitive development of the child, this kind of parental compensatory behavior can play an important role in neutralizing variations of the future income of children

New Electronic Acupuncture System Using Intelligence

Part 13: UMASInternational audienceIn recent years, scientific studies of traditional oriental medicine are accelrating. Furthermore, researches of medical examinations and treatments through collaboration of oriental medicine and western medicine is in progress. This paper will seek for spots on the body suitable for acupuncture using special features that skin impedance values are different. The computer simulation results have shown that Electro-Acupuncture administered by using the medical diagnosis system developed in this study is more effective than the conventional method

Measurement of Intraocular Pressure during Automatic Needle Insertion using Corneal Applanation

Applanation aided automatic needle insertion is a novel insertion method for deep anterior lamellar keratoplasty. The method uses corneal applanation to control insertion depth of needle, but it could increase intraocular pressure. Because high intraocular pressure (IOP) can cause glaucoma, measurement of IOP is required to ensure the safety of the proposed insertion method. This paper describes a design of applanation pad composed of two layered structure and IOP measuring system. For 7 porcine eyeballs, the maximum increase in IOP was 46 ± 10.8 mmHg in average and operation time was 97 seconds in average. Because the amount of IOP increase was lower than IOP increase of other eye surgery, applanation aided needle insertion can be utilized for the precise needle insertion in terms of IOP.2

BK channel density is regulated by endoplasmic reticulum associated degradation and influenced by the SKN-1A/NRF1 transcription factor.

Ion channels are present at specific levels within subcellular compartments of excitable cells. The regulation of ion channel trafficking and targeting is an effective way to control cell excitability. The BK channel is a calcium-activated potassium channel that serves as a negative feedback mechanism at presynaptic axon terminals and sites of muscle excitation. The C. elegans BK channel ortholog, SLO-1, requires an endoplasmic reticulum (ER) membrane protein for efficient anterograde transport to these locations. Here, we found that, in the absence of this ER membrane protein, SLO-1 channels that are seemingly normally folded and expressed at physiological levels undergo SEL-11/HRD1-mediated ER-associated degradation (ERAD). This SLO-1 degradation is also indirectly regulated by a SKN-1A/NRF1-mediated transcriptional mechanism that controls proteasome levels. Therefore, our data indicate that SLO-1 channel density is regulated by the competitive balance between the efficiency of ER trafficking machinery and the capacity of ERAD