Journal of neuroscience methods
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Photoaffinity labeling is a positive function approach that has been used in an effort to identify the cocaine-binding site on the dopamine transporter (DAT). Radioactive and non-radioactive analogs of cocaine and other dopamine uptake blockers are used to irreversibly label the DAT ligand-binding site and the protein is subjected to chemical or enzymatic treatments that cleave at specific amino acid residues. Analysis of cleavage products from radioactively photolabeled DAT using epitope-specific immunoprecipitation, gel electrophoresis, and autoradiography has identified the site of origin in the primary sequence of labeled fragments as small as 4 kDa. ⋯ Fragment retention times are compared to calculated retention times of predicted digest peptides and to chemically or photochemically labeled synthetic peptides. The presence of authentic DAT sequence in HPLC fractions of digests from DAT labeled with non-radioactive ligands is further supported by MALDI and nanoelectrospray mass spectrometry. Using these methods we have identified two distinct regions of DAT that interact with multiple structurally related and diverse irreversible ligands, suggesting that these regions may be involved in the formation of ligand binding sites.
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J. Neurosci. Methods · Mar 2005
Sensitivity of surface EMG-based conduction velocity estimates to local tissue in-homogeneities--influence of the number of channels and inter-channel distance.
The aim of this simulation study was to investigate the influence of local tissue in-homogeneities on the estimates of muscle fiber conduction velocity (CV) from surface EMG signals. A recently developed analytical surface EMG model was used to generate simulated surface EMG signals from a planar layered volume conductor, comprised of the muscle tissue and fat layer, with spheres (1 mm radius) in the fat layer of conductivity different from the surrounding tissue. CV was estimated with a maximum likelihood multi-channel approach, varying the number of channels and the inter-channel distance used for the estimate. ⋯ The maximum percent variation of CV estimates due to the presence of in-homogeneities decreased with increasing number of channels and inter-channel distance: 19.6% (2 channels), 12.1% (3 channels), 6.4% (4 channels), for 5 mm inter-channel distance, and 12.0% (2 channels), 5.2% (3 channels), 2.4% (4 channels), for 10 mm inter-channel distance (for double differential detection). The results were in agreement and explained previous experimental findings. It was concluded that multi-channel methods for CV estimation significantly reduce the sensitivity of CV estimates to tissue in-homogeneities.
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J. Neurosci. Methods · Feb 2005
Comparative StudyA microcapsule technique for long-term conduction block of the sciatic nerve by tetrodotoxin.
Tetrodotoxin (TTX) is a selective blocker of voltage-gated Na+ channels that is used to block action potentials in vitro and in vivo. Maintaining a sufficiently high local concentration of TTX in vivo to block conduction in a peripheral nerve is technically demanding and carries a risk of systemic toxicity. ⋯ The conduction block was reversible and did not cause any obvious nerve injury. Low cost and simple surgical implementation make this new system an interesting alternative to existing long-term drug delivery methods.
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J. Neurosci. Methods · Feb 2005
Comparative StudyAnalysis of behavioral asymmetries in the elevated plus-maze and in the T-maze.
When studying functional asymmetries in normal laboratory rats, several behavioral tests have been applied and proven their utility, including turning in rotometers or open-fields, handedness in paw usage, T-maze alternation, and others. Here, we analyzed male Wistar rats in two tests, namely the elevated plus-maze and the T-maze. In these tests, behavioral asymmetries are rather likely to occur, since the animals have to show several types of turns towards the left or right when ambulating through these environments. ⋯ In addition, it provides new evidence with respect to the elevated plus-maze, indicating that this standard tool of anxiety research may also be useful in research on behavioral asymmetries and their underlying brain mechanisms. Behavioral biases in favor of the right, as shown here, have often been reported before, especially with Wistar rats. Such biases should be taken into account, since they can serve as an approach to study brain/behavior relationships, and since they may affect the outcome of physiological manipulations or behavioral trainings.
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J. Neurosci. Methods · Jan 2005
Chronically implanted electrodes for repeated stimulation and recording of spinal cord potentials.
We have recorded and characterized the spinal cord evoked potentials (SCEPs) from the epidural space in the halothane-anesthetized rats. A group of 11 adult Wistar male rats was chronically implanted with two pairs of epidural electrodes. SCEPs were repeatedly elicited by applying electrical stimuli via bipolar U-shaped electrodes to the dorsal aspect of the spinal cord at C3-4 or Th11-12 levels, respectively. ⋯ We concluded that this technique enables repeated quantitative analysis of the conductivity of the spinal cord white matter in the rat. Our results indicate that SCEPs could be used in long-term experiments for monitoring progressive changes (degeneration/regeneration) in long projection tracts, primarily those occupying the dorsolateral quadrants of the spinal cord. These include projections that are of interest in spinal cord injury studies, i.e. ascending primary afferents, and important descending pathways including corticospinal, rubrospinal, reticulospinal, raphespinal and vestibulospinal tracts.