Journal of Neuroscience Methods

Modeling the vagus nerve system with the Unified Modeling Language.

Publication Date: 2010 Aug 26 PMID: 20801157
Authors: van Beijnum, B. J. - Widya, I. A. - Marani, E.
Journal: J Neurosci Methods

Traditionally, the means of describing anatomical and physiological structures of the autonomic nervous system is natural language, drawings and images as represented in the scientific literature. In behavioral studies of this system, mathematical and electrical models and computer simulation tools are in use. In this article, we propose the use of the Unified Modeling Language to describe and specify the anatomical and physiological structures and indicate how these can be enriched to capture the behavioral view as well. Using the metamodel facilities of the language, we propose a domain specific language that captures the domain concepts, their relationships and constraints. Application of the language is demonstrated by modeling the vagus nerve in part.

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Performance of juvenile mice in a reach-to-grasp task.

Publication Date: 2010 Aug 25 PMID: 20800620
Authors: Marques, J. M. - Olsson, I. A.
Journal: J Neurosci Methods

Reach-to-grasp tasks have been used to study rodent models of motor system damage, stroke, and neurodegenerative disorders such as Parkinson's. These tasks are especially useful as they allow evaluation of bilateral or unilateral damage in different regions of the brain. Performing reach-to-grasp tests in juvenile mice may be important to understand motor disorders of early onset. This study evaluated the performance of juvenile and adolescent mice on a reach-to-grasp task. Male and female C57BL/6J mice (n=93) were tested in a forelimb reaching task at postnatal weeks 4, 5, 7, 9 and 12. At all ages mice could learn the task and improved performance with training. Results show that reach-to-grasp tasks can be used to study skill learning in juvenile and adolescent mice. Results are discussed in terms of adapting methodologies (test protocols and arenas) when performing behaviour tests in young mice.

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Facial affect processing in social anxiety: Tasks and stimuli.

Publication Date: 2010 Aug 26 PMID: 20800619
Authors: Machado-de-Sousa, J. P. - Arrais, K. C. - Alves, N. T. - Chagas, M. H. - de Meneses-Gaya, C. - Crippa, J. A. - Hallak, J. E.
Journal: J Neurosci Methods

Social anxiety (SA) has as its main feature the fear of social situations, being characterized as social phobia or social anxiety disorder when functional impairment emerges as a result of that fear. Although the recognition of the condition has increased in recent years, it is believed that many patients and physicians still take the symptoms of the disorder for personality traits with no need for treatment. There is evidence that people with SA display abnormal patterns or facial emotion processing that could account for the onset and maintenance of the disorder. The objective of this review is to describe, compare, and discuss the methods used to study facial emotion processing in SA with an emphasis on the tasks and stimuli employed. Articles were searched for on online scientific databases. Forty research articles were selected according to the inclusion and exclusion criteria established. The articles were read and information from them was gathered on a comparative table for analysis. Evidence available to date suggests that SA individuals have abnormal patterns of facial information processing characterized by a bias for negative emotions. The results of the articles analyzed have a high degree of concordance, in spite of the variety of tasks and stimuli employed. The similarity between results from non-clinical samples with SA and patients affected by social phobia speaks in favor of the current view that SA occurs as a continuum of severity, rather than a clearly circumscribed nosological entity.

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Remote switching of temperature; gaseous; and aqueous phase in a low-volume interface chamber for brain slices.

Publication Date: 2010 Aug 25 PMID: 20800618
Authors: Wolfer, J. - Speckmann, E. J. - Wassmann, H. - Gorji, A. - Greiner, C.
Journal: J Neurosci Methods

A new remote-controlled interface-type chamber was designed in order to conduct experiments in brain slices involving gas, fluid, and temperature changes with as little tissue manipulation as possible. The chamber allows for extremely quick changes between different fluid and/or gaseous phases and for active cooling as well as heating by using a set of electromechanical valves and Peltier elements. The design drawings are complemented by exemplary tests of temperature and gas changes, and electrophysiological recordings of slices manipulated with gas and fluid alterations were used to test the efficacy and accuracy of the design. Changing between normoxia and anoxia needs less than 30seconds, while the readjustment of the chamber to a new, preset temperature is accomplished in about one minute. Supplementary data provide a proposal for the electronic circuit diagram. This chamber design should simplify data acquisition in interface environments.

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Transcriptome profiling in neurodegenerative disease.

Publication Date: 2010 Aug 25 PMID: 20800617
Authors: Courtney, E. - Kornfeld, S. - Janitz, K. - Janitz, M.
Journal: J Neurosci Methods

Changes in gene expression and splicing patterns (that occur prior to the onset and during the progression of complex diseases) have become a major focus of neurodegenerative disease research. These signature patterns of gene expression provide clues about the mechanisms involved in the molecular pathogenesis of neurodegenerative disease and may facilitate the discovery of novel therapeutic drugs. With the development of array technologies and the very recent RNA-seq technique, our understanding of the pathogenesis of neurodegenerative disease is expanding exponentially. Here, we review the technologies involved in gene expression and splicing analysis and the related literature on three common neurodegenerative diseases: Alzheimer's disease, Parkinson's disease and Huntington's disease.

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Transfection of chicken cerebellar granule neurons used to study glucocorticoid receptor regulation by nuclear receptor 4A (NR4A).

Publication Date: 2010 Aug 19 PMID: 20727911
Authors: Strom, B. O. - Aden, P. - Mathisen, G. H. - Lomo, J. - Davanger, S. - Paulsen, R. E.
Journal: J Neurosci Methods

Transfection is a useful tool for studying molecular signalling pathways. However, neurons have proven hard to transfect. In the present paper we have optimized a new electroporation procedure using the Cellaxess((R)) system for transient transfection of adherent primary neurons from chicken (Gallus gallus) and compared it to a liposome based procedure using Metafectene((R)) Pro. In order to evaluate the two methods, glucocorticoid receptor (GR) function was chosen as a test. GRs are expressed in high amounts in the cerebellum. GR is regulated by another nuclear receptor (NGFI-B, the first member found in the NR4A family). We first showed that forskolin and phorbol ester activated an NR4A-dependent reporter gene indicating that members of the NR4A nuclear receptor family are present endogenously and upregulated by external stimuli. Then, transfected NGFI-B was shown to antagonize the dexamethasone-activated transcriptional activation by endogenous GR, leading to the conclusion that NR4A-family members are important modulators of GR mediated regulatory processes in the cerebellum, as in other cell types. Both transfection methods proved useful. While the electroporation technique yielded small rings with many transfected cells optimal for microscopy studies, the liposome based method resulted in transfected cells evenly distributed in the dish rendering this method well suited for biochemical studies.

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Automatic mosaicking and volume assembly for high-throughput serial-section transmission electron microscopy.

Publication Date: 2010 Aug 13 PMID: 20713087
Authors: Tasdizen, T. - Koshevoy, P. - Grimm, B. C. - Anderson, J. R. - Jones, B. W. - Watt, C. B. - Whitaker, R. T. - Marc, R. E.
Journal: J Neurosci Methods

We describe a computationally efficient and robust fully-automatic method for large-scale electron microscopy image registration. The proposed method is able to construct large image mosaics from thousands of smaller, overlapping tiles with unknown or uncertain positions, and to align sections from a serial section capture into a common coordinate system. The method also accounts for nonlinear deformations both in constructing sections and in aligning sections to each other. The underlying algorithms are based on the Fourier shift property which allows for a computationally efficient and robust method. We demonstrate results on two electron microscopy datasets. We also quantify the accuracy of the algorithm through a simulated image capture experiment. The publicly available software tools include the algorithms and a Graphical User Interface for easy access to the algorithms.

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Autogenic EMG-controlled functional electrical stimulation for ankle dorsiflexion control.

Publication Date: 2010 Aug 14 PMID: 20713086
Authors: Yeom, H. - Chang, Y. H.
Journal: J Neurosci Methods

Our objectives were to develop and test a new system for the potential for stable, real-time cancellation of residual stimulation artefacts (RSA) using surface electrode autogenic electromyography-controlled functional electrical stimulator (aEMGcFES). This type of closed-loop FES could be used to provide more natural, continuous control of lower extremity paretic muscles. We built upon work that has been done in the field of FES with one major technological innovation, an adaptive Gram-Schmidt filtering algorithm, which allowed us to digitally cancel RSA in real-time. This filtering algorithm resulted in a stable real-time estimation of the volitional intent of the stimulated muscle, which then acted as the direct signal for continuously controlling homonymous muscle stimulation. As a first step toward clinical application, we tested the viability of our aEMGcFES system to continuously control ankle dorsiflexion in a healthy subject. Our results indicate positively that an aEMGcFES device with adaptive filtering can respond proportionally to voluntary EMG and activate forceful movements to assist dorsiflexion during controlled isometric activation at the ankle. We also verified that normal ankle joint range of movement could be maintained while using the aEMGcFES system. We suggest that real-time cancellation of both primary and RSA is possible with surface electrode aEMGcFES in healthy subjects and shows promising potential for future clinical application to gait pathologies such as drop foot related to hemiparetic stroke.

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Rotating disk electrode voltammetric measurements of serotonin transporter kinetics in synaptosomes.

Publication Date: 2010 Aug 14 PMID: 20713085
Authors: Hagan, C. E. - Neumaier, J. F. - Schenk, J. O.
Journal: J Neurosci Methods

Altered serotonin (5-HT) signaling is implicated in several neuropsychiatric disorders, including depression, anxiety, obsessive-compulsive disorder, and autism. The 5-HT transporter (SERT) modulates 5-HT neurotransmission strength and duration. This is the first study using rotating disk electrode voltammetry (RDEV) to measure 5-HT clearance. SERT kinetics were measured in whole-brain synaptosomes. Uptake kinetics of exogenous 5-HT were measured using glassy carbon electrodes rotated in 500muL glass chambers containing synaptosomes from SERT-knockout (-/-), heterozygous (+/-), or wild-type (+/+) mice. RDEV detected 5-HT concentrations of 5nM and higher. Initial velocities were kinetically resolved with K(m) and V(max) values of 99+/-35 standard error of regression (SER) nM and 181+/-11 SER fmol/(sxmg protein), respectively in wild-type synaptosomes. The method enables control over drug and chemical concentrations, facilitating interpretation of results. Results are compared in detail to other techniques used to measure SERT kinetics, including tritium labeled assays, chronoamperometry, and fast scan cyclic voltammetry. RDEV exhibits decreased 5-HT detection limits, decreased vulnerability to 5-HT oxidation products that reduce electrode sensitivity, and also overcomes diffusion limitations via forced convection by providing a continuous, kinetically resolved signal. Finally, RDEV distinguishes functional differences between genotypes, notably, between wild-type and heterozygous mice, an experimental problem with other experimental approaches.

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A hardware-algorithm co-design approach to optimize seizure detection algorithms for implantable applications.

Publication Date: 2010 Aug 14 PMID: 20713084
Authors: Raghunathan, S. - Gupta, S. K. - Markandeya, H. S. - Roy, K. - Irazoqui, P. P.
Journal: J Neurosci Methods

Implantable neural prostheses that deliver focal electrical stimulation upon demand are rapidly emerging as an alternate therapy for roughly a third of the epileptic patient population that is medically refractory. Seizure detection algorithms enable feedback mechanisms to provide focally and temporally specific intervention. Real-time feasibility and computational complexity often limit most reported detection algorithms to implementations using computers for bedside monitoring or external devices communicating with the implanted electrodes. A comparison of algorithms based on detection efficacy does not present a complete picture of the feasibility of the algorithm with limited computational power, as is the case with most battery-powered applications. We present a two-dimensional design optimization approach that takes into account both detection efficacy and hardware cost in evaluating algorithms for their feasibility in an implantable application. Detection features are first compared for their ability to detect electrographic seizures from micro-electrode data recorded from kainate-treated rats. Circuit models are then used to estimate the dynamic and leakage power consumption of the compared features. A score is assigned based on detection efficacy and the hardware cost for each of the features, then plotted on a two-dimensional design space. An optimal combination of compared features is used to construct an algorithm that provides maximal detection efficacy per unit hardware cost. The methods presented in this paper would facilitate the development of a common platform to benchmark seizure detection algorithms for comparison and feasibility analysis in the next generation of implantable neuroprosthetic devices to treat epilepsy.

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Production of a monoclonal antibody, against human alpha-synuclein, in a subpopulation of C57BL/6J mice, presenting a deletion of the alpha-synuclein locus.

Publication Date: 2010 Aug 13 PMID: 20709102
Authors: Mougenot, A. L. - Betemps, D. - Hogeveen, K. N. - Kovacs, G. G. - Chouaf-Lakhdar, L. - Milhavet, O. - Lehmann, S. - Legastelois, S. - Pin, J. J. - Baron, T. G.
Journal: J Neurosci Methods

Analyses using antibodies directed against alpha-synuclein play a key role in the understanding of the pathologies associated with neurodegenerative disorders such as Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). However, the generation of antibodies against immunogens with significant sequence similarity to host proteins such as alpha-synuclein is often hindered by host immunotolerance. In contrast to wild-type C57BL/6J and BALB/c mice immunized with recombinant human alpha-synuclein, C57BL/6S Deltasnca mice presenting a natural deletion of the alpha-synuclein locus, bypassed the immunotolerance process which resulted in a much higher polyclonal antibody response. The native or fibrillized conformation of alpha-synuclein used as the immunogen did not have an impact on the amounts of specific antibodies in sera of the host. The immunization protocols resulted in the generation of the IgG AS11, raised against fibrillized recombinant human alpha-synuclein in C57BL/6S Deltasnca mice. This monoclonal antibody, recognizing an N-terminal alpha-synuclein epitope, was selected for its specificity and significant reactivity in Western-blot, immunofluorescence and immunohistochemistry assays. The ability of AS11 to detect both soluble and aggregated forms of alpha-synuclein present in pathological cytoplasmic inclusions was further assessed using analysis of human brains with PD or MSA, transgenic mouse lines expressing A53T human alpha-synuclein, and cellular models expressing human alpha-synuclein. Taken together, our study indicates that novel antibodies helpful to characterize alterations of alpha-synuclein leading to neurodegeneration in PD and related disorders could be efficiently developed using this original immunization strategy.

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Neural influence on cold induced vasodilatation using a new set-up for bilateral measurement in the rat hind limb.

Publication Date: 2010 Aug 13 PMID: 20709101
Authors: Kusters, F. J. - Walbeehm, E. T. - Niehof, S. P.
Journal: J Neurosci Methods

Cold induced vasoconstriction (CIVC) is a way for mammals to reduce heat loss in an effort to maintain body core temperature. As blood flow to a cooled extremity is reduced, the amount of body heat lost at the cooled location is minimised. However, when the extremity temperature gets below a certain threshold, Cold induced vasodilation (CIVD) occurs, a phenomenon that is believed to reduce the risk of local cold injuries. Many theories explaining the mechanism of the CIVD reaction have been postulated, but no consensus has been found. One of these theories is that the CIVD reaction is controlled neurally. To study the effect of neural influence on the vascularisation and rewarming patterns a new experimental set-up was designed. This set-up is able to measure responses in both hind paws simultaneously, creating the opportunity to study the effect of nerve injury on one limb and use the contralateral limb as a control. Ten rats received a sciatic nerve transection and repair of either the left (n=5) or the right (n=5) hind limb. Measurements were performed, 1 day pre-operatively, directly post-operatively, and at days 1, 7, 14, 21 and 49 post-operatively. Although results are not significant, there is a tendency for the CIVD reaction to be reduced in the nerve injured paw until the nerve is regenerated around day 35. Further investigation of neural influence on the CIVD reaction will be necessary; this set-up may prove to be useful in future experiments to elucidate the mechanism of the CIVD reaction.

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Seizure logging: A new approach to synchronized cable-free EEG and video recordings of seizure activity in mice.

Publication Date: 2010 Aug 11 PMID: 20708034
Authors: Etholm, L. - Arabadzisz, D. - Lipp, H. P. - Heggelund, P.
Journal: J Neurosci Methods

We describe a new cable-free, non-telemetric method for synchronized electrophysiological and video recordings of seizure activity in freely moving mice. The electrophysiological recordings were made by a head-mounted 4-channel data-logging device, allowing the mouse to move freely in its cage, and even to be moved from cage to cage under ongoing recording. Seizures were studied in Synapsin I/II double knock-out (SynDKO) mice, a genetically engineered mouse line that shows seizures upon daily handling procedures such as tail lifting during cage changes, much in resemblance to the more studied El mouse. The ability to elicit seizures through daily handling in SynDKO mice undergoing electrophysiological recording is a significant improvement in comparison to the traditional cable-based set-up. Furthermore, with its four channels and a sample rate of up to 500Hz, the data-logging device opens for more varied electrophysiological studies than other available cable-free systems.

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Measuring torsional eye movements by tracking stable iris features.

Publication Date: 2010 Aug 11 PMID: 20708033
Authors: Ong, J. K. - Haslwanter, T.
Journal: J Neurosci Methods

We propose a new method to measure torsional eye movements from videos taken of the eye. In this method, we track iris features that have been identified as Maximally Stable Volumes. These features, which are stable over time, are dark regions with bright borders that are steep in intensity. The advantage of Maximally Stable Volumes is that they are robust to nonuniform illumination and to large changes in eye and camera position. The method performs well even when the iris is partially occluded by reflections or eyelids, and is faster than cross-correlation. In addition, it is possible to use the method on videos of macaque eyes taken in the infrared, where the iris appears almost featureless.

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An experimental paradigm to compare motor performance under laboratory and under everyday-like conditions.

Publication Date: 2010 Aug 10 PMID: 20705100
Authors: Bock, O. - Hagemann, A.
Journal: J Neurosci Methods

Research findings on human motor skills may not necessarily hold in everyday life, since laboratory and everyday scenarios typically differ with respect to the subjects' attention to the skill, their motivation to perform at their best, the goals they try to achieve, and the mode of movement initiation - extrinsic versus intrinsic. Here we present an experimental approach which can be used to substantiate the hypothesized effects of laboratory (L) versus everyday (E) settings on one type of motor skill, i.e., manual prehension. This approach is based on two tasks: In task L, subjects are told that they will participate in an experiment on grasping, and are instructed to seize and move a lever upon appearance of a visual target. In task E, they are told that they will play a computer game, and they have to seize and move the lever in order to proceed from one game level to the next. Both tasks include prehension movements from the same starting position and object to the same terminal position and object; movements differ only in their behavioural context. We exemplify the utility of our approach with a preliminary analysis of kinematic and force data. It shows that the two tasks differ with respect to several performance measures, and that some performance measures make independent contributions to that difference. The existence of independent contributions suggests that behavioural context may influence prehension via several distinct routes. Our approach can be used for comprehensive analyses of the context-dependence of motor skills in various reference groups.

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Nerve conduction block using combined thermoelectric cooling and high frequency electrical stimulation.

Publication Date: 2010 Aug 10 PMID: 20705099
Authors: Ackermann, D. M. - Foldes, E. L. - Bhadra, N. - Kilgore, K. L.
Journal: J Neurosci Methods

Conduction block of peripheral nerves is an important technique for many basic and applied neurophysiology studies. To date, there has not been a technique which provides a quickly initiated and reversible "on-demand" conduction block which is both sustainable for long periods of time and does not generate activity in the nerve at the onset of the conduction block. In this study we evaluated the feasibility of a combined method of nerve block which utilizes two well established nerve blocking techniques in a rat and cat model: nerve cooling and electrical block using high frequency alternating currents (HFAC). This combined method effectively makes use of the contrasting features of both nerve cooling and electrical block using HFAC. The conduction block was initiated using nerve cooling, a technique which does not produce nerve "onset response" firing, a prohibitive drawback of HFAC electrical block. The conduction block was then readily transitioned into an electrical block. A long-term electrical block is likely preferential to a long-term nerve cooling block because nerve cooling block generates large amounts of exhaust heat, does not allow for fiber diameter selectivity and is known to be unsafe for prolonged delivery.

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Detection of input sites in scanning photostimulation data based on spatial correlations.

Publication Date: 2010 Aug 10 PMID: 20705098
Authors: Bendels, M. H. - Beed, P. - Schmitz, D. - Johenning, F. W. - Leibold, C.
Journal: J Neurosci Methods

Scanning photostimulation is a well-established method for studying the functional microcircuitry in brain slices. Light-evoked responses are thereby taken as an indicator for a connected presynaptic partner. Such an approach thus requires a clear distinction between the photo-evoked and the spontaneous responses. Here we show that, for a data set from entorhinal cortex layer II with high spontaneous synaptic rates of up to 10Hz, it is possible to identify presynaptic sites. The underlying detection algorithm is based on the finding that a presynaptic cell has several neighboring activation sites, resulting in the clustered appearance of specific photo-evoked inputs. The main idea behind this approach is to identify "hit" locations at which the number of intracellularly recorded synaptic events is significantly larger as expected from the hypothesis of statistical independence. The algorithm works without making use of EPSC amplitude information and for single trials, i.e., each site is stimulated only once. The hit maps are tested upon reliability by repeated stimulations and by blocking synaptically mediated responses via TTX. Furthermore, based on the hit density of surrogate data, we devise a Bayesian formalism to estimate the number of presynaptic partners. In these simulations we find good agreement between estimated and real number of input cells, which shows that the hit density can be used as a reliable measure for afferent connectivity.

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Genetically encoded Cl-Sensor as a tool for monitoring of Cl-dependent processes in small neuronal compartments.

Publication Date: 2010 Aug 10 PMID: 20705097
Authors: Waseem, T. - Mukhtarov, M. - Buldakova, S. - Medina, I. - Bregestovski, P.
Journal: J Neurosci Methods

Chloride (Cl) participates in a variety of physiological functions. To study processes connected with Cl homeostasis we need effective and quantitative probes allowing measurements of intracellular Cl concentration ([Cl(-)](i)) in different cell types, particularly in specialized small cellular compartments such as dendrites and dendritic spines. Of the different tools proposed for monitoring [Cl(-)](i), the genetically encoded Cl-sensitive indicators are the most promising. Recently, a ratiometric CFP-YFP based construct, termed "Cl-Sensor", with a relatively high sensitivity to Cl has been proposed (Markova et al., 2008). In the present study, we have developed conditions for the efficient expression of Cl-Sensor in tiny neuronal compartments including distal dendrites and spines. We also propose a new approach for the calibration of intracellularly expressed probes using a natural triterpenoid saponin, beta-escin. We have mapped [Cl(-)](i) distribution in different neuronal compartments of cultured hippocampal and spinal cord neurons. The maximum Cl concentration was observed in the soma and it had a tendency to decrease gradually along dendritic branches, reaching minimum values in thin distal dendrites. We have also monitored transient increases in intracellular Cl in dendritic spines caused by glutamate application. These results demonstrate that Cl-Sensor enables non-invasive monitoring of the [Cl(-)](i) distribution in different types of neurons with variable morphology. This probe represents an effective tool for the quantitative estimation of [Cl(-)](i) in various cellular compartments including dendritic spines.

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A FPGA real-time model of single and multiple visual cortex neurons.

Publication Date: 2010 Aug 10 PMID: 20705096
Authors: Li, G. - Talebi, V. - Yoonessi, A. - Baker, C. L. Jr
Journal: J Neurosci Methods

Using a biologically realistic model of a single neuron can be very beneficial for visual physiologists to test their electrophysiology setups, train students in the laboratory, or conduct classroom-teaching demonstrations. Here we present a Field Programmable Gate Array (FPGA)-based spiking model of visual cortex neurons, which has the ability to simulate three independent neurons and output analog spike waveform signals in four channels. To realistically simulate multi-electrode (tetrode) recordings, the independently generated spikes of each simulated neuron has a distinct waveform, and each channel outputs a differentially weighted sum of these waveforms. The model can be easily constructed from a small number of inexpensive commercially available parts, and is straightforward to operate. In response to sinewave grating stimuli, the neurons exhibit biologically realistic simple-cell-like response properties, including highly modulated Poisson spike trains, orientation selectivity, spatial/temporal frequency selectivity, and space-time receptive fields. Users can customize their model neurons by downloading modifications to the FPGA with varying parameter values, particularly desired features, or qualitatively different models of their own design. The source code and documentation are provided to enable users to modify or extend the model's functionality according to their individual needs.

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Comparing levels of biochemical markers in CSF from cannulated and non-cannulated rats.

Publication Date: 2010 Aug 6 PMID: 20692294
Authors: Cassar, S. C. - Tovcimak, A. E. - Rustay, N. R. - Ellis, T. A. - Hooker, B. A. - Witte, D. G. - Li, J. - Buck, W. R. - Scharf, D. - Muller, U. - Jeromin, A. - Wang, K. K. - Waring, J. F.
Journal: J Neurosci Methods

Cerebrospinal fluid (CSF) is commonly used for assessing biomarkers of drug efficacy or disease progression in the central nervous system. Studies of CSF from pre-clinical species can characterize biomarkers for use in clinical trials. However, obtaining CSF from pre-clinical species, particularly rodents, can be challenging due to small body sizes, and consequently, low volumes of CSF. Surgical cannulation of rats is commonly used to allow for CSF withdrawal from the cisterna magna. However, cannulae do not remain patent over multiple days, making chronic studies on the same rats difficult. Moreover, CSF biomarkers may be affected by cannulation. Thus cannulation may contribute confounding factors to the understanding of CSF biomarkers. To determine the potential impact on biomarkers, CSF was analyzed from cannulated rats, surgically implanted with catheters as well as from non-cannulated rats. Brain protein biomarkers (alphaII-spectrin SBDP150 and total tau) and albumin, were measured in the CSF using ELISA assays. Overall, cannulated rat CSF had elevated levels of the biomarkers examined compared to non-cannulated rat CSF. Additionally, the variation in biomarker levels observed among CSF from cannulated rats was greater than that observed for non-cannulated rat CSF. These results demonstrate that in some cases, biomarker assessment using CSF from cannulated rats may differ from that of non-cannulated animals and may contribute confounding factors to biomarker measurements and assay development for clinical use.

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Spinal cord integrity monitoring by adaptive coherence measurement.

Publication Date: 2010 Aug 6 PMID: 20692293
Authors: Sherman, D. L. - Wuyyuru, V. - Brooke, M. J. - Zhang, H. X. - Sepkuty, J. P. - Thakor, N. V. - Natarajan, A. - All, A. H.
Journal: J Neurosci Methods

OBJECTIVE: Injury during routine spinal cord procedures could result in devastating consequences for the surgical patient. Spinal cord monitoring through somatosensory evoked potentials (SEPs) remains a viable method for prevention of serious injury. METHODS: The adaptive coherence estimation (ACE) is a method to iteratively calculate signal match quality through successive filter entrainment. Here we compare the speed of detection with ACE to conventional amplitude measurements. Both absolute magnitude of ACE and amplitude as well as slope change detector algorithm (Farley-Hinich) was run as well to determine the earliest time when a significant change occurred. RESULTS: The standard error for the ACE algorithm is close to one tenth of the amplitude measure, Since the ACE algorithm achieved low variance during baseline measurement, we were able to achieve rapid detection of injury. For absolute magnitude detection ACE was faster than amplitude for the 20g injury weight class. It took an average of 10 epochs to detect the injury with adaptive coherence and nearly 19 with standard amplitude metrics using absolute magnitude changes. Abrupt change detection methods using slope change show that ACE provides more favorable detection capabilities comparable to amplitude. Additionally, there was a significant increase in the ROC curve between ACE and amplitude alone (p<0.05). CONCLUSIONS: Because of its excellent detection capabilities, the adaptive coherence method provides an excellent supplement to traditional amplitude for capturing injury-related changes in SEPs. SIGNIFICANCE: Adaptive coherence remains a viable method for rapidly and accurately detecting spinal injury.

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A new MRI approach for accurately implanting microelectrodes into deep brain structures of the rhesus monkey (Macaca mulatta).

Publication Date: 2010 Aug 5 PMID: 20692292
Authors: Jing, W. - Wenchao, W. - Heng, T. - Huihui, J. - Lin, L. - Li, L. - Rui, S. - Jianhong, W. - Yuanye, M. - Xintian, H.
Journal: J Neurosci Methods

The accurate implantation of microelectrodes is a significant difficulty facing many neurophysiologists. This paper reports on a new method used to promote the precise positioning of electrode implantation through magnetic resonance imaging (MRI), allowing both the relevant brain structure and the MRI-visible external markers anchored on the skull (in this case rigid glass tubes with a 0.5mm internal diameter) to be displayed. By referencing these markers, the coordinates of the brain target were calculated. Using this novel approach, recording electrodes were successfully implanted into the superior colliculus (SC) of rhesus monkeys, with an error <1mm, and its neuronal discharge signals were obtained. This new method allows neurophysiologists to precisely target the small deep brain structures of monkeys and study their electrophysiological characteristics in detail.

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A three-dimensional digital segmented and deformable brain atlas of the domestic pig.

Publication Date: 2010 Aug 6 PMID: 20692291
Authors: Saikali, S. - Meurice, P. - Sauleau, P. - Eliat, P. A. - Bellaud, P. - Randuineau, G. - Verin, M. - Malbert, C. H.
Journal: J Neurosci Methods

We used high-magnetic field (4.7T) magnetic resonance imaging (MRI) to build the first high-resolution (100mumx150mumx100mum) three-dimensional (3D) digital atlas in stereotaxic coordinates of the brain of a female domestic pig (Sus scrofa domesticus). This atlas was constructed from one hemisphere which underwent a symmetrical transformation through the midsagittal plane. Concomitant construction of a 3D histological atlas based on the same scheme facilitated control of deep brain structure delimitation and enabled cortical mapping to be achieved. The atlas contains 178 individual cerebral structures including 42 paired and 9 single deep brain structures, 5 ventricular system areas, 6 paired deep cerebellar nuclei, 12 cerebellar lobules and 28 cortical areas per hemisphere. Given the increasing importance of pig brains in medical research, this atlas should be a useful tool for intersubject normalization in anatomical imaging as well as for precisely localizing brain areas in functional MR studies or electrode implantation trials. The atlas can be freely downloaded from our institution's Website.

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Staining of fluorogold-prelabeled retinal ganglion cells with calcein-AM: A new method for assessing cell vitality.

Publication Date: 2010 Aug 5 PMID: 20691729
Authors: Grieshaber, P. - Lagreze, W. A. - Noack, C. - Boehringer, D. - Biermann, J.
Journal: J Neurosci Methods

PURPOSE: The number of retinal ganglion cells (RGC) is often used as an outcome measure in neuroprotection. The gold standard for staining RGC is retrograde labeling, e.g. with fluorogold (FG). However, this method alone does not permit to differentiate between viable and dead cells, because dying cells only avoid being counted once they have undergone complete microglial-phagocytosis. To differentiate between viable and dead but still existent RGC, we additionally stained FG-labeled RGC with calcein-acetoxymethylester (CAM). METHODS: The left optic nerves of rats were crushed 6 days after stereotactical injection of FG into both superior colliculi. The right eyes served as controls. Retinal whole mounts were prepared 2, 5, 8 or 11 days after optic nerve crush (ONC), and incubated for 30min in culture media containing 0.01% CAM. RGC densities were determined in defined areas at different eccentricities under a fluorescence microscope using the appropriate filters. Twice-positive RGC were counted after merging both filters. RESULTS: The loss of RGC induced by ONC is identified earlier when these cells are detected by FG+CAM rather than by FG-labeling alone. The percentages of FG-positive RGC stained with CAM were 83% in controls, 68% on day 2, 48% on day 5, 26% on day 8, and 9% on day 11 after ONC. The decay rate of FG-prelabeled RGC appears accelerated and becomes more linear when only viable RGC positive for CAM are counted. CONCLUSIONS: The staining of FG-prelabeled RGC with CAM permits the discrimination between dead and viable RGC in retinal whole mounts, which enables to quantify RGC degeneration earlier after injury than by using microglial-phagocytosis-dependant retrograde labeling alone.

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Recordings from human myenteric neurons using voltage-sensitive dyes.

Publication Date: 2010 Aug 5 PMID: 20691728
Authors: Vignali, S. - Peter, N. - Ceyhan, G. - Demir, I. E. - Zeller, F. - Senseman, D. - Michel, K. - Schemann, M.
Journal: J Neurosci Methods

Voltage-sensitive dye (VSD) imaging became a powerful tool to detect neural activity in the enteric nervous system, including its routine use in submucous neurons in freshly dissected human tissue. However, VSD imaging of human myenteric neurons remained a challenge because of limited visibility of the ganglia and dye accessibility. We describe a protocol to apply VSD for recordings of human myenteric neurons in freshly dissected tissue and myenteric neurons in primary cultures. VSD imaging of guinea-pig myenteric neurons was used for reference. Electrical stimulation of interganglionic fiber tracts and exogenous application of nicotine or elevated KCl solution was used to evoke action potentials. Bath application of the VSDs Annine-6Plus, Di-4-ANEPPS, Di-8-ANEPPQ, Di-4-ANEPPDHQ or Di-8-ANEPPS revealed no neural signals in human tissue although most of these VSD worked in guinea-pig tissue. Unlike methylene blue and FM1-43, 4-Di-2-ASP did not influence spike discharge and was used in human tissue to visualize myenteric ganglia as a prerequisite for targeted intraganglionic VSD application. Of all VSDs, only intraganglionic injection of Di-8-ANEPPS by a volume controlled injector revealed neuronal signals in human tissue. Signal-to-noise ratio increased by addition of dipicrylamine to Di-8-ANEPPS (0.98+/-0.16 vs. 2.4+/-0.62). Establishing VSD imaging in primary cultures of human myenteric neurons led to a further improvement of signal-to-noise ratio. This allowed us to routinely record spike discharge after nicotine application. The described protocol enabled reliable VSD recordings from human myenteric neurons but may also be relevant for the use of other fluorescent dyes in human tissues.

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A new rodent behavioral paradigm for studying forelimb movement.

Publication Date: 2010 Aug 5 PMID: 20691727
Authors: Slutzky, M. W. - Jordan, L. R. - Bauman, M. J. - Miller, L. E.
Journal: J Neurosci Methods

The center-out task is a standard paradigm often used to study the neural control of reaching movements in human and non-human primates. However, there are several disadvantages to the use of monkeys, notably costs, infrastructural requirements, and ethical considerations. Here we describe a similar task designed to examine forelimb movements in rats. Rats were trained to grasp a joystick with their forepaw and use it to control the movements of a sipper tube in two dimensions. The rats learned to move the joystick in four directions with at least 70% accuracy after about 45 days of training. In addition, rats were able to learn a reversed mapping between joystick and sipper tube movement. This is a more complicated behavior than has been previously demonstrated for rats, and it could allow more motor behavior studies to be conducted in rodents instead of monkeys. We currently are using this behavior to decode the rats' forelimb movements from their brain signals.

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Voltage- and temperature-dependent gating of heterologously expressed channelrhodopsin-2.

Publication Date: 2010 Aug 4 PMID: 20691205
Authors: Chater, T. E. - Henley, J. M. - Brown, J. T. - Randall, A. D.
Journal: J Neurosci Methods

Channelrhodopsins are light-activated channels originally isolated from algae that are being used increasingly as tools to non-invasively stimulate neurones. Despite their widespread use some aspects of their biophysical properties have not been fully characterised. Here we report detailed investigation of the gating kinetics and voltage-dependence of ChR2 transiently expressed in HEK-293 cells. Currents were elicited using light pulses of defined duration and intensity generated by a blue LED. Datasets were gathered both at room temperature (RT, approximately 22 degrees C) and 37 degrees C. Current responses to light rose rapidly to a peak and then desensitized to a steady state plateau. When illumination was terminated currents rapidly deactivated. Recovery from desensitization at -85mV was slow with half-times of 1.4 and 3.1s at 37 degrees C and approximately 22 degrees C, respectively. At both temperatures, the reversal potential of ChR2 responses was a few mV positive to 0mV. Both the peak and plateau phases of ChR2 responses exhibited strong inward rectification with only small outward currents at positive membrane potentials. The rates of ChR2 activation, deactivation and desensitization were approximately 2 times faster at 37 degrees C than at approximately 22 degrees C. Both the activation and deactivation kinetics of ChR2 were significantly slowed by depolarization at both temperatures. Additionally, the degree of steady state desensitization was greater at more depolarized potentials. The macroscopic desensitization kinetics were not voltage-dependent, but recovery from desensitization was slowed by depolarization. These gating behaviour data provide an important basis for more detailed analysis of the properties and limitations of ChR2 use in more complex systems.

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Characterization of a 3-vessel occlusion model for the induction of complete global cerebral ischemia in mice.

Publication Date: 2010 Aug 3 PMID: 20688105
Authors: Thal, S. C. - Thal, S. E. - Plesnila, N.
Journal: J Neurosci Methods

Existing murine models of global cerebral ischemia are technically challenging thereby hampering the use of genetically engineered mice to study cardiac arrest-induced brain damage. We therefore investigated, if disconnecting the cerebral circulation from vertebral collateral blood flow by proximal occlusion of the basilar artery together with temporary bilateral common carotid artery occlusion (BCCAo) may be a more feasible approach. C57/Bl6 mice were anesthetized and the basilar artery was occluded through a ventral approach. Ten days later BCCAo was performed for 8-14min. Increasing durations of ischemia resulted in enhanced neuronal cell death in cortex, striatum, and hippocampus (22-63%) and increased neurological dysfunction and mortality (0-36%). Following 10min of BCCAo, the duration of global ischemia with the most favorable mortality/neuronal cell death ratio, hippocampal damage started 6h after the insult while cortical and striatal damage was delayed by at least 24h. No further loss of neuronal cells was observed later than 3 days. The proposed two-step approach resulted in complete cerebral ischemia and caused neuronal damage with high reproducibility and small variability. In combination with transgenic and knock-out mice this technically feasible model may help to extend our knowledge on the pathophysiology of cardiac arrest-induced brain damage.

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Fully exploratory network ICA (FENICA) on resting-state fMRI data.

Publication Date: 2010 Aug 3 PMID: 20688104
Authors: Schopf, V. - Kasess, C. H. - Lanzenberger, R. - Fischmeister, F. - Windischberger, C. - Moser, E.
Journal: J Neurosci Methods

Independent component analysis (ICA) is one of the most valuable explorative methods for analyzing resting-state networks (RSNs) in fMRI, representing a data-driven approach that enables decomposition of high-dimensional data into discrete components. Extensions to a group-level suffer from the drawback of evaluating single-subject resting-state components of interest either using a predefined spatial template or via visual inspection. FENICA introduced in the context of group ICA methods is based solely on spatially consistency across subjects directly reflecting similar networks. Therefore, group data can be processed without further visual inspection of the single-subject components or the definition of a template (Schopf et al., 2009). In this study FENICA was applied to fMRI resting-state data from 28 healthy subjects resulting in eight group RSNs. These RSNs resemble the spatial patterns of the following previously described networks: (1) visual network, (2) default mode network, (3) sensorimotor network, (4) dorsolateral prefrontal network, (5) temporal prefrontal network, (6) basal ganglia network, (7) auditory processing network, and (8) working memory network. This novel analysis approach for identifying spatially consistent networks across a group of subjects does not require manual or template-based selection of single-subject components and, therefore, offers a truly explorative procedure of assessing RSNs.

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An implicit measure of olfactory performance for non-human primates reveals aversive and pleasant odor conditioning.

Publication Date: 2010 Aug 3 PMID: 20688103
Authors: Livneh, U. - Paz, R.
Journal: J Neurosci Methods

We have little understanding of how odorants are processed in neural networks of the primate brain. Because chemo-stimuli are harder to control than physical stimuli (e.g. vision, audition), such research was limited by the temporal resolution, accuracy, and reliability of olfactometers (odor producing machines). Recent advances were able to create olfactometers that overcome these limitations, allowing their use together with neuroimaging techniques in humans. From the behavioral point of view, olfaction research requires a behavioral measure that can be used to quantify olfactory performance. This becomes a real problem when working with animals, where, unlike humans, explicit measures are harder to obtain. Furthermore, because odorants are powerful primitive reinforcers, such implicit measures can be beneficial to use in learning paradigms. Here we describe an olfactometer suitable for use in non-human primates, and an end-port design that allows the accurate measure of real-time respiratory modulations that are elicited in response to odor presentation. We demonstrate that this implicit measure is differentially modulated when experiencing pleasant or aversive odors. We then present an experimental paradigm in which monkeys learn to associate tones with odors, and show that the time delay from the conditioned stimuli to the next breath can be used to measure learning and memory expression in this paradigm. Using this construct, we reveal olfactory performance during acquisition and extinction of odor conditioning. These techniques can be used in electrophysiological recordings from relevant brain areas to shed light on neural networks involved in odor processing and reinforcement-learning.

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A simple method of in vitro electroporation allows visualization, recording, and calcium imaging of local neuronal circuits.

Publication Date: 2010 Aug 15 PMID: 20669363
Authors: Hovis, K. R. - Padmanabhan, K. - Urban, N. N.
Journal: J Neurosci Methods

Since Cajal's early drawings, the characterization of neuronal architecture has been paramount in understanding neuronal function. With the development of electrophysiological techniques that provide unprecedented access to the physiology of these cells, experimental questions of neuronal function have also become more tractable. Fluorescent tracers that can label the anatomy of individual or populations of neurons have opened the door to linking anatomy with physiology. Experimentally however, current techniques for bulk labeling of cells in vitro often affect neuronal function creating a barrier for exploring structure-function questions. Here we describe a new technique for highly localized electroporation within a cell or cell population that enables the introduction of membrane impermeable charged dyes including dextran-conjugated fluorophores, hydrazide tracers, and calcium indicator dyes in vitro. We demonstrate that this technique is highly versatile, allowing for labeling of large or small areas of tissue, allowing for the investigation of both cellular morphology and physiological activity in identified neuronal circuits in acute brain slices. Furthermore, this approach allows subsequent targeted whole-cell patch recording based on well-defined connectivity as well as assessment of physiological activity in targeted circuits on a fast time scale.

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The issue of multiple univariate comparisons in the context of neuroelectric brain mapping: an application in a neuromarketing experiment.

Publication Date: 2010 Aug 30 PMID: 20637802
Authors: Vecchiato, G. - De Vico Fallani, F. - Astolfi, L. - Toppi, J. - Cincotti, F. - Mattia, D. - Salinari, S. - Babiloni, F.
Journal: J Neurosci Methods

This paper presents some considerations about the use of adequate statistical techniques in the framework of the neuroelectromagnetic brain mapping. With the use of advanced EEG/MEG recording setup involving hundred of sensors, the issue of the protection against the type I errors that could occur during the execution of hundred of univariate statistical tests, has gained interest. In the present experiment, we investigated the EEG signals from a mannequin acting as an experimental subject. Data have been collected while performing a neuromarketing experiment and analyzed with state of the art computational tools adopted in specialized literature. Results showed that electric data from the mannequin's head presents statistical significant differences in power spectra during the visualization of a commercial advertising when compared to the power spectra gathered during a documentary, when no adjustments were made on the alpha level of the multiple univariate tests performed. The use of the Bonferroni or Bonferroni-Holm adjustments returned correctly no differences between the signals gathered from the mannequin in the two experimental conditions. An partial sample of recently published literature on different neuroscience journals suggested that at least the 30% of the papers do not use statistical protection for the type I errors. While the occurrence of type I errors could be easily managed with appropriate statistical techniques, the use of such techniques is still not so largely adopted in the literature.

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Electrophysiological and theoretical analysis of melatonin in peripheral nerve crush injury.

Publication Date: 2010 Aug 30 PMID: 20637233
Authors: Zencirci, S. G. - Bilgin, M. D. - Yaraneri, H.
Journal: J Neurosci Methods

Electrophysiological and theoretical studies have been performed to investigate peripheral nerve injuries and nerve regeneration. The aim of this work is to evaluate the effect of melatonin functionally and electrophysiologically on peripheral nerve crush injury. Adult male Wistar rats (n=32, 200+/-50 g) were randomly allocated into four groups. Sciatic crush was constituted on left sciatic nerves. Treatment groups received intraperitoneal melatonin at doses of 5 and 20mg/kg for 21 days. Functional nerve recovery was evaluated using sciatic functional index (SFI) every week during the experiment. In vivo electrophysiological measurements were performed at the end of the treatment. The electrophysiological data were also analyzed by wavelet analysis. Melatonin treatments increased the SFI values in the injured sciatic nerves. In vivo electrophysiological measurements showed that melatonin increased the conduction velocities and also decreased the latency values. The wavelet analysis showed that melatonin treatment reduced the densities of high frequency components of compound muscle action potential (CMAP). These results suggest that melatonin application is a promising strategy for the treatment of peripheral nerve crush injuries. Furthermore, analysis of EMG data with wavelet methods seems to give more reliable results to evaluate the nerve recovery.

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Extra-intracranial blood shunt mimicking aneurysm rupture: intracranial-pressure-controlled rabbit subarachnoid hemorrhage model.

Publication Date: 2010 Aug 30 PMID: 20624427
Authors: Marbacher, S. - Sherif, C. - Neuschmelting, V. - Schlappi, J. A. - Takala, J. - Jakob, S. M. - Fandino, J.
Journal: J Neurosci Methods

INTRODUCTION: The achieved degree of delayed cerebral vasospasm (DCVS) in the rabbits most frequently applied cistern magna blood injection model is often mild. The aim of this study was to characterize and evaluate the feasibility of an experimental SAH technique that mimics pathophysiological mechanisms and triggers higher degrees of DCVS. MATERIALS AND METHODS: SAH was induced by extracranial-intracranial (EC/IC) shunting of blood from the subclavian artery into the great cerebral cistern. Intracranial pressure (ICP), arterial blood pressure, heart rate, arterial blood gas analysis, and neurological status were monitored throughout the experiments. The magnitude of spasm was determined by comparison of pre-SAH (day 0) and post-SAH (day 3) angiograms and postmortem morphometric analysis of the basilar artery. RESULTS: A total of 13 experiments (SAH, n=11; controls, n=2) were performed. Two animals died after initiation of the EC/IC blood shunt in respiratory arrest. In SAH animals, ICP (baseline: 12+/-1 [mean+/-SD]; peak: 51+/-4; steady-state level: 15+/-2 mm Hg) rose to diastolic blood pressure levels (56+/-3 mm Hg) within 98+/-20s, and fell to a steady state within 186+/-41 s. SAH-induced vasoconstriction of the basilar artery was 53.1+/-2.8% on day 3 compared to baseline (P<0.05) and histology confirmed marked vasoconstriction. CONCLUSIONS: This novel technique of SAH induction closely mimics the pathophysiological sequelae of aneurysm rupture and triggers constant higher degrees of delayed cerebral vasospasm than previously described rabbit models. The severity of vasospasm attained offers a unique opportunity to evaluate future therapeutic treatment options.

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An interactive tool for visualization of spike train synchronization.

Publication Date: 2010 Aug 15 PMID: 20621707
Authors: Terry, K.
Journal: J Neurosci Methods

A number of studies have examined the synchronization of central and peripheral spike trains by applying signal analysis techniques in the time and frequency domains. These analyses can reveal the presence of one or more common neural inputs that produce synchronization. However, synchronization measurements can fluctuate significantly due to the inherent variability of neural discharges and a finite data record length. Moreover, the effect of these natural variations is further compounded by the number of parameters available for calculating coherence in the frequency domain and the number of indices used to quantify short-term synchronization (STS) in the time domain. The computational tool presented here provides the user with an interactive environment that dynamically calculates and displays spike train properties along with STS and coherence indices to show how these factors interact. It is intended for a broad range of users, from those who are new to synchronization to experienced researchers who want to develop more meaningful and effective computational and experimental studies. To ensure this freely available tool meets the needs of all users, there are two versions. The first is a stand-alone version for educational use that can run on any computer. The second version can be modified and expanded by researchers who want to explore more in-depth questions about synchronization. Therefore, the distribution and use of this tool should both improve the understanding of fundamental spike train synchronization dynamics and produce more efficient and meaningful synchronization studies.

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A sensitive method to analyse the effect of putative regulatory ligands on the release of glycoprotein from primary cultures of dispersed bovine subcommissural organ cells.

Publication Date: 2010 Aug 30 PMID: 20619293
Authors: Bermudez-Silva, F. J. - Perez, J. - Cifuentes, M. - Perez-Martin, M. - Grondona, J. M. - Lopez-Avalos, M. D. - Estivill-Torrus, G. - Fernandez-Llebrez, P.
Journal: J Neurosci Methods

The subcommissural organ (SCO) releases into the cerebrospinal fluid (CSF) large glycoproteins that polymerize forming the Reissner's fibre (RF), which is involved in CSF circulation and homeostasis. We obtained high purity primary cultures of bovine secretory SCO cells and measured glycoprotein release by a reliable and sensitive ELISA method. We also analysed the effect of regulatory ligands known to control the secretory activity of the SCO. Cells cultured for short time (4h) released a high amount of glycoproteins that decreased with time. In young cultures, ATP increased and serotonin inhibited secretion rate. By contrast the acetylcholine agonist carbachol and high potassium did not evoke any detectable change in SCO glycoprotein release. These results support not only the suitability of the methodological approach but an important role of both ATP and serotonin in regulating SCO secretory activity as well.

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A facile method for immunofluorescence microscopy of highly autofluorescent human retinal sections using nanoparticles with large Stokes shifts.

Publication Date: 2010 Aug 30 PMID: 20619292
Authors: Petty, H. R. - Elner, V. M. - Kawaji, T. - Clark, A. - Thompson, D. - Yang, D. L.
Journal: J Neurosci Methods

The human retina is rich in autofluorescent species, such as lipofuscin and melanin. Consequently, it is difficult to localize antigens in the human retina using immunofluorescence microscopy. To address this issue, we have developed a methodology to tag retinal antigens using quantum dot nanoparticles that absorb in the ultraviolet and emit in the infrared, thereby avoiding the visible spectrum. This protocol dramatically improves signal-to-background autofluorescence ratios of immunofluorescence images of human retinal sections, thus enhancing the specific fluorescence in microscopic studies. Of particular note is the ability to detect antigens within the brightly autofluorescent RPE cell layer.

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Detecting changes in human cerebral blood flow after acute exercise using arterial spin labeling: implications for fMRI.

Publication Date: 2010 Aug 30 PMID: 20603148
Authors: Smith, J. C. - Paulson, E. S. - Cook, D. B. - Verber, M. D. - Tian, Q.
Journal: J Neurosci Methods

The use of arterial spin labeling to measure cerebral blood flow (CBF) after acute exercise has not been reported. The aims of this study were to examine: (1) the optimal inversion time to detect changes in CBF after acute exercise and (2) if acute exercise alters CBF in the motor cortex at rest or during finger-tapping. Subjects (n=5) performed 30 min of moderate intensity exercise on an electronically braked cycle ergometer (perceived exertion 'somewhat hard'). Before and after exercise, relative CBF was measured using multiple inversion time (TI) pulsed arterial spin labeling (PASL). Two multiple TI runs were obtained at rest and during 4 Hz finger-tapping. Four inversion times (675, 975, 1275, and 1,575 ms) were acquired per run, with 20 interleaved pairs of tag and control images per inversion time (320 s run). The results indicated that global CBF increased approximately 20% following exercise, with significant differences observed at an inversion time of 1,575 ms (p<.05). Finger-tapping induced CBF in the motor cortex significantly increased from before to after exercise at TI=1,575 ms (p<.01). These findings suggest changes in human cerebral blood flow that result from acute moderate intensity exercise can be detected afterwards using PASL at 3T with an inversion time of 1,575 ms. The effect of prior acute exercise to increase motor cortex CBF during the performance of a motor task suggests future use of indices of functional activation should account for exercise-induced changes in cardio-pulmonary physiology and CBF.

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A spatial paradigm, the allothetic place avoidance alternation task, for testing visuospatial working memory and skill learning in rats.

Publication Date: 2010 Aug 30 PMID: 20603147
Authors: Dockery, C. A. - Wesierska, M. J.
Journal: J Neurosci Methods

We present a paradigm for assessing visuospatial working memory and skill learning in a rodent model, based on the place avoidance test. In our allothetic place avoidance alternation task (APAAT) the paradigm is comprised of minimal training sessions, tests various aspects of learning and memory and provides a rich set of parameters. A single working memory session consists of four conditions: habituation (no shock), two place avoidance training intervals (shock activated) and a retrieval test (shock inactivated). The location of the shock sector is alternated for each training day which initially requires extinction of previous representations and further working memory to achieve effective place avoidance across sessions. Visuospatial skill memory was evaluated by the shock/entrance ratio by tracking locomotor activity which is essential to execute a place avoidance strategy. For each day rats learned to avoid a new place with shock, as shown by a decreased number of entrances, and an increased time to the first entrance and maximum avoidance time. Skill learning improved according to the decreased number of shocks per entrance across conditions. These results indicate that complex cognitive functions are captured by this behavioral method. This APAAT paradigm expands and complements existing tools for studying hippocampal-prefrontal dependent functions to support development of treatment interventions.

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Controlled water intake: a method for objectively evaluating thirst and hydration state in monkeys by the measurement of blood osmolality.

Publication Date: 2010 Aug 15 PMID: 20600323
Authors: Yamada, H. - Louie, K. - Glimcher, P. W.
Journal: J Neurosci Methods

Standard methods for behavioral and neurophysiological experiments in the non-human primate rely on controlled water access as a means for motivating subject performance. It is, however, still not clear whether animals are able to regulate their fluid balance appropriately under these experimental settings. Further, the physical state associated with a subject monkey's thirst has not yet been objectively assessed under these conditions. Both of these deficiencies arise from the lack of a method for independently evaluating the hydration state of these subjects during experimental testing. To address these limitations, we measured the blood osmolality, the most widely used hematological index of hydration status, of three rhesus monkeys under conditions of controlled water access while they participated in a standard reinforced behavioral task for fluid rewards. We found that day-to-day hydration levels, as measured by serum osmolality, appears to be well regulated in a narrow range of values (300-320 mOsmo/kg H(2)O) by experimental subjects under these conditions: animals work harder and longer to earn more water rewards on a day when they are in a lower hydration state (higher osmolality) than when they are in a higher hydration state (lower osmolality). We also found that osmolality level decreases almost immediately after water intake, within 30 min, in a surprisingly linear manner. Osmolality thus seems to provide a fairly precise reflection of the monkeys' hydration state on a timescale of minutes. This evidence suggests that osmolality can be used as a tool for monitoring the hydration level of experimental subjects.

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Characterization of the hemodynamic response in the rat lumbar spinal cord using intrinsic optical imaging and laser speckle.

Publication Date: 2010 Aug 30 PMID: 20600322
Authors: Brieu, N. - Beaumont, E. - Dubeau, S. - Cohen-Adad, J. - Lesage, F.
Journal: J Neurosci Methods

Quantifying spinal cord functions is crucial for understanding neurophysiological mechanisms governing the intact and the injured spinal cord. Intrinsic optical imaging (IOI) and laser speckle provides measures of deoxyhemoglobin (HbR) and oxyhemoglobin (HbO(2)) concentrations, blood volume (BV) and blood flow (BF) at high spatial and temporal resolution. In this study we used IOI and laser speckle to characterize the hemodynamic response to neuronal activation in the lumbar spinal cord of anaesthetized rats (N=9). We report consistent temporal variations of HbR, HbO(2), BV and BF located ipsilaterally at L3-L5. Responses were significantly higher when stimulation intensity was increased. Vascular changes extended several millimetres from the epicenter, supporting the venous drainage observed in functional magnetic resonance imaging studies.

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Semi-automatic behavior analysis using robot/insect mixed society and video tracking.

Publication Date: 2010 Aug 15 PMID: 20600321
Authors: Guerra Rda, S. - Aonuma, H. - Hosoda, K. - Asada, M.
Journal: J Neurosci Methods

This paper proposes a novel robot/insect mixed society setup which enhances the possibilities for insect behavioral research and can be used as a powerful tool for interdisciplinary studies on insect behavior. Micro-robots are equipped with decoys so as to allow a controlled dynamic interaction with crickets, Gryllus bimaculatus. A camera records the interaction and the video is later processed for the automatic tracking of each encounter between cricket and robot. A novelty of our method lies in using the robots as tools for the controlled evoking of specific insect behaviors rather than trying to build an insect-like robot. The possibility for performing controlled repeatable movements allows the stimulation of certain insect behaviors that are usually difficult to trigger using insects alone, allowing consistent behavioral research. A set of experiments were performed in order to validate the proposed setup. We also demonstrate the use of our setup for stimulating agonistic behavior during an electromyography recording session.

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A surgical technique of spinal cord cell transplantation in amyotrophic lateral sclerosis.

Publication Date: 2010 Aug 30 PMID: 20600320
Authors: Blanquer, M. - Perez-Espejo, M. A. - Martinez-Lage, J. F. - Iniesta, F. - Martinez, S. - Moraleda, J. M.
Journal: J Neurosci Methods

We report an original method for implanting bone marrow stem cells within the spinal cord parenchyma. This method was used for the experimental treatment of patients diagnosed with amyotrophic lateral sclerosis. The methodology is reproducible and devoid of major complications even in patients showing significant spinal atrophy. Therefore, this report describes a surgical procedure that could be used in other experimental treatments involving the intraspinal delivery of stem cells.

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Identification of neuromuscular junctions by correlative confocal and transmission electron microscopy.

Publication Date: 2010 Aug 30 PMID: 20600319
Authors: Modla, S. - Mendonca, J. - Czymmek, K. J. - Akins, R. E.
Journal: J Neurosci Methods

The physiological processes regulating neuromuscular transmission are highly dependent on the structural features of the motor neuron and motor endplate, and detailing the structure of neuromuscular junctions (NMJs) in muscle biopsies is a powerful method for research and diagnostics. The observation of NMJ ultrastructure, however, is complicated by the difficulty in locating NMJs for analysis by electron microscopy. Consequently, a correlative confocal-transmission electron microscopy method was developed. Fixed muscle samples were cryo-protected in sucrose, sectioned on a cryostat, and stained with fluorescent alpha-bungarotoxin for confocal microscopy. Sections containing junctions were mapped and then processed for transmission electron microscopy (TEM). Cryostat sections allowed large expanses of muscle tissue to be rapidly screened and enabled specific junctions to be targeted for TEM. The morphology of the junctions was well preserved with all essential features of the pre- and postsynaptic elements readily identifiable without freeze damage. Unlike NMJ correlative methods using histochemical stains and DAB photo-oxidation, no electron dense precipitate was deposited over the NMJ, enabling an unobstructed view of the pre- and postsynaptic structures.

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Achieving precise display timing in visual neuroscience experiments.

Publication Date: 2010 Aug 30 PMID: 20600318
Authors: Elze, T.
Journal: J Neurosci Methods

In experimental visual neuroscience brief presentations of visual stimuli are often required. Accurate knowledge of the durations of visual stimuli and their signal shapes is important in psychophysical experiments with humans and in neuronal recordings with animals. In this study we measure and analyze the changes in luminance of visual stimuli on standard computer monitors. Signal properties of the two most frequently used monitor technologies, cathode ray tube (CRT) and liquid crystal display (LCD) monitors, are compared, and the effects of the signal shapes on the stated durations of visual stimuli are analyzed. The fundamental differences between CRT and LCD signals require different methods for the specification of durations, especially for brief stimulus presentations. In addition, stimulus durations on LCD monitors vary over different monitor models and are not even homogeneous with respect to different luminance levels on a single monitor. The use of LCD technology for brief stimulus presentation requires extensive display measurements prior to the experiment.

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Cross-correlation of instantaneous amplitudes of field potential oscillations: a straightforward method to estimate the directionality and lag between brain areas.

Publication Date: 2010 Aug 30 PMID: 20600317
Authors: Adhikari, A. - Sigurdsson, T. - Topiwala, M. A. - Gordon, J. A.
Journal: J Neurosci Methods

Researchers performing multi-site recordings are often interested in identifying the directionality of functional connectivity and estimating lags between sites. Current techniques for determining directionality require spike trains or involve multivariate autoregressive modeling. However, it is often difficult to sample large numbers of spikes from multiple areas simultaneously, and modeling can be sensitive to noise. A simple, model-independent method to estimate directionality and lag using local field potentials (LFPs) would be of general interest. Here we describe such a method using the cross-correlation of the instantaneous amplitudes of filtered LFPs. The method involves four steps. First, LFPs are band-pass filtered; second, the instantaneous amplitude of the filtered signals is calculated; third, these amplitudes are cross-correlated and the lag at which the cross-correlation peak occurs is determined; fourth, the distribution of lags obtained is tested to determine if it differs from zero. This method was applied to LFPs recorded from the ventral hippocampus and the medial prefrontal cortex in awake behaving mice. The results demonstrate that the hippocampus leads the mPFC, in good agreement with the time lag calculated from the phase locking of mPFC spikes to vHPC LFP oscillations in the same dataset. We also compare the amplitude cross-correlation method to partial directed coherence, a commonly used multivariate autoregressive model-dependent method, and find that the former is more robust to the effects of noise. These data suggest that the cross-correlation of instantaneous amplitude of filtered LFPs is a valid method to study the direction of flow of information across brain areas.

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Efficacy of fluorescent tracers in retrograde labeling of cutaneous afferent neurons in the rat.

Publication Date: 2010 Aug 30 PMID: 20600316
Authors: Zele, T. - Sketelj, J. - Bajrovic, F. F.
Journal: J Neurosci Methods

BACKGROUND: In the present study, the labeling efficacy of tracers Fluoro-ruby (FR), Fluoro-emerald (FE), True Blue (TB), Fluoro-Gold (FG), Diamidino Yellow (DY) and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) to retrogradely label the cutaneous afferent neurons in the rat was examined. METHODS: The proximal stump of the transected sural nerve was exposed for 1 hour either to one of the examined dyes (FR, FE, TB, FG, DY and DiI group) in single labeling experiments, or to mixtures of two dyes (TB-FG, FG-DiI, TB-DY and TB-DiI group) in double labeling experiments (n=5 for each group). After 10 days, dorsal root ganglia (DRGs) L3-S1 were harvested, cut to 20 microm thick longitudinal sections and all labeled neurons were counted. RESULTS: The average numbers of labeled DRG neurons in FR group (1063+/-158; mean+/-SD) and FE group (1067+/-203) were statistically significantly lower than those in TB group (2831+/-379), FG group (2802+/-134), DY group (2888+/-262) or DiI group (2900+/-278) (p<0.05). In double labeling experiments, the average number of double labeled neurons in TB-DY group (2208+/-207) was statistically significantly lower than those in TB-FG group (2775+/-316), FG-DiI group (2921+/-419), or TB-DiI group (2805+/-179) (p<0.05). CONCLUSIONS: Among examined tracers, TB, FG, DY and DiI, have the highest and similar labeling efficacy for retrograde labeling of cutaneous afferent neurons in the rat. The tracers TB, DiI and FG effectively label the same neuronal population in double labeling, therefore, their combinations are most suitable for double retrograde labeling studies of cutaneous afferent neurons in the rat.

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Extensive scarring induced by chronic intrathecal tubing augmented cord tissue damage and worsened functional recovery after rat spinal cord injury.

Publication Date: 2010 Aug 30 PMID: 20600315
Authors: Zhang, S. X. - Huang, F. - Gates, M. - White, J. - Holmberg, E. G.
Journal: J Neurosci Methods

Intrathecal infusion has been widely used to directly deliver drugs or neurotrophins to a lesion site following spinal cord injury. Evidence shows that intrathecal infusion is efficient for 7 days but is markedly reduced after 14 days, due to time dependent occlusion. In addition, extensive fibrotic scarring is commonly observed with intrathecal infusion. These anomalies need to be clearly elucidated in histology. In the present study, all adult Long-Evans rats received a 25 mm contusion injury on spinal cord T10 produced using the NYU impactor device. Immediately after injury, catheter tubing with an outer diameter of 0.38 mm was inserted through a small dural opening at L3 into the subdural space with the tubing tip positioned near the injury site. The tubing was connected to an Alzet mini pump, which was filled with saline solution and was placed subcutaneously. Injured rats without tubing served as control. Rats were behaviorally tested for 6 weeks using the BBB locomotor rating scale and histologically assessed for tissue scarring. Six weeks later, we found that the intrathecal tubing caused extensive scarring and inflammation, related to neutrophils, macrophages and plasma cells. The tubing's tip was occluded by scar tissue and inflammatory cells. The scar tissue surrounding the tubing consists of 20-70 layers of fibroblasts and densely compacted collagen fibers, seriously compressing and damaging the cord tissue. BBB scores of rats with intrathecal tubing were significantly lower than control rats (p<0.01) from 2 weeks after injury, implying serious impairment of functional recovery caused by the scarring.

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Assaying multiple biochemical variables from the same tissue sample.

Publication Date: 2010 Aug 30 PMID: 20600314
Authors: Leak, R. K. - Castro, S. L. - Jaumotte, J. D. - Smith, A. D. - Zigmond, M. J.
Journal: J Neurosci Methods

Experiments often involve multiple analyses, such as assays of neurotransmitters and proteins, and this can require different initial sample preparations. Typically, this is accomplished by using different animals or different tissue samples from the same animal. Either approach renders comparisons between assays more variable and greatly increases the effort and/or cost. Using tissue collected from rat striatum and molecules of special relevance to studies of Parkinson's disease, we show that tissue sonication in water prior to aliquoting into the appropriate concentrated solutions (e.g. HClO(4) and lysis buffers) permits several types of measurements to be made from the same initial samples. Dopamine and its metabolite homovanillic acid, serotonin and its metabolite 5-hydroxyindoleacetic acid, tyrosine hydroxylase and its phosphorylation at Ser19 and Ser31, and the dopamine transporter were unaffected. However, phospho-Akt levels fell slightly and phospho-ERK1/2 tended to drop. We also present a simple technique to preserve phosphorylation state of proteins such as ERK1/2 by perfusing animals through the heart with a phosphatase inhibitor, NaF. Dopamine metabolite dihydroxyphenyl acetic acid (DOPAC) levels were raised with both techniques, however. The general principles reported here are likely to apply to other brain regions, facilitate multiple comparisons of variables, increase efficiency, and decrease costs.

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The non-separability of physiologic noise in functional connectivity MRI with spatial ICA at 3T.

Publication Date: 2010 Aug 30 PMID: 20600313
Authors: Beall, E. B. - Lowe, M. J.
Journal: J Neurosci Methods

The impact of physiologic noise on spatial ICA analyses of resting state BOLD-weighted MRI data is investigated. Using FastICA and Infomax ICA, two common ICA algorithms, we apply a group spatial ICA method across multiple subjects. We compare the spatial maps from five commonly identified functional networks and show that physiologic noise correction techniques introduce significant changes in the spatial ICA decomposition of all five networks, greater than the changes introduced by either algorithmic indeterminacy (re-running ICA) or the changes introduced by decreasing the decomposition dimensionality due to physiologic noise removal. In addition, we demonstrate that the sources associated with these components have significant temporal correlation to parallel measures of cardiac and respiratory rates, and these are reduced after correction. We conclude that ICA decomposition is significantly affected by physiologic noise and the ICA process alone is not sufficient to separate physiologic noise effects in the brain. It is recommended that physiologic noise correction be applied to timeseries data prior to ICA decomposition.

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Long-term histological and behavioural characterisation of a collagenase-induced model of intracerebral haemorrhage in rats.

Publication Date: 2010 Aug 30 PMID: 20600312
Authors: Beray-Berthat, V. - Delifer, C. - Besson, V. C. - Girgis, H. - Coqueran, B. - Plotkine, M. - Marchand-Leroux, C. - Margaill, I.
Journal: J Neurosci Methods

Although intracerebral haemorrhage (ICH) entails the highest rates of mortality and disability of all stroke subtypes, efficient neuroprotective therapy is still needed. As functional recovery is a major endpoint in clinical trials, preclinical studies must demonstrate the potential of drugs to improve the sensorimotor and cognitive function of animals. In addition, behavioural studies should be performed on the long-term in order to truly mimic clinical needs. The aim of our study was to characterise a model of intracerebral haemorrhage using both histology and long-term behaviour. ICH was induced in rats by an intrastriatal injection of collagenase. Histology was performed 24h, 7 days and 2 months after ICH. Among a set of sensorimotor tests, we discriminate those able to reveal long-term deficits (up to 2 months) after cerebral haemorrhage. Our five behavioural tests (a neurological score, an adhesive removal test, two beam-walking tests and ipsilateral circling induced by dexamphetamine) proved to be effective in revealing sensorimotor deficits up to 35 days or more after cerebral haemorrhage. In conclusion, these behavioural tests appear of particular interest to screen protective agents that may exhibit benefits in patients who suffer ICH.

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Feasibility of prefronto-caudate pathway tractography using high resolution diffusion tensor tractography data at 3T.

Publication Date: 2010 Aug 30 PMID: 20600311
Authors: Kamali, A. - Kramer, L. A. - Hasan, K. M.
Journal: J Neurosci Methods

Mapping the human brain frontostriatal pathways using noninvasive diffusion tensor imaging (DTI) has been hampered by the inadequate imaging sensitivity, poor spatial resolution, lower tensor anisotropy within gray matter, increased partial volume averaging effects and poor signal-to-noise ratio. We investigated for the first time the utility of high spatial resolution DTI-based fiber-tractography using the fiber assignment by continuous tracking (FACT) to reconstruct and quantify bilaterally the prefronto-caudo-thalamic connections within the human brain at 3T. Five healthy right-handed men (age range 24-37 years) were studied. We traced the anterior thalamic radiation and prefronto-caudo-thalamic pathways bilaterally and measured the volume of each tract and the corresponding diffusion tensor metrics in all subjects. The anterior thalamic radiation tract volume and corresponding fractional anisotropy (FA) were significantly larger bilaterally than prefronto-caudate pathway, whereas the mean diffusivity (D(av)) values were similar (p>0.7). For both anterior thalamic radiation and prefronto-caudate pathway the tract volume and corresponding DTI metrics (FA, D(av)) were not significantly different between the two hemispheres (p>0.2). Our DTI acquisition protocol and analysis permitted the reconstruction of the connectivity of the caudate with the thalamus as well as with the prefrontal cortex and allowed tracking of the whole trajectory of the prefronto-caudo-thalamic pathway.

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Proteomic investigations of the ventriculo-lumbar gradient in human CSF.

Publication Date: 2010 Aug 30 PMID: 20599557
Authors: Simonsen, A. H. - Bech, S. - Laursen, I. - Salvesen, L. - Winge, K. - Waldemar, G. - Werdelin, L. - Nielsen, J. E. - McGuire, J. N. - Hjermind, L. E.
Journal: J Neurosci Methods

Cerebrospinal fluid (CSF) is an ideal biological material in which to search for new biomarkers for improved diagnosis of neurological diseases. During a lumbar puncture between 5 and 15 mL of CSF are obtained. Previous studies have assessed the ventriculo-lumbar concentration gradient of a number of specific proteins. In the present study we took a proteomics approach to investigate the possible concentration gradient of a panel of proteins and peptides in the CSF of 16 patients with neurodegenerative diseases. Using two different mass spectrometry techniques, matrix assisted laser desorption ionization time of flight (MALDI-TOF) and surface enhanced laser desorption ionization time of flight (SELDI-TOF), we found that only one of the investigated proteins, apolipoprotein CI, was significantly decreased between the 1st and the 10th mL of CSF. Furthermore, we confirmed previous results showing a significant decrease in albumin concentration from the first to the last CSF aliquots. In conclusion, we found a significant gradient effect for only two of the measured proteins. However, a standardized procedure for CSF collection for diagnostic and research purposes is crucial to allow comparisons of results between patient groups and between laboratories. This is especially important since CSF is usually collected at several centres and variation in sampled CSF due to pre-analytical factors could complicate the interpretation of the results.

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Distributional properties and variance-stabilizing transformations for measures of uncontrolled manifold effects.

Publication Date: 2010 Aug 30 PMID: 20599556
Authors: Verrel, J.
Journal: J Neurosci Methods

In the uncontrolled manifold (UCM) approach, variability in elemental variables (such as joint angles or muscle modes) is decomposed into goal-equivalent and non-goal-equivalent variability components (GEV, NGEV) with regard to a specified task variable. A UCM effect is present, when GEV exceeds NGEV, and different indices have been proposed to quantify the strength of UCM effects. We propose variance-stabilizing transformations for each of these measures. Our results indicate that the variability components should be log-transformed prior to statistical analysis, to reduce non-normality and inhomogeneity of variances. Moreover, it is formally shown that the UCM indices are identical after appropriate variance-stabilizing transformations. The theoretical analysis is illustrated by empirical and simulated data from a study on manual pointing.

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Automatic epileptic seizure detection in EEGs based on line length feature and artificial neural networks.

Publication Date: 2010 Aug 15 PMID: 20595035
Authors: Guo, L. - Rivero, D. - Dorado, J. - Rabunal, J. R. - Pazos, A.
Journal: J Neurosci Methods

About 1% of the people in the world suffer from epilepsy. The main characteristic of epilepsy is the recurrent seizures. Careful analysis of the electroencephalogram (EEG) recordings can provide valuable information for understanding the mechanisms behind epileptic disorders. Since epileptic seizures occur irregularly and unpredictably, automatic seizure detection in EEG recordings is highly required. Wavelet transform (WT) is an effective analysis tool for non-stationary signals, such as EEGs. The line length feature reflects the waveform dimensionality changes and is a measure sensitive to variation of the signal amplitude and frequency. This paper presents a novel method for automatic epileptic seizure detection, which uses line length features based on wavelet transform multiresolution decomposition and combines with an artificial neural network (ANN) to classify the EEG signals regarding the existence of seizure or not. To the knowledge of the authors, there exists no similar work in the literature. A famous public dataset was used to evaluate the proposed method. The high accuracy obtained for three different classification problems testified the great success of the method.

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A method to study global spatial patterns related to sensory perception in scalp EEG.

Publication Date: 2010 Aug 15 PMID: 20595034
Authors: Ruiz, Y. - Pockett, S. - Freeman, W. J. - Gonzalez, E. - Li, G.
Journal: J Neurosci Methods

Prior studies of multichannel ECoG from animals showed that beta and gamma oscillations carried perceptual information in both local and global spatial patterns of amplitude modulation, when the subjects were trained to discriminate conditioned stimuli (CS). Here the hypothesis was tested that similar patterns could be found in the scalp EEG human subjects trained to discriminate simultaneous visual-auditory CS. Signals were continuously recorded from 64 equispaced scalp electrodes and band-pass filtered. The Hilbert transform gave the analytic phase, which segmented the EEG into temporal frames, and the analytic amplitude, which expressed the pattern in each frame as a feature vector. Methods applied to the ECoG were adapted to the EEG for systematic search of the beta-gamma spectrum, the time period after CS onset, and the scalp surface to locate patterns that could be classified with respect to type of CS. Spatial patterns of EEG amplitude modulation were found from all subjects that could be classified with respect to stimulus combination type significantly above chance levels. The patterns were found in the beta range (15-22 Hz) but not in the gamma range. They occurred in three short bursts following CS onset. They were non-local, occupying the entire array. Our results suggest that the scalp EEG can yield information about the timing of episodically synchronized brain activity in higher cognitive function, so that future studies in brain-computer interfacing can be better focused. Our methods may be most valuable for analyzing data from dense arrays with very high spatial and temporal sampling rates.

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Development of an apparatus and methodology for conducting functional magnetic resonance imaging (fMRI) with pharmacological stimuli in conscious rhesus monkeys.

Publication Date: 2010 Aug 15 PMID: 20566353
Authors: Murnane, K. S. - Howell, L. L.
Journal: J Neurosci Methods

Functional magnetic resonance imaging (fMRI) is a technique with significant potential to advance our understanding of multiple brain systems. However, when human subjects undergo fMRI studies they are typically conscious whereas pre-clinical fMRI studies typically utilize anesthesia, which complicates comparisons across studies. Therefore, we have developed an apparatus suitable for imaging conscious rhesus monkeys. In order to minimize subject stress and spatial motion, each subject was acclimated to the necessary procedures over several months. The effectiveness of this process was then evaluated, in fully trained subjects, by quantifying objective physiological measures. These physiological metrics were stable both within and across sessions and did not differ from when these same subjects were immobilized using standard primate handling procedures. Subject motion and blood oxygenation level dependent (BOLD) fMRI measurements were then evaluated by scanning subjects under three different conditions: the absence of stimulation, presentation of a visual stimulus, or administration of intravenous (i.v.) cocaine (0.3mg/kg). Spatial motion differed neither by condition nor along the three principal axes. In addition, maximum translational and rotational motion never exceeded one half of the voxel size (0.75 mm) or 1.5 degrees, respectively. Furthermore, the localization of changes in blood oxygenation closely matched those reported in previous studies using similar stimuli. These findings document the feasibility of fMRI data collection in conscious rhesus monkeys using these procedures and allow for the further study of the neural effects of psychoactive drugs.

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Automated analysis of antidepressants' effect in the forced swim test.

Publication Date: 2010 Aug 15 PMID: 20566352
Authors: Kulikov, A. V. - Morozova, M. V. - Kulikov, V. A. - Kirichuk, V. S. - Popova, N. K.
Journal: J Neurosci Methods

The forced swim test (FST) is a commonly used procedure of preclinical screening of drugs for the antidepressant activity. It has high predictive validity for a large group of antidepressant drugs blocking serotonin and noradrenaline reuptakes and improvement of immobility time evaluation in the FST is an important problem of preclinical psychopharmacology. Here a new automated version of the FST was developed. This version includes 4 inventions: (1) transmitted lighting instead of reflected lighting, (2) mouse silhouette tracking, (3) automated choice of immobility threshold and (4) the permutation test of drug's effect. Experiment was carried out on adult males of C57BL/6J and BALB/cJ mouse strains. The mice were treated with tricyclic antidepressant imipramine (15 and 30 mg/kg, i.p.). Mouse was placed in water tank, its movements were recorded by a rater and the silhouette alterations were automatically tracked. The sequence of silhouette alterations was scanned for immobility bouts with a threshold algorithm. Threshold was gradually altered and the value which maximized the difference between control and treated groups was chosen. The immobility values obtained with the procedure were compared with the permutation test. The data obtained with this procedure did not differ from those obtained by the rater. Imipramine dose dependently attenuated immobility time in C57BL/6 mice without any effect on BALB/c mice. The new procedure has been implemented in the EthoStudio software. It provides an objective automated evaluation of immobility time in the FST.

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Analyzing spatio-temporal patterns of genuine cross-correlations.

Publication Date: 2010 Aug 15 PMID: 20566351
Authors: Rummel, C. - Muller, M. - Baier, G. - Amor, F. - Schindler, K.
Journal: J Neurosci Methods

In multivariate time series analysis, the equal-time cross-correlation is a classic and computationally efficient measure for quantifying linear interrelations between data channels. When the cross-correlation coefficient is estimated using a finite amount of data points, its non-random part may be strongly contaminated by a sizable random contribution, such that no reliable conclusion can be drawn about genuine mutual interdependencies. The random correlations are determined by the signals' frequency content and the amount of data points used. Here, we introduce adjusted correlation matrices that can be employed to disentangle random from non-random contributions to each matrix element independently of the signal frequencies. Extending our previous work these matrices allow analyzing spatial patterns of genuine cross-correlation in multivariate data regardless of confounding influences. The performance is illustrated by example of model systems with known interdependence patterns. Finally, we apply the methods to electroencephalographic (EEG) data with epileptic seizure activity.

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In vivo visualization and functional characterization of primary somatic neurons.

Publication Date: 2010 Aug 15 PMID: 20558205
Authors: Ma, C. - Donnelly, D. F. - LaMotte, R. H.
Journal: J Neurosci Methods

In vivo electrophysiological recordings from cell bodies of primary sensory neurons are used to determine sensory function but are commonly performed blindly and without access to voltage- (patch-clamp) electrophysiology or optical imaging. We present a procedure to visualize and patch-clamp the neuronal cell body in the dorsal root ganglion, in vivo, manipulate its chemical environment, determine its receptive field properties, and remove it either to obtain subsequent molecular analyses or to gain access to deeper lying cells. This method allows the association of the peripheral transduction capacities of a sensory neuron with the biophysical and chemical characteristics of its cell body.

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A multiplexed quantitative proteomics approach for investigating protein expression in the developing central nervous system.

Publication Date: 2010 Aug 15 PMID: 20558204
Authors: Orme, R. P. - Gates, M. A. - Fricker-Gates, R. A.
Journal: J Neurosci Methods

Cell transplantation using stem cell-derived neurons is commonly viewed as a candidate therapy for neurodegenerative diseases. However, methods for differentiating stem cells into homogenous populations of neurons suitable for transplant remain elusive. This suggests that there are as yet unknown signalling factors working in vivo to specify neuronal cell fate during development. These factors could be manipulated to better differentiate stem cells into neural populations useful for therapeutic transplantation. Here a quantitative proteomics approach is described for investigating cell signalling in the developing central nervous system (CNS), using the embryonic ventral mesencephalon as a model. Briefly, total protein was extracted from embryonic ventral midbrain tissue before, during and after the birth of dopaminergic neurons, and digested using trypsin. Two-dimensional liquid chromatography, coupled with tandem mass spectrometry, was then used to identify proteins from the tryptic peptides. Isobaric tagging for relative and absolute quantification (iTRAQ) reagents were used to label the tryptic peptides and facilitate relative quantitative analysis. The success of the experiment was confirmed by the identification of proteins known to be expressed in the developing ventral midbrain, as well as by Western blotting, and immunolabelling of embryonic tissue sections. This method of protein discovery improves upon previous attempts to identify novel signalling factors through microarray analysis. Importantly, the methods described here could be applied to virtually any aspect of development.

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Revealing voxel correlation cliques by functional holography analysis of fMRI.

Publication Date: 2010 Aug 15 PMID: 20558203
Authors: Jacob, Y. - Rapson, A. - Kafri, M. - Baruchi, I. - Hendler, T. - Jacob, E. B.
Journal: J Neurosci Methods

Data acquired by functional brain imaging are of a multivariate and complex nature. Selecting relevant topographically specific information for system-level analysis is a highly non-trivial task. This challenge has traditionally been addressed by hypothesis-driven approaches. Recently, data-driven methods making no a priori assumptions about the signal were developed. Here, we present a hybrid approach, selecting data-driven voxels in paradigm-driven measurements in order to identify functional connectivity motifs in the voxel correlations. Our tool is the functional holography (FH) method, originally developed for analyzing electrophysiological recordings and based on analyzing the voxel-voxel correlation matrices. The algorithm selects the relevant voxels using a dendrogram clustering method combined with a unique standard deviation (STD) filter, identifying the voxels with high STD correlations. Functional connectivity motifs are revealed through a dimension-reduction procedure by principal component analysis (PCA) allowing for a reduced three-dimensional holographic presentation space. Information loss due to PCA is retrieved by connecting voxels in the reduced space with lines that are color-coded according to the correlations. Our results show that the FH analysis performed for a single trial reveals interesting motifs, even in a simple motor task: unilateral hand movements yielded two clusters, one in the contralateral M1 region showing neuronal activation and one in the ipsilateral homologues region showing deactivation. Thus, according to a single trial level analysis, of 12-time points alone, we can determine which hand the subject moved. Moreover, using cluster quantification based on eigenvalue entropy calculation, we obtained good separation between right- and left-handed subjects.

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Recording and analysis of electrically evoked compound action potentials (ECAPs) with MED-EL cochlear implants and different artifact reduction strategies in Matlab.

Publication Date: 2010 Aug 15 PMID: 20558202
Authors: Bahmer, A. - Peter, O. - Baumann, U.
Journal: J Neurosci Methods

Electrically evoked compound action potentials (ECAPs) are used in auditory research to evaluate the response of the auditory nerve to electrical stimulation. Animal preparations are typically used for the recording. With the introduction of a new generation of cochlear implants, however it is possible to record the response of the auditory nerve to electrical stimulation in humans as well, which is used in the clinic to test whether the implant works properly and whether the auditory nerve is responsive. Currently, ECAPs are used to estimate thresholds for speech processor programs. In addition, ECAPs recordings allow new research to be addressed, e.g., to evaluate enhanced electrical stimulation patterns. Research platforms are required to test user-defined stimuli and algorithms for the ECAPs analysis. Clinical fitting software that records ECAPs is not flexible enough for this purpose. To enable a larger group of scientists to pursue research in this field, we introduce a flexible setup that allows to change stimulation and recording parameters. ECAP recording and analysis software was developed in Matlab (The Mathworks, Inc.) for standard PC, using a National instruments (PCI-6533, National Instruments, Austin, TX) card and a Research Interface Box 2 (RIB2, Department of Ion Physics and Applied Physics at the University of Innsbruck, Innsbruck, Austria) for MED-EL cochlear implants. ECAP recordings of a human subject with three different artifact reduction methods (alternating, Miller modified masker-probe, triphasic pulses) are presented and compared.

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Modeling upper eyelid kinematics during spontaneous and reflex blinks.

Publication Date: 2010 Aug 15 PMID: 20547184
Authors: Malbouisson, J. M. - Messias, A. - Garcia, D. M. - Cechetti Sde, P. - Barbosa, J. C. - Cruz, A. A.
Journal: J Neurosci Methods

To test a mathematical model for measuring blinking kinematics. Spontaneous and reflex blinks of 23 healthy subjects were recorded with two different temporal resolutions. A magnetic search coil was used to record 77 blinks sampled at 200 Hz and 2 kHz in 13 subjects. A video system with low temporal resolution (30 Hz) was employed to register 60 blinks of 10 other subjects. The experimental data points were fitted with a model that assumes that the upper eyelid movement can be divided into two parts: an impulsive accelerated motion followed by a damped harmonic oscillation. All spontaneous and reflex blinks, including those recorded with low resolution, were well fitted by the model with a median coefficient of determination of 0.990. No significant difference was observed when the parameters of the blinks were estimated with the under-damped or critically damped solutions of the harmonic oscillator. On the other hand, the over-damped solution was not applicable to fit any movement. There was good agreement between the model and numerical estimation of the amplitude but not of maximum velocity. Spontaneous and reflex blinks can be mathematically described as consisting of two different phases. The down-phase is mainly an accelerated movement followed by a short time that represents the initial part of the damped harmonic oscillation. The latter is entirely responsible for the up-phase of the movement. Depending on the instantaneous characteristics of each movement, the under-damped or critically damped oscillation is better suited to describe the second phase of the blink.

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Data-driven approach to the estimation of connectivity and time delays in the coupling of interacting neuronal subsystems.

Publication Date: 2010 Aug 15 PMID: 20542060
Authors: Silchenko, A. N. - Adamchic, I. - Pawelczyk, N. - Hauptmann, C. - Maarouf, M. - Sturm, V. - Tass, P. A.
Journal: J Neurosci Methods

One of the challenges in neuroscience is the detection of directionality between signals reflecting neural activity. To reveal the directionality of coupling and time delays between interacting multi-scale signals, we use a combination of a data-driven technique called empirical mode decomposition (EMD) and partial directed coherence (PDC) together with the instantaneous causality test (ICT). EMD is used to separate multiple processes associated with different frequency bands, while PDC and ICT allow to explore directionality and characteristic time delays, respectively. We computationally validate our approach for the cases of both stochastic and chaotic oscillatory systems with different types of coupling. Moreover, we apply our approach to the analysis of the connectivity in different frequency bands between local field potentials (LFPs) bilaterally recorded from the left and right of subthalamic nucleus (STN) in patients with Parkinson's disease (PD). We reveal a bidirectional coupling between the left and right STN in the beta-band (10-30 Hz) for an akinetic PD patient and in the tremor band (3-5 Hz) for a tremor-dominant PD patient. We detect a short time delay, most probably reflecting the inter-hemispheric transmission time. Additionally, in both patients we observe a long time delay of approximately a mean period of the beta-band activity in the akinetic PD patient or the tremor band activity in the tremor-dominant PD patient. These long delays may emerge in subcortico-thalamic loops or longer pathways, comprising reflex loops, respectively. We show that the replacement of EMD by conventional bandpass filtering complicates the detection of directionality and leads to a spurious detection of time delays.

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Generalized framework for stimulus artifact removal.

Publication Date: 2010 Aug 15 PMID: 20542059
Authors: Erez, Y. - Tischler, H. - Moran, A. - Bar-Gad, I.
Journal: J Neurosci Methods

Stimulation is extensively used in neuroscience research in diverse fields ranging from cognitive to clinical. Studying the effect of electrical and magnetic stimulation on neuronal activity is complicated by large stimulation-derived artifacts on the recording electrodes, which mask the spiking activity. Multiple studies have suggested a variety of solutions for the removal of artifacts and were typically directed at specific stimulation setups. In this study we introduce a generalized framework for stimulus artifacts removal, the Stimulus Artifact Removal Graphical Environment (SARGE). The framework provides an encapsulated environment for a multi-stage removal process, starting from the stimulus pulse detection, through estimation of the artifacts and their removal, and finally to signal reconstruction and the assessment of removal quality. The framework provides the user with subjective graphical and objective quantitative tools for assessing the resulting signal, and the ability to adjust the process to optimize the results. This extendable publicly available framework supports different types of stimulation, stimulation patterns and shapes, and a variety of artifact estimation methods. We exemplify the removal of artifacts generated by electrical micro- and macro-stimulation and magnetic stimulation and different stimulation protocols. The use of different estimation methods, such as averaging and function fitting is demonstrated, and the differences between them are discussed. Finally, the quality of removal is assessed and validated using quantitative measures and combined experimental-simulation studies. The framework marks a shift from "algorithm" and "data" centric approach to a "workflow" centric approach, thus introducing an innovative concept to the artifact removal process.

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Optimization of a cAMP response element signal pathway reporter system.

Publication Date: 2010 Aug 15 PMID: 20540964
Authors: Shan, Q. - Storm, D. R.
Journal: J Neurosci Methods

A sensitive cAMP response element (CRE) reporter system is essential for studying the cAMP/protein kinase A/cAMP response element binding protein signal pathway. Here we have tested a few CRE promoters and found one with high sensitivity to external stimuli. Using this optimal CRE promoter and the enhanced green fluorescent protein as the reporter, we have established a CRE reporter cell line. This cell line can be used to study the signal pathway by fluorescent microscope, fluorescence-activated cell analysis and luciferase assay. This cell line's sensitivity to forskolin, using the technique of fluorescence-activated cell sorting, was increased to approximately seven times that of its parental HEK 293 cell line, which is currently the most commonly used cell line in the field for the signal pathway study. Therefore, this newly created cell line is potentially useful for studying the signal pathway's modulators, which generally have weaker effect than its mediators. Our research has also established a general procedure for optimizing transcription-based reporter cell lines, which might be useful in performing the same task when studying many other transcription-based signal pathways.

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High-throughput study of synaptic transmission at the neuromuscular junction enabled by optogenetics and microfluidics.

Publication Date: 2010 Aug 15 PMID: 20538016
Authors: Stirman, J. N. - Brauner, M. - Gottschalk, A. - Lu, H.
Journal: J Neurosci Methods

Over the past several years, optogenetic techniques have become widely used to help elucidate a variety of neuroscience problems. The unique optical control of neurons within a variety of organisms provided by optogenetics allows researchers to probe neural circuits and investigate neuronal function in a highly specific and controllable fashion. Recently, optogenetic techniques have been introduced to investigate synaptic transmission in the nematode Caenorhabditis elegans. For synaptic transmission studies, although quantitative, this technique is manual and very low-throughput. As it is, it is difficult to apply this technique to genetic studies. In this paper, we enhance this new tool by combining it with microfluidics technology and computer automation. This allows us to increase the assay throughput by several orders of magnitude as compared to the current standard approach, as well as improving standardization and consistency in data gathering. We also demonstrate the ability to infuse drugs to worms during optogenetic experiments using microfluidics. Together, these technologies will enable high-throughput genetic studies such as those of synaptic function.

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