Journal of Neuroscience Methods

Multi-modal pain measurements in infants.

Publication Date: 2012 Jan 24 PMID: 22285660
Authors: Worley, A. - Fabrizi, L. - Boyd, S. - Slater, R.
Journal: J Neurosci Methods

A non-invasive integrated method was developed to measure neural and behavioural responses to peripheral sensory and noxious stimulation in human infants. The introduction of a novel event-detection interface allows synchronous recording of: (i) muscle and central nervous system activity with surface electromyography (EMG), scalp electroencephalography (EEG) and near-infrared spectroscopy (NIRS); (ii) behavioural responses with video-recording and (iii) autonomic responses (heart rate, oxygen saturation, respiratory rate and cardiovascular activity) with electrocardiography (ECG) and pulse oximetry. The system can detect noxious heel lance and touch stimuli with precision (33mus and 624mus respectively) and accuracy (523mus and 256mus) and has 100% sensitivity and specificity for both types of stimulation. Its ability to detect response latencies accurately was demonstrated by a shift in latency of the vertex potential of 20.7+/-15.7ms (n=6 infants), following touch of the heel and of the shoulder, reflecting the distance between the two sites. This integrated system has provided reliable and reproducible measurements of responses to sensory and noxious stimulation in human infants on more than 100 test occasions.

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A novel approach to study motor neurons from zebrafish embryos and larvae in culture.

Publication Date: 2012 Jan 20 PMID: 22285259
Authors: Sakowski, S. A. - Lunn, J. S. - Busta, A. S. - Palmer, M. - Dowling, J. J. - Feldman, E. L.
Journal: J Neurosci Methods

Zebrafish are becoming increasingly popular models for examining the mechanisms of and treatments for neurological diseases. The available methods and technology to examine disease processes in vivo are increasing, however, detailed observations of subcellular structures and processes are complex in whole organisms. To address this need, we developed a primary motor neuron (MN) culture technique for utilization with zebrafish neurological disease models. Our protocol enables the culturing of cells from embryos older than 24h post-fertilization, at points after MN axonal development and outgrowth begins, which enables MN axons to develop in vivo in the context of the normal endogenous cues of the model organism, while also providing the accessibility of an in vitro system. When utilized with the increasing number of genetically modified or transgenic models of neurological diseases, this approach provides a novel tool for the examination of cellular and subcellular disease mechanisms, and offers a new platform for therapeutic discoveries in zebrafish.

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The statistical analysis of partially confounded covariates important to neural spiking.

Publication Date: 2012 Jan 17 PMID: 22281297
Authors: Lepage, K. Q. - Macdonald, C. J. - Eichenbaum, H. - Eden, U. T.
Journal: J Neurosci Methods

A method is presented capable of disambiguating the relative influence of statistical covariates upon neural spiking activity. The method, an extension of the generalized linear model (GLM) methodology introduced in Truccolo et al. (2005) to analyze neural spiking data, exploits projection operations motivated by a geometry present in the Fisher information of the GLM maximum likelihood parameter estimator. By exploiting these projections, neural activity can be divided into three categories. These three categories, neural activity due solely to a set of covariates of interest, neural activity due solely to a set of uninteresting, or nuisance, covariates, and neural activity that cannot be unequivocally assigned to either set of covariates, can be associated with physical variables such as time, position, head-direction and velocity. This association allows the analysis of neural activity that can, for example, be due solely to temporal influence, irrespective of other, identified, influences. The method is applied in simulation to a rat exploring a temporally modulated place field. A portion of the analysis reported in MacDonald et al. (2011), using the methodology described herein, is reproduced. This analysis demonstrates the temporal bridging of a delay period in a sequential memory task by firing activity of cells present in the rodent hippocampus that cannot be explained by rodent position, head direction or velocity.

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Animal models of cerebral arterial gas embolism.

Publication Date: 2012 Jan 17 PMID: 22281296
Authors: Weenink, R. P. - Hollmann, M. W. - van Hulst, R. A.
Journal: J Neurosci Methods

Cerebral arterial gas embolism is a dreaded complication of diving and invasive medical procedures. Many different animal models have been used in research on cerebral arterial gas embolism. This review provides an overview of the most important characteristics of these animal models. The properties discussed are species, cerebrovascular anatomy, method of air embolization, amount of air, bubble size, outcome parameters, anesthesia, blood glucose, body temperature and blood pressure.

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A combined brain-computer interface based on P300 potentials and motion-onset visual evoked potentials.

Publication Date: 2012 Jan 16 PMID: 22269596
Authors: Jin, J. - Allison, B. Z. - Wang, X. - Neuper, C.
Journal: J Neurosci Methods

Brain-computer interfaces (BCIs) allow users to communicate via brain activity alone. Many BCIs rely on the P300 and other event-related potentials (ERPs) that are elicited when target stimuli flash. Although there have been considerable research exploring ways to improve P300 BCIs, surprisingly little work has focused on new ways to change visual stimuli to elicit more recognizable ERPs. In this paper, we introduce a "combined" BCI based on P300 potentials and motion-onset visual evoked potentials (M-VEPs) and compare it with BCIs based on each simple approach (P300 and M-VEP). Offline data suggested that performance would be best in the combined paradigm. Online tests with adaptive BCIs confirmed that our combined approach is practical in an online BCI, and yielded better performance than the other two approaches (P<0.05) without annoying or overburdening the subject. The highest mean classification accuracy (96%) and practical bit rate (26.7bit/s) were obtained from the combined condition.

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Validity and inter-rater reliability of inertial gait measurements in Parkinson's disease: A pilot study.

Publication Date: 2012 Jan 16 PMID: 22269595
Authors: Esser, P. - Dawes, H. - Collett, J. - Feltham, M. G. - Howells, K.
Journal: J Neurosci Methods

Walking models driven by centre of mass (CoM) data obtained from inertial measurement units (IMU) or optical motion capture systems (OMCS) can be used to objectively measure gait. However current models have only been validated within typical developed adults (TDA). The purpose of this study was to compare the projected CoM movement within Parkinson's disease (PD) measured by an IMU with data collected from an OMCS after which spatio-temporal gait measures were derived using an inverted pendulum model. The inter-rater reliability of spatio-temporal parameters was explored between expert researchers and clinicians using the IMU processed data. Participants walked 10m with an IMU attached over their centre of mass which was simultaneously recorded by an OMCS. Data was collected on two occasions, each by an expert researcher and clinician. Ten people with PD showed no difference (p=0.13) for vertical, translatory acceleration, velocity and relative position of the projected centre of mass between IMU and OMCS data. Furthermore no difference (p=0.18) was found for the derived step time, stride length and walking speed for people with PD. Measurements of step time (p=0.299), stride length (p=0.883) and walking speed (p=0.751) did not differ between experts and clinicians. There was good inter-rater reliability for these parameters (ICC3.1=0.979, ICC3.1=0.958 and ICC3.1=0.978, respectively). The findings are encouraging and support the use of IMUs by clinicians to measure CoM movement in people with PD.

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A novel high electrode count spike recording array using an 81,920 pixel transimpedance amplifier-based imaging chip.

Publication Date: 2012 Jan 12 PMID: 22266817
Authors: Johnson, L. J. - Cohen, E. - Ilg, D. - Klein, R. - Skeath, P. - Scribner, D. A.
Journal: J Neurosci Methods

Microelectrode recording arrays of 60-100 electrodes are commonly used to record neuronal biopotentials, and these have aided our understanding of brain function, development and pathology. However, higher density microelectrode recording arrays of larger area are needed to study neuronal function over broader brain regions such as in cerebral cortex or hippocampal slices. Here, we present a novel design of a high electrode count picocurrent imaging array (PIA), based on an 81,920 pixel Indigo ISC9809 readout integrated circuit camera chip. While originally developed for interfacing to infrared photodetector arrays, we have adapted the chip for neuron recording by bonding it to microwire glass resulting in an array with an inter-electrode pixel spacing of 30mum. In a high density electrode array, the ability to selectively record neural regions at high speed and with good signal to noise ratio are both functionally important. A critical feature of our PIA is that each pixel contains a dedicated low noise transimpedance amplifier ( approximately 0.32pA rms) which allows recording high signal to noise ratio biocurrents comparable to single electrode voltage amplifier recordings. Using selective sampling of 256 pixel subarray regions, we recorded the extracellular biocurrents of rabbit retinal ganglion cell spikes at sampling rates up to 7.2kHz. Full array local electroretinogram currents could also be recorded at frame rates up to 100Hz. A PIA with a full complement of 4 readout circuits would span 1cm and could acquire simultaneous data from selected regions of 1024 electrodes at sampling rates up to 9.3kHz.

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Calcium imaging of multiple neurons in freely behaving C. elegans.

Publication Date: 2012 Jan 11 PMID: 22260981
Authors: Zheng, M. - Cao, P. - Yang, J. - Xu, X. Z. - Feng, Z.
Journal: J Neurosci Methods

Caenorhabditis elegans is a popular model organism to study how neural circuits and genes regulate behavior. To reliably correlate circuit function with behavior, it is important to record neuronal activity in freely behaving worms. As neural circuits are composed of multiple neurons that cooperate to process information, it is highly desirable to simultaneously record the activity of multiple neurons in the circuitry. However, such a system has not been available in C. elegans. Here, we report the CARIBN II (Calcium Ratiometric Imaging of Behaving Nematodes version II) system. This system provides smoother data collection and more importantly permits simultaneous imaging of calcium transients from multiple neurons in freely behaving worms. Using this system, we imaged the activity of AVA and RIM, two key neurons in the locomotion circuitry that regulate backward movement (reversal) in locomotion behavior. We found that AVA activity increases while RIM activity decreases during the same reversal events in spontaneous locomotion, consistent with the recent report that the AVA and RIM are involved in promoting the initiation of reversals. The CARIBN II system provides a valuable tool for dissecting the neural basis of behavior in C. elegans.

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An in vitro injury model for SH-SY5Y neuroblastoma cells: Effect of strain and strain rate.

Publication Date: 2012 Jan 12 PMID: 22257521
Authors: Skotak, M. - Wang, F. - Chandra, N.
Journal: J Neurosci Methods

There is a great need to have in vitro cell injury model wherein a wide range of strain (varepsilon) and strain rate (epsilon ) can be precisely and independently applied. Such a model will enable exploration of various biomechanical loading conditions cells normally encounter during either blunt or blast impact-induced traumatic brain injuries (TBIs). In combination with a highly automated data acquisition and analysis system, this method can quickly generate a large data set of experimental results to yield identification of bio-mechanical and chemical sequelae following injury. A proper understanding of these sequelae will enable the discovery of the time window of opportunity available for pharmacological interventions. In this study we present such an injury model, a modified version of the Cultured Axonal Injury (CAI) device, and demonstrate its efficacy through viability of SH-SY5Y cells at different ranges of strain (0-140%) and strain rate (15-68s(-1)). We identified three different regimes in the stretch-induced dose-response of curves of SH-SY5Y cells, with a very sharp decline from live to dead in a narrow range of strain (30-55%). The effect of strain rate is minimal when the final strain in the cells was fixed at 50%. The model further shows that time-after-injury plays a vital role in the determination of recovery-deterioration pathways and the biological selection depends on the severity of initial injury. These data point out the initial strain level is vital to the cell fate and emphasize the need to study the various mechanisms triggered by different magnitudes of initial injuries.

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Lentiviral vectors carrying enhancer elements of Hb9 promoter drive selective transgene expression in mouse spinal cord motor neurons.

Publication Date: 2012 Jan 8 PMID: 22245491
Authors: Peviani, M. - Kurosaki, M. - Terao, M. - Lidonnici, D. - Gensano, F. - Battaglia, E. - Tortarolo, M. - Piva, R. - Bendotti, C.
Journal: J Neurosci Methods

Recombinant lentiviral vectors (rLVs) have emerged as versatile tools for gene delivery applications due to a number of favorable features, such as the possibility to maintain long-term transgene expression, the flexibility in the design of the expression cassettes and recent improvements in their biosafety profile. Since rLVs are able to infect multiple cell types including post-mitotic cells such as neurons and skeletal muscle cells, several studies have been exploring their application for the study and cure of neurodegenerative diseases. In particular, the introduction of rLVs carrying cell-type specific promoters could restrict the transgene expression either to neuronal or glial cells, thus helping to better dissect in vivo the role played by these cell populations in several neurodegenerative processes. In this study we developed rLVs carrying motor neuron specific regulatory sequences derived from the promoter of homeobox gene Hb9, and demonstrated that these constructs can represent a suitable platform for selective gene-targeting of murine spinal cord motor neurons, in vivo. This tool could be instrumental in the dissection of the molecular mechanisms involved in the selective degeneration of motor neurons occurring in Motor Neuron Diseases.

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A rule-based automatic sleep staging method.

Publication Date: 2012 Jan 9 PMID: 22245090
Authors: Liang, S. F. - Kuo, C. E. - Hu, Y. H. - Cheng, Y. S.
Journal: J Neurosci Methods

In this paper, a rule-based automatic sleep staging method was proposed. Twelve features including temporal and spectrum analyses of the EEG, EOG, and EMG signals were utilized. Normalization was applied to each feature to eliminating individual differences. A hierarchical decision tree with fourteen rules was constructed for sleep stage classification. Finally, a smoothing process considering the temporal contextual information was applied for the continuity. The overall agreement and kappa coefficient of the proposed method applied to the all night polysomnography (PSG) of seventeen healthy subjects compared with the manual scorings by R&K rules can reach 86.68% and 0.79, respectively. This method can integrate with portable PSG system for sleep evaluation at-home in the near future.

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Feasibility of approaches combining sensor and source features in brain-computer interface.

Publication Date: 2012 Feb 15 PMID: 22108142
Authors: Ahn, M. - Hong, J. H. - Jun, S. C.
Journal: J Neurosci Methods

Brain-computer interface (BCI) provides a new channel for communication between brain and computers through brain signals. Cost-effective EEG provides good temporal resolution, but its spatial resolution is poor and sensor information is blurred by inherent noise. To overcome these issues, spatial filtering and feature extraction techniques have been developed. Source imaging, transformation of sensor signals into the source space through source localizer, has gained attention as a new approach for BCI. It has been reported that the source imaging yields some improvement of BCI performance. However, there exists no thorough investigation on how source imaging information overlaps with, and is complementary to, sensor information. Information (visible information) from the source space may overlap as well as be exclusive to information from the sensor space is hypothesized. Therefore, we can extract more information from the sensor and source spaces if our hypothesis is true, thereby contributing to more accurate BCI systems. In this work, features from each space (sensor or source), and two strategies combining sensor and source features are assessed. The information distribution among the sensor, source, and combined spaces is discussed through a Venn diagram for 18 motor imagery datasets. Additional 5 motor imagery datasets from the BCI Competition III site were examined. The results showed that the addition of source information yielded about 3.8% classification improvement for 18 motor imagery datasets and showed an average accuracy of 75.56% for BCI Competition data. Our proposed approach is promising, and improved performance may be possible with better head model.

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Extraocular muscle motor units characterized by spike-triggered averaging in alert monkey.

Publication Date: 2012 Feb 15 PMID: 22108141
Authors: Gamlin, P. D. - Miller, J. M.
Journal: J Neurosci Methods

Single-unit recording in macaque monkeys has been widely used to study extraocular motoneuron behavior during eye movements. However, primate extraocular motor units have only been studied using electrical stimulation in anesthetized animals. To study motor units in alert, behaving macaques, we combined chronic muscle force transducer (MFT) and single-unit extracellular motoneuron recordings. During steady fixation with low motoneuron firing rates, we used motoneuron spike-triggered averaging of MFT signals (STA-MFT) to extract individual motor unit twitches, thereby characterizing each motor unit in terms of twitch force and dynamics. It is then possible, as in conventional studies, to determine motoneuron activity during eye movements, but now with knowledge of underlying motor unit characteristics. We demonstrate the STA-MFT technique for medial rectus motor units. Recordings from 33 medial rectus motoneurons in three animals identified 20 motor units, which had peak twitch tensions of 0.5-5.25mg, initial twitch delays averaging 2.4ms, and time to peak contraction averaging 9.3ms. These twitch tensions are consistent with those reported in unanesthetized rabbits, and with estimates of the total number of medial rectus motoneurons and twitch tension generated by whole-nerve stimulation in monkey, but are substantially lower than those reported for lateral rectus motor units in anesthetized squirrel monkey. Motor units were recruited in order of twitch tension magnitude with stronger motor units reaching threshold further in the muscle's ON-direction, showing that, as in other skeletal muscles, medial rectus motor units are recruited according to the "size principle".

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Automated quantification of synapses by fluorescence microscopy.

Publication Date: 2012 Feb 15 PMID: 22108140
Authors: Schatzle, P. - Wuttke, R. - Ziegler, U. - Sonderegger, P.
Journal: J Neurosci Methods

The quantification of synapses in neuronal cultures is essential in studies of the molecular mechanisms underlying synaptogenesis and synaptic plasticity. Conventional counting of synapses based on morphological or immunocytochemical criteria is extremely work-intensive. We developed a fully automated method which quantifies synaptic elements and complete synapses based on immunocytochemistry. Pre- and postsynaptic elements are detected by their corresponding fluorescence signals and their proximity to dendrites. Synapses are defined as the combination of a pre- and postsynaptic element within a given distance. The analysis is performed in three dimensions and all parameters required for quantification can be easily adjusted by a graphical user interface. The integrated batch processing enables the analysis of large datasets without any further user interaction and is therefore efficient and timesaving. The potential of this method was demonstrated by an extensive quantification of synapses in neuronal cultures from DIV 7 to DIV 21. The method can be applied to all datasets containing a pre- and postsynaptic labeling plus a dendritic or cell surface marker.

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A review of multivariate methods for multimodal fusion of brain imaging data.

Publication Date: 2012 Feb 15 PMID: 22108139
Authors: Sui, J. - Adali, T. - Yu, Q. - Chen, J. - Calhoun, V. D.
Journal: J Neurosci Methods

The development of various neuroimaging techniques is rapidly improving the measurements of brain function/structure. However, despite improvements in individual modalities, it is becoming increasingly clear that the most effective research approaches will utilize multi-modal fusion, which takes advantage of the fact that each modality provides a limited view of the brain. The goal of multi-modal fusion is to capitalize on the strength of each modality in a joint analysis, rather than a separate analysis of each. This is a more complicated endeavor that must be approached more carefully and efficient methods should be developed to draw generalized and valid conclusions from high dimensional data with a limited number of subjects. Numerous research efforts have been reported in the field based on various statistical approaches, e.g. independent component analysis (ICA), canonical correlation analysis (CCA) and partial least squares (PLS). In this review paper, we survey a number of multivariate methods appearing in previous multimodal fusion reports, mostly fMRI with other modality, which were performed with or without prior information. A table for comparing optimization assumptions, purpose of the analysis, the need of priors, dimension reduction strategies and input data types is provided, which may serve as a valuable reference that helps readers understand the trade-offs of the 7 methods comprehensively. Finally, we evaluate 3 representative methods via simulation and give some suggestions on how to select an appropriate method based on a given research.

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Pitfalls using tyramide signal amplification (TSA) in the mouse gastrointestinal tract: Endogenous streptavidin-binding sites lead to false positive staining.

Publication Date: 2012 Feb 15 PMID: 22101195
Authors: Horling, L. - Neuhuber, W. L. - Raab, M.
Journal: J Neurosci Methods

Highly sensitive immunohistochemical detection systems such as tyramide signal amplification (TSA) are widely used, since they allow using two primary antibodies raised in the same species. Most of them are based on the streptavidin-biotin-peroxidase system and include streptavidin-coupled secondary antibodies. Using TSA in cryostat-sectioned tissues of mouse esophagus, we were puzzled by negative controls with unexpected staining mostly in the ganglionic areas. This prompted us to search for the causing agent and to include also other parts of the mouse gastrointestinal tract for comparison. Streptavidin-coupled antibodies bound to endogenous binding sites yet to be characterized, which are present throughout the mouse intestines. Staining was mainly localized around neuronal cell bodies of enteric ganglia. Thus, caution is warranted when applying streptavidin-coupled antibodies in the mouse gastrointestinal tract. The use of endogenous biotin-blocking kits combined with a prolonged post-fixation time could significantly reduce unintentional staining.

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A method for generating high-yield enriched neuronal cultures from P19 embryonal carcinoma cells.

Publication Date: 2012 Feb 15 PMID: 22101194
Authors: Monzo, H. J. - Park, T. I. - Montgomery, J. M. - Faull, R. L. - Dragunow, M. - Curtis, M. A.
Journal: J Neurosci Methods

P19 embryonal carcinoma (EC) cells are an invaluable tool for approximating the mechanisms that govern neuronal differentiation but with an enormous degree of simplification and have primarily been used to model the early stages of neurogenesis. However, they are often cultured under conditions that promote unrestricted non-neuronal growth that compromises neuronal viability. In this study we report an improved method to differentiate P19 EC cells that gives rise to high yields of functionally and morphologically mature neurons while significantly reducing the over-growth of non-neuronal cells in the cultures. In this protocol, P19 EC cells are induced in Minimum Essential Medium alpha supplemented with all-trans retinoic acid (RA) and 2.5% serum, and cultured as a monolayer. After RA-induction, cells are cultured on Matrigel coated-plates using defined media comprised of Neurobasal-A medium temporally supplemented with N2 and then B-27 for the remaining culture period. By treating the culture with Cytosine beta-d-arabinofuranoside and 2'-Deoxycytidine for five days, the cultures are reliably promoted toward the neuronal differentiation vs non-neuronal differentiation, this accounting for a progressive neuronal enrichment of the cultures reaching 56% after 20 days of culture. P19-derived neural progenitor cells progressively expressed neuronal markers such as NeuN, Calretinin, Calbindin and Synapsin I in close resemblance to that occurring in vivo in the central nervous system (CNS). Furthermore, RA-induced P19 EC cells progressively acquired functional neuronal traits and after approximately 3 weeks in culture revealed mature neurophysiological properties, characteristics of CNS neurons. This protocol allows for a more specific assessment of the neuronal differentiation processes in vitro.

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In situ prolyl oligopeptidase activity assay in neural cell cultures.

Publication Date: 2012 Feb 15 PMID: 22101193
Authors: Klimaviciusa, L. - Jain, R. K. - Jaako, K. - Van Elzen, R. - Gerard, M. - van Der Veken, P. - Lambeir, A. M. - Zharkovsky, A.
Journal: J Neurosci Methods

Prolyl oligopeptidase (PREP, E.C.3.4.21.26) is a cytosolic serine protease that hydrolyzes small (<3kDa), proline-containing peptides on the carboxyl terminal side of proline residues, and is widely distributed in the brain. High PREP activity, due to aging or neurodegenerative disease, has been hypothesised to lead to an increased breakdown of neuropeptides, resulting in a decline of cognitive functions and an acceleration of neurodegeneration. Recent data have suggested that PREP involvement in neurodegeneration cannot be explained by its extracellular space proteolytic activity alone, but may involve intracellular PREP activities as well. In order to test this, appropriate methods for measuring PREP intracellular activity must first be developed. In the present study, we developed and validated an in situ PREP intracellular activity assay in primary rat cortical neurons, using nitroblue tetrazolium chloride salt (NBT) and a PREP specific substrate (S)-benzyl 2-(2-(4-hydroxynaphthalen-l-ylcarbanoyl)pyrrolidin-l-yl)-2-oxoethylcarbama te (UAMC-00682). This novel in situ PREP activity assay was further validated in neuroblastoma SH-SY5Y cells, under conditions of PREP overexpression and inhibited PREP expression. Using this assay, we demonstrated that PREP inhibitors, Z-Pro-Pro-aldehyde-dimethylacetal, Boc-Asn-Phe-Pro-aldehyde, and (S)-1-((S)-1-(4-phenylbutanoyl)-pyrrolidine-2-carbonyl)pyrrolidine-2-carbo nitrile (KYP-2047), were able to inhibit intracellular PREP activity in primary rat cortical neurons. KYP-2047 was the most potent PREP inhibitor in all assay systems tested. The validated assay enables localization and quantification of in situ PREP activity in primary rat cortical neurons and neuroblastoma SH-SY5Y cells, as well allows testing cell permeability and efficiency of novel PREP inhibitors.

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Three-dimensional alteration of microvasculature in a rat model of traumatic spinal cord injury.

Publication Date: 2012 Feb 15 PMID: 22101144
Authors: Hu, J. Z. - Wu, T. D. - Zhang, T. - Zhao, Y. F. - Pang, J. - Lu, H. B.
Journal: J Neurosci Methods

Acute spinal cord injury (SCI) always leads to severe destruction of the microvascular networks. To investigate the three-dimensional (3D) alterations of microvasculature following SCI, we utilized an established rat SCI model. Based on the hypothesis that the spinal cord would undergo reorganization and postinjury modification of the vascular networks after SCI, we reconstructed the normal and injured angioarchitecture using micro-CT images of silicone rubber microsphere-perfused specimens. Several morphometric parameters were used to study the 3D vascular alterations in the SCI rat model, including the casting-based vessel volume fraction, connectivity density, separation, thickness and thickness distribution. Our results indicated that the microvascular spatial conformations were significantly different between the normal and injured spinal cord segments. The morphometric changes showed an increase of the vessel volume fraction and separation and a decrease of vessel connectivity density during the vascular healing process after SCI. Our results may contribute to elucidation of the mechanisms of compensatory vascular reconstitution in traumatized spinal cord. The method used here has the potential to improve our understanding of changes in the spatial architecture of vascular networks after SCI compared to the conventional histomorphology techniques. In summary, we developed a new methodology to analyze neurovascular pathology based on 3D vascular network patterns and features in an experimental rat SCI model. This technique could be used as a complementary tool to investigate the efficacy and side effects of therapeutic drugs or rehabilitation regimens.

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BSMac: A MATLAB toolbox implementing a Bayesian spatial model for brain activation and connectivity.

Publication Date: 2012 Feb 15 PMID: 22101143
Authors: Zhang, L. - Agravat, S. - Derado, G. - Chen, S. - McIntosh, B. J. - Bowman, F. D.
Journal: J Neurosci Methods

We present a statistical and graphical visualization MATLAB toolbox for the analysis of functional magnetic resonance imaging (fMRI) data, called the Bayesian Spatial Model for activation and connectivity (BSMac). BSMac simultaneously performs whole-brain activation analyses at the voxel and region of interest (ROI) levels as well as task-related functional connectivity (FC) analyses using a flexible Bayesian modeling framework (Bowman et al., 2008). BSMac allows for inputting data in either Analyze or Nifti file formats. The user provides information pertaining to subgroup memberships, scanning sessions, and experimental tasks (stimuli), from which the design matrix is constructed. BSMac then performs parameter estimation based on Markov Chain Monte Carlo (MCMC) methods and generates plots for activation and FC, such as interactive 2D maps of voxel and region-level task-related changes in neural activity and animated 3D graphics of the FC results. The toolbox can be downloaded from http://www.sph.emory.edu/bios/CBIS/. We illustrate the BSMac toolbox through an application to an fMRI study of working memory in patients with schizophrenia.

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A comparison of MR imaging of a mouse model of glioma at 0.2T and 9.4T.

Publication Date: 2012 Feb 15 PMID: 22101142
Authors: Blasiak, B. - Volotovskyy, V. - Deng, C. - Foniok, T. - Tomanek, B.
Journal: J Neurosci Methods

Both 0.2T and 9.4T MRI systems were used to image a mouse model of glioma. RF coils were designed for both fields. A spin-echo, multi-echo pulse sequence was used to determine T(2) relaxation times of both brain and tumor tissues. Contrast-to-noise ratio was calculated based on the selected echo time. The results showed that 0.2T is suitable for mouse model imaging, however total scan time must be long to achieve high enough SNR. T(2) relaxation times of the tumor and brain tissues can be measured at 0.2T and are 2.1 and 1.8 times respectively longer at 0.2T than at 9.4T. Contrast to noise ratio for tumor and brain was better at high field than at the low field. We concluded that 0.2T may be used to study mouse model of glioma using spin echo pulse sequence, yet the total scan time is long (about 40min), resolution is lower ( approximately 250mumx250mum) and slice thickness (3mm) must be large enough to obtain sufficient SNR.

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NeuroQuest: A comprehensive analysis tool for extracellular neural ensemble recordings.

Publication Date: 2012 Feb 15 PMID: 22101141
Authors: Kwon, K. Y. - Eldawlatly, S. - Oweiss, K.
Journal: J Neurosci Methods

Analyzing the massive amounts of neural data collected using microelectrodes to extract biologically relevant information is a major challenge. Many scientific findings rest on the ability to overcome these challenges and to standardize experimental analysis across labs. This can be facilitated in part through comprehensive, efficient and practical software tools disseminated to the community at large. We have developed a comprehensive, MATLAB-based software package - entitled NeuroQuest - that bundles together a number of advanced neural signal processing algorithms in a user-friendly environment. Results demonstrate the efficiency and reliability of the software compared to other software packages, and versatility over a wide range of experimental conditions.

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Questioning the necessity of food- and fluid regimes: Reply to Prescott and colleagues' response.

Publication Date: 2012 Feb 15 PMID: 22100901
Authors: Westlund, K.
Journal: J Neurosci Methods

In this reply, I contest the reply of the NC3R's Working Group to my critique of their report on refinement in behavioural neuroscience research involving food or fluid control. I argue that much of their hesitation to my suggested techniques is unfounded. I question the default use of deprivation regimes, suggest alternatives and insist that proper trainer skill (seemingly an overlooked variable) is pivotal in successfully accomplishing the desired performance goals.

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Repeated assessment of orthotopic glioma pO(2) by multi-site EPR oximetry: A technique with the potential to guide therapeutic optimization by repeated measurements of oxygen.

Publication Date: 2012 Feb 15 PMID: 22079559
Authors: Khan, N. - Mupparaju, S. - Hou, H. - Williams, B. B. - Swartz, H.
Journal: J Neurosci Methods

Tumor hypoxia plays a vital role in therapeutic resistance. Consequently, measurements of tumor pO(2) could be used to optimize the outcome of oxygen-dependent therapies, such as, chemoradiation. However, the potential optimizations are restricted by the lack of methods to repeatedly and quantitatively assess tumor pO(2) during therapies, particularly in gliomas. We describe the procedures for repeated measurements of orthotopic glioma pO(2) by multi-site electron paramagnetic resonance (EPR) oximetry. This oximetry approach provides simultaneous measurements of pO(2) at more than one site in the glioma and contralateral cerebral tissue. The pO(2) of intracerebral 9L, C6, F98 and U251 tumors, as well as contralateral brain, were measured repeatedly for five consecutive days. The 9L glioma was well oxygenated with pO(2) of 27-36mm Hg, while C6, F98 and U251 glioma were hypoxic with pO(2) of 7-12mm Hg. The potential of multi-site EPR oximetry to assess temporal changes in tissue pO(2) was investigated in rats breathing 100% O(2). A significant increase in F98 tumor and contralateral brain pO(2) was observed on day 1 and day 2, however, glioma oxygenation declined on subsequent days. In conclusion, EPR oximetry provides the capability to repeatedly assess temporal changes in orthotopic glioma pO(2). This information could be used to test and optimize the methods being developed to modulate tumor hypoxia. Furthermore, EPR oximetry could be potentially used to enhance the outcome of chemoradiation by scheduling treatments at times of increase in glioma pO(2).

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Lipschitz-Killing curvature based expected Euler characteristics for p-value correction in fNIRS.

Publication Date: 2012 Feb 15 PMID: 22074819
Authors: Li, H. - Tak, S. - Ye, J. C.
Journal: J Neurosci Methods

Functional near-infrared spectroscopy (fNIRS) is a non-invasive imaging approach for measuring brain activities based on changes in the cerebral concentrations of hemoglobin. Recently, statistical analysis based on a general linear model (GLM) has become popular. Here, to impose statistical significance on the activation detected by fNIRS, family-wise error (FWE) rate control is important. However, unlike fMRI, in which measurements are densely sampled on a regular lattice and Gaussian smoothing makes the resulting random field homogeneous, the random fields from fNIRS are inhomogeneous due to the interpolation from sparsely and irregularly distributed optode locations. Thus, tube formula based correction has been proposed to address this issue. However, Sun's tube formula cannot be used for general random fields such as F-statistics. To overcome these difficulties, we employ the expected Euler characteristic approach based on Lipschitz-Killing curvature (LKC) to control the family-wise error rate. We compared this correction method with Sun's tube formula for t-statistics to confirm the existing method. Based on this comparison, we show that covariance estimation should be modified to consider channel-wise least-square residual correlation. These new results supplement the existing tool of statistical parameter mapping for fNIRS.

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Validation of an air-puff passive-avoidance paradigm for assessment of aversive learning and memory in rat models of chronic pain.

Publication Date: 2012 Feb 15 PMID: 22074818
Authors: Moriarty, O. - Roche, M. - McGuire, B. E. - Finn, D. P.
Journal: J Neurosci Methods

Chronic pain is associated with cognitive deficits. Considerable overlap in brain regions involved in pain and aversion suggests that aversive learning and memory may be affected during chronic pain. Passive-avoidance paradigms traditionally use foot-shock to induce context-conditioned avoidance and may be unsuitable for use in animal models of chronic pain, which are commonly associated with hypersensitivity of the hind-paws. The aim of the present study was to develop and validate a novel passive-avoidance paradigm in rats, employing air-puff as the aversive stimulus, and to use this paradigm to assess aversive learning and memory in rat models of chronic inflammatory and neuropathic pain. Air-puff exposure produced a significant passive-avoidance and this response was attenuated following administration of scopolamine. Nerve-ligated rats and rats injected with complete Freund's adjuvant developed allodynia and hyperalgesia. Air-puff produced a significant passive-avoidance response in both chronic pain models. However, there was no difference in the response between either model and its respective control group. Thus, air-puff can be used as an alternative to foot-shock to induce a passive-avoidance response. The data generated using this model suggest that aversive learning and memory remain intact in the rat spinal nerve ligation and complete Freund's adjuvant models of chronic neuropathic and inflammatory pain, respectively.

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A robust experimental protocol for pharmacological fMRI in rats and mice.

Publication Date: 2012 Feb 15 PMID: 22068031
Authors: Ferrari, L. - Turrini, G. - Crestan, V. - Bertani, S. - Cristofori, P. - Bifone, A. - Gozzi, A.
Journal: J Neurosci Methods

Pharmacological Magnetic Resonance Imaging (phMRI) methods have significantly expanded the stimulation repertoire available to preclinical fMRI research, by allowing to selectively probe the activity of specific brain circuitries and neurotransmitter systems. However, the application of phMRI to animal models is constrained by a number of experimental factors. Firstly, in order to prevent motion artefacts and reduce restraint-induced stress, phMRI studies are typically performed under anaesthesia. Moreover, several psychoactive drugs produce blood pressure changes and alterations in respiratory frequency that may perturb central haemodynamic readouts of brain function. Hence, the quality and outcome of phMRI studies is critically dependent on the ability to monitor and control peripheral physiological parameters (i.e. blood pressure, arterial blood gases) that could alter phMRI readouts. Here we provide a thorough methodological description of a robust protocol to measure drug-induced cerebral blood volume changes in anaesthetised rats and mice. We show that the protocol ensures stable physiological parameters and robust phMRI response to the psychostimulant drug d-amphetamine in three different rat strains. We also document the successful application of the protocol to map the central effects produced by d-amphetamine in C57Bl/6J mice, a strain commonly used as background for the generation of transgenic lines, thus paving the way to the implementation of phMRI in genetically engineered animals.

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Neurons on Parafilm: Versatile elastic substrates for neuronal cell cultures.

Publication Date: 2012 Feb 15 PMID: 22068030
Authors: Yoo, S. J. - Nam, Y.
Journal: J Neurosci Methods

A variety of materials has been applied to neuronal cell culture substrates to improve the efficiency of the culture and to provide pertinent cell growth environment. Here we report the application of Parafilm((R)) M ('Parafilm') as a novel substrate for neuronal culture and patterning. Cell culture results show that elastic Parafilm had effects on cell viability, length and number of neurites, and soma spreading. Parafilm was also an effective substrate to obtain patterned neuronal cultures using a conventional micro-contract printing (muCP) technique. Polylysine micropatterns in line or grid forms were readily transferred from PDMS stamp to bare Parafilm surfaces and spatially confined neuronal cultures were successfully maintained for over three weeks. We also demonstrate that batch-processing cell culture substrates can be easily fabricated using a piece of Parafilm. The softness, plasticity, and hydrophobicity were main features that made it attractive for Parafilm to be considered as a practical cell culture platform. The results can be extended to develop an inexpensive and practical neuronal culture substrates in tissue engineering and biochip applications.

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The human brain in 1700 pieces: Design and development of a three-dimensional, interactive and reference atlas.

Publication Date: 2012 Feb 15 PMID: 22062451
Authors: Nowinski, W. L. - Chua, B. C. - Qian, G. Y. - Nowinska, N. G.
Journal: J Neurosci Methods

As the human brain is the most complex living organ, constructing its detailed model with exploration capabilities in a form of an atlas is a challenge. Our overall goal is to construct an advanced, detailed, parcellated, labeled, accurate, interactive, three-dimensional (3D), and scalable whole human brain atlas of structure, vasculature, tracts and systems. The objectives of this work are three-fold; to present: (1) method of atlas design and development including design principles, accuracy requirements, atlas content, architecture, functionality, user interface, and customized tools; (2) creation of an atlas of structure and systems including its modeling method and validation; and (3) integration of this atlas with the cerebrovasculature and tracts created earlier. The atlas is created from multiple in vivo 3/7T scans. Its design based on "pyramidal principle" enables scalability while preserving design principles and exploits interaction paradigm "from blocks to brain". The atlas contains (1) navigator with modules for system/object/object state management, interaction, user interfacing, and rendering; and (2) brain model with cerebrum, cerebellum, brainstem, spinal cord, white matter, deep structures, systems, ventricles, arteries, veins, sinuses, and tracts. The brain model is parcellated, labeled, consistent, realistic, of high resolution, polygonal/volumetric, dissectible, extendable, and deformable. It has over 1700 3D components. The atlas has sub-voxel accuracy of 0.1mm and the smallest vessels of 80mum. Brain exploration includes dynamic scene composition, manipulation-independent 3D labeling, interaction combined with animation, meta-labeling, and quantification. This atlas is useful in education, research, and clinical applications. It can potentially be foundation for a multi-level molecular-cellular-anatomical-physiological-behavioral platform.

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Single particle tracking of acyl carrier protein (ACP)-tagged TrkA receptors in PC12nnr5 cells.

Publication Date: 2012 Feb 15 PMID: 22061422
Authors: Callegari, A. - Luin, S. - Marchetti, L. - Duci, A. - Cattaneo, A. - Beltram, F.
Journal: J Neurosci Methods

There is a wide interest in studying the membrane mobility of Nerve Growth Factor (NGF) tropomyosin receptor kinase A (TrkA) at the single molecule level, in order to elucidate its diverse signaling responses related to different receptor functions. Here we present an experimental strategy based on the acyl carrier protein (ACP) tag in order to study the dynamics of the high-affinity NGF receptor TrkA in the membrane of PC12nnr5 cells. We present a single-particle tracking (SPT) study using highly photostable semiconductor quantum dots (Qdots) conjugated to ACP-tagged TrkA receptors. We demonstrate that ACP-TrkA shows biochemical and biological properties identical to those of its unmodified counterpart and that single receptor molecules in living cells display distinct diffusive regimes and a highly heterogeneous dynamics.

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Automatic analysis of EMG during clonus.

Publication Date: 2012 Feb 15 PMID: 22057220
Authors: Mummidisetty, C. K. - Bohorquez, J. - Thomas, C. K.
Journal: J Neurosci Methods

Clonus can disrupt daily activities after spinal cord injury. Here an algorithm was developed to automatically detect contractions during clonus in 24h electromyographic (EMG) records. Filters were created by non-linearly scaling a Mother (Morlet) wavelet to envelope the EMG using different frequency bands. The envelope for the intermediate band followed the EMG best (74.8-193.9Hz). Threshold and time constraints were used to reduce the envelope peaks to one per contraction. Energy in the EMG was measured 50ms either side of each envelope (contraction) peak. Energy values at 5% and 95% maximal defined EMG start and end time, respectively. The algorithm was as good as a person at identifying contractions during clonus (p=0.946, n=31 spasms, 7 subjects with cervical spinal cord injury), and marking start and end times to determine clonus frequency (intra class correlation coefficient, alpha: 0.949), contraction intensity using root mean square EMG (alpha: 0.997) and EMG duration (alpha: 0.852). On average the algorithm was 574 times faster than manual analysis performed independently by two people (p
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Signals from intraventricular depth electrodes can control a brain-computer interface.

Publication Date: 2012 Jan 30 PMID: 22044847
Authors: Shih, J. J. - Krusienski, D. J.
Journal: J Neurosci Methods

A brain-computer interface (BCI) is a device that enables severely disabled people to communicate and interact with their environments using their brain waves. Most research investigating BCI in humans have used scalp-recorded electroencephalography (EEG). We have recently demonstrated that signals from intracranial electrocorticography (ECoG) and stereotactic depth electrodes (SDE) in the hippocampus can be used to control a BCI P300 Speller paradigm. We report a case in which stereotactic depth electrodes positioned in the ventricle were able to obtain viable signals for a BCI. Our results demonstrate that event-related potentials from intraventricular electrodes can be used to reliably control the P300 Speller BCI paradigm.

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Robust modeling based on optimized EEG bands for functional brain state inference.

Publication Date: 2012 Jan 30 PMID: 22044846
Authors: Podlipsky, I. - Ben-Simon, E. - Hendler, T. - Intrator, N.
Journal: J Neurosci Methods

The need to infer brain states in a data driven approach is crucial for BCI applications as well as for neuroscience research. In this work we present a novel classification framework based on Regularized Linear Regression classifier constructed from time-frequency decomposition of an EEG (electro-encephalography) signal. The regression is then used to derive a model of frequency distributions that identifies brain states. The process of classifier construction, preprocessing and selection of optimal regularization parameter by means of cross-validation is presented and discussed. The framework and the feature selection technique are demonstrated on EEG data recorded from 10 healthy subjects while requested to open and close their eyes every 30 s. This paradigm is well known in inducing Alpha power modulations that differ from low power (during eyes opened) to high (during eyes closed). The classifier was trained to infer eyes opened or eyes closed states and achieved higher than 90% classification accuracy. Furthermore, our findings reveal interesting patterns of relations between experimental conditions, EEG frequencies, regularization parameters and classifier choice. This viable tool enables identification of the most contributing frequency bands to any given brain state and their optimal combination in inferring this state. These features allow for much greater detail than the standard Fourier Transform power analysis, making it an essential method for both BCI proposes and neuroimaging research.

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Performance comparison of extracellular spike sorting algorithms for single-channel recordings.

Publication Date: 2012 Jan 30 PMID: 22037595
Authors: Wild, J. - Prekopcsak, Z. - Sieger, T. - Novak, D. - Jech, R.
Journal: J Neurosci Methods

Proper classification of action potentials from extracellular recordings is essential for making an accurate study of neuronal behavior. Many spike sorting algorithms have been presented in the technical literature. However, no comparative analysis has hitherto been performed. In our study, three widely-used publicly-available spike sorting algorithms (WaveClus, KlustaKwik, OSort) were compared with regard to their parameter settings. The algorithms were evaluated using 112 artificial signals (publicly available online) with 2-9 different neurons and varying noise levels between 0.00 and 0.60. An optimization technique based on Adjusted Mutual Information was employed to find near-optimal parameter settings for a given artificial signal and algorithm. All three algorithms performed significantly better (p<0.01) with optimized parameters than with the default ones. WaveClus was the most accurate spike sorting algorithm, receiving the best evaluation score for 60% of all signals. OSort operated at almost five times the speed of the other algorithms. In terms of accuracy, OSort performed significantly less well (p<0.01) than WaveClus for signals with a noise level in the range 0.15-0.30. KlustaKwik achieved similar scores to WaveClus for signals with low noise level 0.00-0.15 and was worse otherwise. In conclusion, none of the three compared algorithms was optimal in general. The accuracy of the algorithms depended on proper choice of the algorithm parameters and also on specific properties of the examined signal.

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Classifying heterogeneity of spontaneous up-states: a method for revealing variations in firing probability, engaged neurons and Fano factor.

Publication Date: 2012 Jan 30 PMID: 22037594
Authors: Gullo, F. - Maffezzoli, A. - Dossi, E. - Lecchi, M. - Wanke, E.
Journal: J Neurosci Methods

The dynamics of spontaneous and sensory-evoked up-states have been recently compared, in multi-site recordings in vivo and found to have similarities and differences. Also in vitro, this is evident because we here describe a novel computational method to classify into statistically different states the spontaneous reverberating activity recorded from long-term (12-18 days-in vitro) cultured cortical neurons (from 60-site multi-electrode arrays, MEA). State classification was performed by spike number time histograms (SNTH, or other burst features) of excitatory and inhibitory neuron clusters and revealed that in novel identified states the number of engaged neurons or up-state duration can change. To improve the characterization of each state we also computed the firing spike histograms (FSH) which revealed a new facet of the firing probability of clusters. In exemplary functional experiments we show that: (i) up to 6-7 states can be safely categorized during several hours of recordings without observing spike rate changes, (ii) they disappear after a short pharmacological stimulation being replaced with novel states active and living up to 6-8 h, (iii) antagonists in the nM range can split the activity of a homogeneous network into the chronological coexistence of 2 states, one completely different and one not significantly different from control state. In conclusion, we believe that this novel procedure better characterizes the number of functional states of a network and opens up the possibility of predicting the elementary "vocabulary" used by small networks of neurons.

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Evaluation of the performances of different P300 based brain-computer interfaces by means of the Efficiency metric.

Publication Date: 2012 Jan 30 PMID: 22027493
Authors: Quitadamo, L. R. - Abbafati, M. - Cardarilli, G. C. - Mattia, D. - Cincotti, F. - Babiloni, F. - Marciani, M. G. - Bianchi, L.
Journal: J Neurosci Methods

The aim of this paper is to show how to use the Efficiency, a brain-computer interface (BCI) performance indicator, to evaluate the performances of a wide range of BCI systems. Unlike the most used metrics in the BCI research field, the Efficiency takes into account the penalties and the strategies to recover errors and this makes it a reliable instrument to describe the behavior of real BCIs. The Efficiency is compared with the accuracy and the information transfer rate, both in the Wolpaw and Nykopp definitions. The comparison covers four widely used classifiers and different stimulation sequences. Results show that the Efficiency is able to predict if the communication will not be possible, because the time spent to correct mistakes is longer than the time needed to generate a correct selection, and therefore it provides a much more realistic evaluation of a system. It can also be easily adapted to evaluate different applications, so it reveals a more general and versatile indicator for BCI systems.

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Use of vivo-morpholinos for control of protein expression in the adult rat brain.

Publication Date: 2012 Jan 30 PMID: 22027492
Authors: Reissner, K. J. - Sartor, G. C. - Vazey, E. M. - Dunn, T. E. - Aston-Jones, G. - Kalivas, P. W.
Journal: J Neurosci Methods

Vivo-morpholinos are commercially available morpholino oligomers with a terminal octa-guanidinium dendrimer for enhanced cell-permeability. Existing evidence from systemically delivered vivo-morpholinos indicate that genetic suppression can last from days to weeks without evidence of cellular toxicity. However, intravenously delivered vivo-morpholinos are ineffective at protein suppression in the brain, and no evidence is available regarding whether intracranially delivered vivo-morpholinos effectively reduce target protein levels, or do so without inducing neurotoxicity. Here we report examples in which in vivo microinjection of antisense vivo-morpholinos directed against three different targets (xCT, GLT1, orexin) in two different brain regions resulted in significant suppression of protein expression without neurotoxicity. Expression was significantly suppressed at six to seven days post-administration, but returned to baseline levels within fourteen days. These results indicate that direct intracranial administration of vivo-morpholinos provides an effective means by which to suppress protein expression in the brain for one to two weeks.

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An implantable triple-function device for local drug delivery, cerebrospinal fluid removal and EEG recording in the cranial subdural/subarachnoid space of primates.

Publication Date: 2012 Jan 30 PMID: 22027491
Authors: Ludvig, N. - Medveczky, G. - Rizzolo, R. - Tang, H. M. - Baptiste, S. L. - Doyle, W. K. - Devinsky, O. - Carlson, C. - French, J. A. - Kral, J. G. - Charchaflieh, J. - Kuzniecky, R. I.
Journal: J Neurosci Methods

Transmeningeal pharmacotherapy for cerebral cortical disorders requires drug delivery through the subdural/subarachnoid space, ideally with a feedback controlled mechanism. We have developed a device suitable for this function. The first novel component of the apparatus is a silicone rubber strip equipped with (a) fluid-exchange ports for both drug delivery and local cerebrospinal fluid (CSF) removal, and (b) EEG recording electrode contacts. This strip can be positioned between the dura and pia maters. The second novel component is an implantable dual minipump that directs fluid movement to and from the silicone strip and is accessible for refilling and emptying the drug and CSF reservoirs, respectively. This minipump is regulated by a battery-powered microcontroller integrating a bi-directional radiofrequency (RF) communication module. The entire apparatus was implanted in 5 macaque monkeys, with the subdural strip positioned over the frontal cortex and the minipump assembly secured to the cranium under a protective cap. The system was successfully tested for up to 8 months for (1) transmeningeal drug delivery using acetylcholine (ACh) and muscimol as test compounds, (2) RF-transmission of neocortical EEG data to assess the efficacy of drug delivery, and (3) local CSF removal for subsequent diagnostic analyses. The device can be used for (a) monitoring neocortical electrophysiology and neurochemistry in freely behaving nonhuman primates for more than 6 months, (b) determining the neurobiological impact of subdural/subarachnoid drug delivery interfaces, (c) obtaining novel neuropharmacological data on the effects of central nervous system (CNS) drugs, and (d) performing translational studies to develop subdural pharmacotherapy devices.

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A transgenic rat expressing green fluorescent protein (GFP) in peripheral nerves provides a new hindlimb model for the study of nerve injury and regeneration.

Publication Date: 2012 Feb 15 PMID: 22027490
Authors: Moore, A. M. - Borschel, G. H. - Santosa, K. A. - Flagg, E. R. - Tong, A. Y. - Kasukurthi, R. - Newton, P. - Yan, Y. - Hunter, D. A. - Johnson, P. J. - Mackinnon, S. E.
Journal: J Neurosci Methods

BACKGROUND: In order to evaluate nerve regeneration in clinically relevant hindlimb surgical paradigms not feasible in fluorescent mice models, we developed a rat that expresses green fluorescent protein (GFP) in neural tissue. METHODS: Transgenic Sprague-Dawley rat lines were created using pronuclear injection of a transgene expressing GFP under the control of the thy1 gene. Nerves were imaged under fluorescence microscopy and muscles were imaged with confocal microscopy to determine GFP expression following sciatic nerve crush, transection and direct suturing, and transection followed by repair with a nerve isograft from nonexpressing littermates. RESULTS: In each surgical paradigm, fluorescence microscopy demonstrated the loss and reappearance of fluorescence with regeneration of axons following injury. Nerve regeneration was confirmed with imaging of Wallerian degeneration followed by reinnervation of extensor digitorum longus (EDL) muscle motor endplates using confocal microscopy. CONCLUSION: The generation of a novel transgenic rat model expressing GFP in neural tissue allows in vivo imaging of nerve regeneration and visualization of motor endplate reinnervation. This rat provides a new model for studying peripheral nerve injury and regeneration over surgically relevant distances.

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Granger causality analysis implementation on MATLAB: a graphic user interface toolkit for fMRI data processing.

Publication Date: 2012 Jan 30 PMID: 22020117
Authors: Zang, Z. X. - Yan, C. G. - Dong, Z. Y. - Huang, J. - Zang, Y. F.
Journal: J Neurosci Methods

A lot of functional magnetic resonance imaging (fMRI) studies have indicated that Granger causality analysis (GCA) is a suitable method to reveal causal effect among brain regions. Based on another MATLAB GUI toolkit, Resting State fMRI Data Analysis Toolkit (REST), we implemented GCA on MATLAB as a graphical user interface (GUI) toolkit. This toolkit, namely REST-GCA, could output both the residual-based F and the signed-path coefficient. REST-GCA also intergrates a programme that could transform the distribution of residual-based F to approximately normal distribution and then permit parametric statistical inference at group level. Using REST-GCA, we tested the causal effect of the right frontal-insular cortex (rFIC) onto each voxel in the whole brain, and vice versa, each voxel in the whole brain on the rFIC, in a voxel-wise way in a resting-state fMRI dataset from 30 healthy college students. Using Jarque-Bera goodness-of-fit test and the Lilliefors goodness-of-fit test, we found that the transformation from F to F' and the further standardization from F' to Z score substantially improved the normality. The results of one sample t-tests on Z score showed bi-directional positive causal effect between rFIC and the dorsal anterior cingulate cortex (dACC). One sample t-tests on the signed-path coefficients showed positive causal effect from rFIC to dACC but negative from dACC to rFIC. All these results indicate that REST-GCA may be useful toolkit for caudal analysis of fMRI data.

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Synaptosomal protein synthesis in P2 and Ficoll purified fractions.

Publication Date: 2012 Jan 30 PMID: 22020116
Authors: Eyman, M. - Cefaliello, C. - Bruno, A. - Crispino, M. - Giuditta, A.
Journal: J Neurosci Methods

Cytoplasmic protein synthesis of brain synaptosomes has generally been determined in the Ficoll purified fraction which contains fewer contaminating mitochondria, microsomes and myelin fragments than the parent P2 fraction. Using a highly selective assay of this activity we have compared the total translation activity and the specific activity of the proteins synthesized by either fraction in control rats and in rats trained for a two-way active avoidance task. In control rats the specific activity remained essentially the same in both fractions but in trained rats the value of the Ficoll fraction was markedly lower (38.5%) than in the P2 fraction. Furthermore, the total translation activity of the Ficoll fraction was 30% lower than in the P2 fraction in control rats and 62% lower in trained rats. These decrements indicate that a large proportion of active synaptosomes present in the P2 fraction is not recovered in the Ficoll fraction, notably in rats undergoing plastic brain changes. We conclude that cytoplasmic protein synthesis of brain synaptosomes is better preserved in the P2 fraction.

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Effect of individual anatomy on resting motor threshold--computed electric field as a measure of cortical excitability.

Publication Date: 2012 Jan 30 PMID: 22019330
Authors: Danner, N. - Kononen, M. - Saisanen, L. - Laitinen, R. - Mervaala, E. - Julkunen, P.
Journal: J Neurosci Methods

INTRODUCTION: Transcranial magnetic stimulation (TMS) is used for assessing the excitability of cortical neurons and corticospinal pathways by determining the subject-specific motor threshold (MT). However, the MT is dependent on the TMS instrumentation and exhibits large variation. We hypothesized that between-subject differences in scalp-to-cortex distance could account for the variation in the MT. Computational electric field (EF) estimation could theoretically be applied to reduce the effect of anatomical differences, since it provides a more direct measure of corticospinal excitability. METHODS: The resting MT of the thenar musculature of 50 healthy subjects (24 male and 26 female, 22-69 years) was determined bilaterally at the primary motor cortex with MRI-navigated TMS using monophasic and biphasic stimulation. The TMS-induced maximum EF was computed at a depth of 25 mm from the scalp (EF(25 mm)) and at the individual depth of the motor cortex (EF(cortex)) determined from MRI-scans. RESULTS: All excitability parameters (MT, EF(25 mm) and EF(cortex)) correlated significantly with each other (p<0.001). EF(cortex) at MT intensity was 95+/-20 V/m for biphasic and 120+/-24 V/m for monophasic stimulation. The MT did not correlate with the anatomical scalp-to-cortex distance, whereas the coil-to-cortex distance was found to correlate positively with the MT and negatively with EF(cortex) (p<0.05). DISCUSSION: In healthy subjects, the scalp-to-cortex distance is not a significant determinant of the MT, and thus the use of EF(cortex) does not offer substantial advantages. However, it provides a purposeful and promising tool for studying non-motor cortical areas or patient groups with possible disease-related anatomical alterations.

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High throughput object-based image analysis of beta-amyloid plaques in human and transgenic mouse brain.

Publication Date: 2012 Feb 15 PMID: 22019329
Authors: Samaroo, H. D. - Opsahl, A. C. - Schreiber, J. - O'Neill, S. M. - Marconi, M. - Qian, J. - Carvajal-Gonzalez, S. - Tate, B. - Milici, A. J. - Bales, K. R. - Stephenson, D. T.
Journal: J Neurosci Methods

Advances in imaging technology have enabled automated approaches for quantitative image analysis. In this study, a high content object based image analysis method was developed for quantification of beta-amyloid (Abeta) plaques in postmortem brains of Alzheimer's disease (AD) subjects and in transgenic mice over overexpressing Abeta. Digital images acquired from immunohistochemically stained sections of the superior frontal gyrus were analyzed for Abeta plaque burden using a Definiens object-based segmentation approach. Blinded evaluation of Abeta stained sections from AD and aged matched human subjects accurately identified AD cases with one exception. Brains from transgenic mice overexpressing Abeta (PS1APP mice) were also evaluated by our Definiens object based image analysis approach. We observed an age-dependent increase in the amount of Abeta plaque load that we quantified in both the hippocampus and cortex. From the contralateral hemisphere, we measured the amount of Abeta in brain homogenates biochemically and observed a significant correlation between our biochemical measurements and those that we measured by our object based Definiens system in the hippocampus. Assessment of Abeta plaque load in PS1APP mice using a manual segmentation technique (Image-Pro Plus) confirmed the results of our object-based image analysis approach. Image acquisition and analysis of 32 stained human slides and 100 mouse slides were executed in 8h and 22h, respectively supporting the relatively high throughput features of the Definiens platform. The data show that digital imaging combined with object based image analysis is a reliable and efficient approach to quantifying Abeta plaques in human and mouse brain.

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Noninvasive in vivo assessment of muscle impairment in the mdx mouse model--a comparison of two common wire hanging methods with two different results.

Publication Date: 2012 Jan 30 PMID: 22015600
Authors: Klein, S. M. - Vykoukal, J. - Lechler, P. - Zeitler, K. - Gehmert, S. - Schreml, S. - Alt, E. - Bogdahn, U. - Prantl, L.
Journal: J Neurosci Methods

Duchenne muscular dystrophy (DMD) is an X-chromosome-linked disorder that arises from a mutation in the gene for the cytoskeletal protein dystrophin, normally expressed in the myofibres. The most widely applied animal model in DMD basic research is the C57BL/10ScSn-mdx/J mouse, commonly referred to as the "mdx mouse". The potential benefit of novel interventions in this in vivo model is often assessed by functioning tests, as the improvement of muscle impairment is the final goal of all approaches to treat DMD. In this study we compared two (TWHT) and four limb wire hanging tests (FWHT) for utility in evaluating muscle impairment in the mdx-mouse relative to its C57BL/10 wild-type counterpart. Our objective was to determine an optimal approach to perform wire hanging measurements in this model system such that latency to fall is indicative of the dystrophic phenotype that provides a quantitative measure of its presentation, and can be used to assess functional improvements that result from therapeutic intervention. Surprisingly the results of the latency times in the TWHT did not allow discrimination between the mdx population and their healthy counterparts, whereas hanging times in the FWHT enabled ready discrimination between the muscle function of mutant and wild-type animals. Furthermore, we analyzed confounding factors that explain the strengths and weaknesses of each wire hanging test configuration. The results of this study are of relevance for investigators who rely on pre clinical function tests to assess potential therapies in DMD.

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The development of an implantable catheter system for chronic or intermittent convection-enhanced delivery.

Publication Date: 2012 Jan 30 PMID: 22015599
Authors: Bienemann, A. - White, E. - Woolley, M. - Castrique, E. - Johnson, D. E. - Wyatt, M. - Murray, G. - Taylor, H. - Barua, N. - Gill, S. S.
Journal: J Neurosci Methods

Convection-enhanced delivery (CED) is a promising technique for the administration of therapeutic agents such as cytotoxics, neurotrophins and enzymes to the brain. In this study we describe the development of an implantable catheter system that is compatible with long-term intermittent CED. Catheters made from fused silica, PEEK or carbothane, and of various internal and external diameters were implanted in the striatum of rats and assessed for patency at 21 or 28 days. A high-rate of catheter blockage was observed with all fused silica and PEEK catheters. Carbothane catheters with an outer diameter of 0.6mm and an inner diameter of 0.35 mm had significantly lower rates of blockage (P
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Simultaneous LC-MS/MS analysis of the biomarkers cAMP and cGMP in plasma, CSF and brain tissue.

Publication Date: 2012 Jan 30 PMID: 22001223
Authors: Oeckl, P. - Ferger, B.
Journal: J Neurosci Methods

The cyclic nucleotides cyclic adenosine-3',5'-monophosphate (cAMP) and cyclic guanosine-3',5'-monophosphate (cGMP) are important second messengers. They are useful biomarkers to indicate biological activity of drugs such as phosphodiesterase (PDE) inhibitors which block the degradation of these nucleotides. Here, we established a fast and sensitive method for the simultaneous analysis of cAMP and cGMP by LC-MS/MS with broad applicability. The limit of detection is 50 pM. Linearity is given in a range of 0.5-500 nM for both nucleotides, with a high intra- and inter-assay precision and accuracy and an analysis time of 3.5 min. We validated the suitability of the method by pharmacological modulation of cAMP or cGMP concentrations in mice with the PDE4 inhibitor rolipram and the PDE5 inhibitor zaprinast. Rolipram significantly increased cAMP concentrations in plasma, CSF and brain tissue. Zaprinast increased cGMP concentrations in plasma but not in brain tissue, which is in accordance with its blood brain barrier permeability. In conclusion, the LC-MS/MS method described here could be a valuable analytical tool for investigating pharmacodynamic effects of PDE inhibitors and to monitor disease-related changes of cAMP and cGMP in the periphery as well as in the central nervous system.

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Mapping the human connectome at multiple scales with diffusion spectrum MRI.

Publication Date: 2012 Jan 30 PMID: 22001222
Authors: Cammoun, L. - Gigandet, X. - Meskaldji, D. - Thiran, J. P. - Sporns, O. - Do, K. Q. - Maeder, P. - Meuli, R. - Hagmann, P.
Journal: J Neurosci Methods

The global structural connectivity of the brain, the human connectome, is now accessible at millimeter scale with the use of MRI. In this paper, we describe an approach to map the connectome by constructing normalized whole-brain structural connection matrices derived from diffusion MRI tractography at 5 different scales. Using a template-based approach to match cortical landmarks of different subjects, we propose a robust method that allows (a) the selection of identical cortical regions of interest of desired size and location in different subjects with identification of the associated fiber tracts (b) straightforward construction and interpretation of anatomically organized whole-brain connection matrices and (c) statistical inter-subject comparison of brain connectivity at various scales. The fully automated post-processing steps necessary to build such matrices are detailed in this paper. Extensive validation tests are performed to assess the reproducibility of the method in a group of 5 healthy subjects and its reliability is as well considerably discussed in a group of 20 healthy subjects.

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A novel computerized system for thermal stimulation of tooth in ferrets.

Publication Date: 2012 Jan 30 PMID: 22001221
Authors: Ahn, D. K. - Monbureau, O. - Narhi, M. - Maixner, W.
Journal: J Neurosci Methods

A dual thermal and electrical stimulator was developed to examine the central pathways that transmit noxious stimuli for intact dentition. This system allows computer-controlled stimulation of the canines of ferrets with either noxious heat or electrical stimuli. A set of in vitro studies demonstrated that the application of thermal stimuli to an intact tooth can produce pulpal temperatures above 43 degrees C, which is perceived as a painful stimulus in humans. In a set of in vivo studies, it was demonstrated that heating an intact tooth at temperatures of at least 40 degrees C, excited trigeminal brainstem neurons. Only 15% of the neurons activated by electrical stimulation responded to noxious heat applied to the canine. Eight of the 23 neurons were classified as nociceptive specific neurons and responded only to noxious stimulation of their cutaneous receptive fields. Fifteen of the 23 neurons were classified as wide dynamic range neurons and responded to both noxious and non-noxious stimulation applied to their cutaneous receptive fields. This new device can accurately deliver both thermal and electrical stimuli to an intact tooth, which allows an evaluation of the central neural circuits that respond to noxious stimulation of the dentition.

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Hippocampal networks on reliable patterned substrates.

Publication Date: 2012 Jan 30 PMID: 21985763
Authors: Boehler, M. D. - Leondopulos, S. S. - Wheeler, B. C. - Brewer, G. J.
Journal: J Neurosci Methods

Toward the goal of reproducible live neuronal networks, we investigated the influence of substrate patterns on neuron compliance and network activity. We optimized process parameters of micro-contact printing for reproducible geometric patterns of 10 mum wide lines of polylysine with 4, 6, or 8 connections at a constant square array of nodes overlying the recording electrodes of a multielectrode array (MEA). We hypothesized that an increase in node connections would give the network more inputs resulting in higher neuronal outputs as network spike rates. We also chronically stimulated these networks during development and added astroglia to enhance network activity. Our results show that despite frequent localization of neuron somata over the electrodes, the number of spontaneously active electrodes was reduced 3-fold compared to random networks, independent of pattern complexity. Of the electrodes active, the overall spike rate was independent of pattern complexity, consistent with homeostasis of activity. Lower mean burst rates were seen with higher levels of pattern complexity; however, burst durations increased 1.6-fold with pattern complexity (n=6027 bursts, p<0.001). Inter-burst interval and percentage of active electrodes displaying bursts also increased with pattern complexity. The extra-burst (non-burst or isolated) spike rate increased 4-fold with pattern complexity, but this relationship was reversed with either chronic stimulation or astroglia addition. These studies suggest for the first time that patterns which limit the distribution of branches and inputs are deleterious to activity in a hippocampal network, but that higher levels of pattern complexity promote non-burst activity and favor longer lasting, but fewer bursts.

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Measuring CaMKII concentration in dendritic spines.

Publication Date: 2012 Jan 15 PMID: 21985762
Authors: Otmakhov, N. - Lisman, J.
Journal: J Neurosci Methods

Here, we present a method for measuring the concentration of endogenous protein in cellular compartments. Importantly, the method is applicable to compartments such as dendritic spines with dimensions often close to the resolution limit of optical microscopy. To our knowledge, a method with such capabilities has not yet been described. The method utilizes overexpression of the protein of interest, which is tagged with fluorescent protein. This is followed by immunostaining of both overexpressed and endogenous proteins. Expression of a volume marker is also required. We applied this method to measure the concentration of Ca/calmodulin kinase II (CaMKII) in different cellular compartments of hippocampal pyramidal neurons. It was found that the concentrations of CaMKIIalpha subunits in cell bodies, proximal dendrites, and spines on these dendrites are 71, 46, and 103 muM, respectively. Considering the 3:1 ratio of alpha to beta CaMKII subunits in the hippocampus, the concentrations of total (alpha+beta) CaMKII subunits in these compartments are 94, 61, and 138 muM, respectively.

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Establishing a new rat model of central venous sinus thrombosis and analyzing its pathophysiological and apoptotic changes.

Publication Date: 2012 Jan 15 PMID: 21985761
Authors: Yang, H. - Meng, Z. - Zhang, C. - Zhang, P. - Wang, Q.
Journal: J Neurosci Methods

This study aims to establish a new animal model of cerebral venous sinus thrombosis (CVST) and to investigate the pathophysiological and apoptotic changes of CVST. Fifty-eight adult Sprague-Dawley rats were used in the present study. They were randomized into three groups, namely control (n=6), embolism (n=26) and sham-operated (n=26) groups. In the embolism group, a self-made plastic graft was inserted in the superior sagittal sinus of each rat during the operation. Each plastic graft had a total length of 0.4 cm, its conical anterior segment had the largest diameter of 0.12 cm and its posterior segment became gradually thin and flat with a width of 0.2 cm and length of 0.1cm. In both the embolism and the sham-operation groups, the rat brains were resected at 6h, 1, 3, and 5 days post-surgery. Gross observation and water content of samples were assessed. Immunohistochemistry for caspase 3, Bcl-2 and Bax were also performed. In the embolism group, cerebral edema reached a peak level at 6h after surgery and then gradually recovered. Significant elevated positive rates of caspase 3, Bcl-2 and Bax after embolism operation were observed, reaching peaks at 1 day time point. Yet, Bcl-2/Bax decrease significantly during the follow-up, indicating apoptosis induction in embolism operation group. The new rat CVST model made by inserting a solid graft into the superior sagittal sinus is reproducible and reliable. Apoptosis plays a crucial role during the development of CVST pathophysiology.

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Mechanomyographic activity in the human lateral pterygoid muscle during mandibular movement.

Publication Date: 2012 Jan 15 PMID: 21985760
Authors: Kawakami, S. - Kodama, N. - Maeda, N. - Sakamoto, S. - Oki, K. - Yanagi, Y. - Asaumi, J. - Maeda, T. - Minagi, S.
Journal: J Neurosci Methods

The activity of the lateral pterygoid muscle has been regarded to be related to the pathological condition of the temporomandibular joint (TMJ) in the craniomandibular disorders. Because the lateral pterygoid muscle is a deep muscle, a needle electrode is necessary for EMG recordings. The purpose of this study was to establish a non-invasive method for the evaluation of muscle activity of the lateral pterygoid muscle using mechanomyogram (MMG). In three male subjects, surface electromyogram (EMG) in the left masseter muscle, left anterior and posterior belly of the temporal muscle, left anterior belly of the digastric muscle and needle EMG of the inferior head of the lateral pterygoid were recorded during mandibular movement tasks simultaneously with the MMG derived from a condenser microphone in the external ear canal. There were significant positive correlations between the needle EMG signal of the lateral pterygoid muscle and the MMG signal for the tasks of static jaw opened position of 30 mm of interincisal distance (p=0.000, R(2)=0.725), static jaw opened position of 40 mm of interincisal distance (p=0.000, R(2)=0.753), 5mm protruded mandibular position (p=0.000, R(2)=0.653), the most protruded mandibular position (p=0.000, R(2)=0803). On the contrary, for the task of maximal clenching, there was no significant correlation between the EMG signal of the lateral pterygoid muscle and the MMG signal. These results suggest that the activity of the lateral pterygoid muscle could be evaluated by the MMG signals recorded in the external ear canal, unless jaw closing major muscles show active contraction.

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Bayesian nonparametric analysis of neuronal intensity rates.

Publication Date: 2012 Jan 15 PMID: 21983110
Authors: Kottas, A. - Behseta, S. - Moorman, D. E. - Poynor, V. - Olson, C. R.
Journal: J Neurosci Methods

We propose a flexible hierarchical Bayesian nonparametric modeling approach to compare the spiking patterns of neurons recorded under multiple experimental conditions. In particular, we showcase the application of our statistical methodology using neurons recorded from the supplementary eye field region of the brains of two macaque monkeys trained to make delayed eye movements to three different types of targets. The proposed Bayesian methodology can be used to perform either a global analysis, allowing for the construction of posterior comparative intervals over the entire experimental time window, or a pointwise analysis for comparing the spiking patterns locally, in a predetermined portion of the experimental time window. By developing our nonparametric Bayesian model we are able to analyze neuronal data from three or more conditions while avoiding the computational expenses typically associated with more traditional analysis of physiological data.

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Brain oxygen tension monitoring following penetrating ballistic-like brain injury in rats.

Publication Date: 2012 Jan 15 PMID: 21983109
Authors: Murakami, Y. - Wei, G. - Yang, X. - Lu, X. C. - Leung, L. Y. - Shear, D. A. - Tortella, F. C.
Journal: J Neurosci Methods

While brain oxygen tension (PbtO(2)) monitoring is an important parameter for evaluating injury severity and therapeutic efficiency in severe traumatic brain injury (TBI) patients, many factors affect the monitoring. The goal of this study was to identify the effects of FiO(2) (fraction of inspired oxygen) on PbtO(2) in uninjured anesthetized rats and measure the changes in PbtO(2) following penetrating ballistic-like brain injury (PBBI). Continuous PbtO(2) monitoring in uninjured anesthetized rats showed that PbtO(2) response was positively correlated with FiO(2) (0.21-0.35) but PbtO(2) remained stable when FiO(2) was maintained at approximately 0.26. Importantly, although increasing FiO(2) from 0.21 to 0.35 improved P(a)O(2), it concomitantly reduced pH levels and elevated P(a)CO(2) values out of the normal range. However, when the FiO(2) was maintained between 0.26 and 0.30, the pH and P(a)O(2) levels remained within the normal or clinically acceptable range. In PBBI rats, PbtO(2) was significantly reduced by approximately 40% (16.9 +/- 1.2 mm Hg) in the peri-lesional region immediately following unilateral, frontal 10% PBBI compared to sham rats (28.6 +/- 1.7 mm Hg; mean +/- SEM, p<0.05) and the PBBI-induced reductions in PbtO(2) were sustained for at least 150 min post-PBBI. Collectively, these results demonstrate that FiO(2) affects PbtO(2) and that PBBI produces acute and sustained hypoxia in the peri-lesional region of the brain injury. This study provides important information for the management of PbtO(2) monitoring in this brain injury model and may offer insight for therapeutic strategies targeted to improve the hypoxia/ischemia state in the penetrating-type brain injury.

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Mannitol-facilitated perfusion staining with 2,3,5-triphenyltetrazolium chloride (TTC) for detection of experimental cerebral infarction and biochemical analysis.

Publication Date: 2012 Jan 15 PMID: 21982741
Authors: Sun, Y. Y. - Yang, D. - Kuan, C. Y.
Journal: J Neurosci Methods

A simple method to quantify cerebral infarction has great value for mechanistic and therapeutic studies in experimental stroke research. Immersion staining of unfixed brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) is a popular method to determine cerebral infarction in preclinical studies. However, it is often difficult to apply immersion TTC-labeling to severely injured or soft newborn brains in rodents. Here we report an in vivo TTC perfusion-labeling method based on osmotic opening of blood-brain-barrier with mannitol-pretreatment. This new method delineates cortical infarction correlated with the boundary of morphological cell injury, differentiates the induction or subcellular redistribution of apoptosis-related factors between viable and damaged areas, and easily determines the size of cerebral infarction in both adult and newborn mice. Using this method, we confirmed that administration of lipopolysaccharide 72 h before hypoxia-ischemia increases the damage in neonatal mouse brains, in contrast to its effect of protective preconditioning in adults. These results demonstrate a fast and inexpensive method that simplifies the task of quantifying cerebral infarction in small or severely injured brains and assists biochemical analysis of experimental cerebral ischemia.

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The effects of apparatus design and test procedure on learning and memory performance of C57BL/6J mice on the Barnes maze.

Publication Date: 2012 Jan 30 PMID: 21982740
Authors: O'Leary, T. P. - Brown, R. E.
Journal: J Neurosci Methods

The Barnes maze is a visuo-spatial learning and memory test originally designed for use with rats, and later adapted for use with mice. The Barnes maze design and test procedure vary across studies using mice, but the effects of variation in Barnes maze design and test procedure on learning and memory in mice have not yet been investigated. Therefore the present experiment investigates whether test procedures, such as the number of habituation trials and parameters of the probe trial (correct zone size and trial length) influence learning and memory performance on three Barnes maze designs that differed in size and the presence of a wall with intra-maze visual cues. Performance was compared across the three mazes to determine how apparatus design influences visuo-spatial cue use. The number of habituation trials and parameters of the probe trial had small effects on learning and memory performance. Apparatus design, had little effect on acquisition performance but had a significant effect on memory performance. Mice on a maze with a small diameter, external wall and intra-maze visual cues had very poor visuo-spatial memory relative to mice tested on small and large diameter mazes without a wall or intra-maze visual cues. Assessment of visuo-spatial cue use indicated that mice do not rely on visuo-spatial cues to locate the escape hole on the small-diameter maze with a wall and intra-maze visual cues, but show reliable visuo-spatial cue use on small or large diameter mazes with no wall. These results indicate that apparatus design influences search strategy use and memory performance on the Barnes maze, and that including a wall around the edge of the Barnes maze decreases visuo-spatial cue use.

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Feasibility of home-based automated Parkinson's disease motor assessment.

Publication Date: 2012 Jan 15 PMID: 21978487
Authors: Mera, T. O. - Heldman, D. A. - Espay, A. J. - Payne, M. - Giuffrida, J. P.
Journal: J Neurosci Methods

Patients with Parkinson's disease (PD) receive therapies aimed at addressing a diverse range of motor symptoms. Motor complications in the form of symptom fluctuations and dyskinesias that commonly occur with chronic PD medication use may not be effectively captured by Unified Parkinson's Disease Rating Scale (UPDRS) assessments performed in the clinic. Therefore, home monitoring may be a viable adjunct tool to provide insight into PD motor symptom response to treatment. In this pilot study, we sought to evaluate the feasibility of capturing PD motor symptoms at home using a computer-based assessment system. Ten subjects diagnosed with idiopathic PD used the system at home and ten non-PD control subjects used the system in a laboratory. The Kinesia system consists of a wireless finger-worn motion sensor and a laptop computer with software for automated tremor and bradykinesia severity score assessments. Data from control subjects were used to develop compliance algorithms for rejecting motor tasks performed incorrectly. These algorithms were then applied to data collected from the PD subjects who used the Kinesia system at home to complete motor exams 3-6 times per day over 3-6 days. Motor tasks not rejected by the compliance algorithms were further processed for symptom severity. PD subjects successfully completed motor assessments at home, with approximately 97% of all motor task data files (1222/1260) accepted. These findings suggest that objective home monitoring of PD motor fluctuations is feasible.

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Optimization of seed density in DTI tractography for structural networks.

Publication Date: 2012 Jan 15 PMID: 21978486
Authors: Cheng, H. - Wang, Y. - Sheng, J. - Sporns, O. - Kronenberger, W. G. - Mathews, V. P. - Hummer, T. A. - Saykin, A. J.
Journal: J Neurosci Methods

Diffusion tensor imaging (DTI) has been used for mapping the structural network of the human brain. The network can be constructed by choosing various brain regions as nodes and fiber tracts connecting those regions as links. The structural network generated from DTI data can be affected by noise in the scans and the choice of tractography algorithm. This study aimed to examine the effect of the number of seeds in tractography on the variance of structural networks. The variance of the network was characterized using an approach similar to the National Electrical Manufacturers Association (NEMA) standards for measurement of image noise. It was shown that the variance of the network is inversely related to the square root of seed density. Consequently, the number of seeds has a large impact on local characteristics and metrics of the network architecture. As the number of seeds increased, increased stability of structural network metrics was observed. However, more seeds can also lead to more spurious fibers and thus affect nodal degrees and edge weights, and proper thresholding may be necessary to create an appropriate weighted network. Because the variance of the network is also influenced by other imaging factors, further increase in the number of seeds has little effect in reducing the network variance. The selection of the seed number should be a balance between the network variance and computational effort.

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Knockdown of cortical transthyretin expression around implanted neural prosthetic devices using intraventricular siRNA injection in the brain.

Publication Date: 2012 Jan 30 PMID: 21978485
Authors: Smith, K. L. - Herron, B. - Dowell-Mesfin, N. - Wu, H. - Kim, S. J. - Shain, W. - Hynd, M. R.
Journal: J Neurosci Methods

Neural prosthetic devices are showing increasing clinical use for the treatment of a variety of neurological disorders. However the functions on these devices are often limited due to an inability to effectively and chronically interface with neural tissue. The insertion of devices has been shown to result in significant cellular and vascular trauma surrounding the insertion site. In particular, the up-regulation of genes involved in neuronal degeneration are believed to contribute to the loss of neuronal tissue. RNA interference is a novel technique for the development of antisense therapeutics for the post-transcriptional silencing of specific genes. In order to demonstrate the feasibility of RNA interference for gene-specific silencing in vivo, a short interfering RNA targeting transthyretin, was infused prior to unilateral device insertion. Injection of siRNA was found to significantly reduce the expression of transthyretin mRNA when expression was assessed at 1 week following device insertion. Concomitant decreases in transthyretin protein levels were also observed. These data demonstrate the feasibility of using RNA interference to modulate the initial reactive cellular responses that occur in the brain following insertion of neural prosthetic devices.

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Reduction of autofluorescence at the microelectrode-cortical tissue interface improves antibody detection.

Publication Date: 2012 Jan 15 PMID: 21978484
Authors: Potter, K. A. - Simon, J. S. - Velagapudi, B. - Capadona, J. R.
Journal: J Neurosci Methods

Immunohistochemistry (IHC) remains among the most utilized methods for detection of inflammatory events occurring at the microelectrode-cortical tissue interface. It has further become a standard protocol to quantify the intensity of this resulting fluorescent signal, normalized to "background", as a measurement of the extent of inflammatory events. Unfortunately, several sources of autofluorescence could result in variations in this user-defined "background". Notably, we found that the presence of hemosiderin-laden macrophages (HLMs) at the interface resulted in a variable source of background in both green and red fluorescent channels. The HLM-derived autofluorescence prevented the reproducible detection of presumably low-level antigens at the interface. Here we show that treatment of the native cortical tissue for no less than 10 min, with a minimum of 0.5mM copper sulfate, resulted in at least a 70% reduction in native HLM autofluorescence in both green and red fluorescent channels. In the case of highly expressed antigens, such as glial fibrillar acidic protein (GFAP), treatment of immuno-labeled tissue with copper sulfate reduced tissue background, compared to standard IHC methodology, but did not result in significant differences in the quantification of normalized signal intensity. However, treatment with copper sulfate substantially enhanced the detection efficiency of weakly expressed antigens at the device-tissue interface. This study demonstrates that the inclusion of copper sulfate incubation during IHC tissue preparation significantly reduced HLM-derived autofluorescence, and allowed for more accurate detection and quantification of faintly expressed inflammatory markers at the device-tissue interface.

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A multivariate, multitaper approach to detecting and estimating harmonic response in cortical optical imaging data.

Publication Date: 2012 Jan 15 PMID: 21970814
Authors: Sornborger, A. T. - Yokoo, T.
Journal: J Neurosci Methods

The efficiency and accuracy of cortical maps from optical imaging experiments have been improved using periodic stimulation protocols. The resulting data analysis requires the detection and estimation of periodic information in a multivariate dataset. To date, these analyses have relied on discrete Fourier transform (DFT) sinusoid estimates. Multitaper methods have become common statistical tools in the analysis of univariate time series that can give improved estimates. Here, we extend univariate multitaper harmonic analysis methods to the multivariate, imaging context. Given the hypothesis that a coherent oscillation across many pixels exists within a specified bandwidth, we investigate the problem of its detection and estimation in noisy data by constructing Hotelling's generalized T(2)-test. We then extend the investigation of this problem in two contexts, that of standard canonical variate analysis (CVA) and that of generalized indicator function analysis (GIFA) which is often more robust in extracting a signal in spatially correlated noise. We provide detailed information on the fidelities of the mean estimates found with our methods and comparison with DFT estimates. Our results indicate that GIFA provides particularly good estimates of harmonic signals in spatially correlated noise and is useful for detecting small amplitude harmonic signals in applications such as biological imaging measurements where spatially correlated noise is common. We demonstrate the power of our methods with an optical imaging dataset of the periodic response to a periodically rotating oriented drifting grating stimulus experiment in cat visual cortex.

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Plate reader-based assays for measuring cell viability, neuroprotection and calcium in primary neuronal cultures.

Publication Date: 2012 Jan 15 PMID: 21968036
Authors: Burroughs, S. L. - Duncan, R. S. - Rayudu, P. - Kandula, P. - Payne, A. J. - Clark, J. L. - Koulen, P. - Kaja, S.
Journal: J Neurosci Methods

Drug discovery and development efforts critically rely on cell-based assays for high-throughput screening. These assay systems mostly utilize immortalized cell lines, such as human embryonic kidney cells, and can provide information on cytotoxicity and cell viability, permeability and uptake of compounds as well as receptor pharmacology. While this approach has proven extremely useful for single-target pharmacology, there is an urgent need for neuropharmacological studies to screen novel drug candidates in a cellular environment resembles neurons in vivo more closely, in order to gain insight into the involvement of multiple signaling pathways. Primary cultured neuronal cells, such as cortical neurons, have long been used for basic research and low-throughput screening and assay development, and may thus be suitable candidates for the development of neuropharmacological high-throughput screening approaches. We here developed and optimized protocols for the use of primary cortical neuronal cells in high-throughput assays for neuropharmacology and neuroprotection, including calcium mobilization, cytotoxicity and viability as well as ion channel pharmacology. Our data show low inter-experimental variability and similar reproducibility as conventional cell line assays. We conclude that primary neuronal cultures provide a viable alternative to cell lines in high-throughput assay systems by providing a cellular environment more closely resembling physiological conditions in the central nervous system.

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Understanding neuronal systems in movement control using Wiener/Volterra kernels: a dominant feature analysis.

Publication Date: 2012 Jan 15 PMID: 21963576
Authors: Jing, X. - Simpson, D. M. - Allen, R. - Newland, P. L.
Journal: J Neurosci Methods

Although Volterra kernels have been extensively applied in modelling and analysis of biological systems, the relationship between the kernel characteristics and physiologically important features under study is still not revealed clearly. In this study, the link between Volterra kernels and dynamic response of neural systems which control animal movements was investigated and demonstrated using a dominant feature analysis. The new results show an effective but simplified method to use Volterra or Wiener kernels to understand and classify the neural systems which are responsible for the fundamental movements such as flexion and extension of animal limbs, and importantly demonstrate how the neuron pathways in locusts control joint activities of low and high frequency and perform fundamental joint movements such as position, velocity and acceleration. These results provide a useful insight into the nonlinear characteristics of neural systems in movement control and show a useful approach to the analysis of physiological systems using Volterra/Wiener kernels.

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A novel experimental in vivo model of cerebral immunomodulation induced by inactivated Staphylococcus epidermidis.

Publication Date: 2012 Jan 15 PMID: 21963575
Authors: Lohr, M. - Molcanyi, M. - Stenzel, W. - Seifert, H. - Tzouras, G. - Rohn, G. - Mohseni, D. - Hampl, J. A.
Journal: J Neurosci Methods

The genesis and appropriate treatment of neuroinflammation in various infectious and non-infectious disorders of the central nervous system is still a matter of debate. We introduce an alternative and simple experimental model for the investigation of the cellular inflammatory response to bacterial antigens by stereotactic intracerebral injection of heat-inactivated Staphylococcus epidermidis (HISE). HISE-injection resulted in well-circumscribed intraparenchymal deposits encompassed by an early micro- and astroglial response and a selective but sustained opening of the blood-brain barrier (BBB). After 24h, the HISE collections were densely infiltrated by granulocytes and few circumjacent macrophages that became the predominating immunocompetent cell type from day 4 on. CD8a+ lymphocytes peaked at day 4, whereas CD4+ and CD20+ lymphocytes increased gradually in number, developing a scattered infiltrate until day 17, indicating the initiation of an adaptive immune response. MHC class II presenting cells were abundantly recruited from day 1 and eventually shaped an increasingly dense accumulation within the lesion. Intracerebral HISE administration provides a controlled, highly reproducible and well defined influx of immunocompetent cells across the BBB leading to a distinct and condensed inflammatory reaction. The technique is straightforward, easily feasible and may significantly enable further investigations of the initiation, maintenance and therapeutic modulation of acute neuroinflammation.

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A new (semantic) reflexive brain-computer interface: in search for a suitable classifier.

Publication Date: 2012 Jan 15 PMID: 21963400
Authors: Furdea, A. - Ruf, C. A. - Halder, S. - De Massari, D. - Bogdan, M. - Rosenstiel, W. - Matuz, T. - Birbaumer, N.
Journal: J Neurosci Methods

The goal of the current study is to find a suitable classifier for electroencephalogram (EEG) data derived from a new learning paradigm which aims at communication in paralysis. A reflexive semantic classical (Pavlovian) conditioning paradigm is explored as an alternative to the operant learning paradigms, currently used in most brain-computer interfaces (BCIs). Comparable with a lie-detection experiment, subjects are presented with true and false statements. The EEG activity following true and false statements was classified with the aim to separate covert 'yes' from covert 'no' responses. Four classification algorithms are compared for classifying off-line data collected from a group of 14 healthy participants: (i) stepwise linear discriminant analysis (SWLDA), (ii) shrinkage linear discriminant analysis (SLDA), (iii) linear support vector machine (LIN-SVM) and (iv) radial basis function kernel support vector machine (RBF-SVM). The results indicate that all classifiers perform at chance level when separating conditioned 'yes' from conditioned 'no' responses. However, single conditioned reactions could be successfully classified on a single-trial basis (single conditioned reaction against a baseline interval). All of the four investigated classification methods achieve comparable performance, however results with RBF-SVM show the highest single-trial classification accuracy of 68.8%. The results suggest that the proposed paradigm may allow affirmative and negative (disapproving negative) communication in a BCI experiment.

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Simultaneous measurement of membrane potential changes in multiple pattern generating neurons using voltage sensitive dye imaging.

Publication Date: 2012 Jan 15 PMID: 21963367
Authors: Stadele, C. - Andras, P. - Stein, W.
Journal: J Neurosci Methods

Optical imaging using voltage-sensitive dyes (VSDs) is a promising technique for the simultaneous activity recording of many individual neurons. While such simultaneous recordings are critical for the understanding of the integral functionality of neural systems, functional interpretations on a single neuron level are difficult without knowledge of the connectivity of the underlying circuit. Central pattern generating circuits, such as the pyloric and gastric mill circuits in the stomatogastric ganglion (STG) of crustaceans, allow such investigations due to their well-known connectivities and have already contributed much to our understanding of general neuronal mechanisms. Here we present for the first time simultaneous optical recordings of the pattern generating neurons in the STG of two crustacean species using bulk loading of the VSD di-4-ANEPPS. We demonstrate the recording of firing activities and synaptic interactions of the circuit neurons as well as inter-circuit interactions in their functional context, i.e. without artificial stimulation. Neurons could be uniquely identified using simple event-triggered averaging. We tested this technique in two different species of crustaceans (lobsters and crabs), since several crustacean species are used for studying motor pattern generation. The signal-to-noise ratio of the optical signal was high enough in both species to derive phase-relationship between the network neurons, as well as action potentials and excitatory and inhibitory postsynaptic potentials. We argue that imaging of neural networks with identifiable neurons with well-known connectivity, like in the STG, is crucial for the understanding of emergence of network functionality.

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Impedance measurement for real time detection of neuronal cell death.

Publication Date: 2012 Jan 15 PMID: 21963366
Authors: Diemert, S. - Dolga, A. M. - Tobaben, S. - Grohm, J. - Pfeifer, S. - Oexler, E. - Culmsee, C.
Journal: J Neurosci Methods

Detection of neuronal cell death is a standard requirement in cell culture models of neurodegenerative diseases. Although plenty of viability assays are available for in vitro applications, most of these are endpoint measurements providing only little information on the kinetics of cell death. Here, we validated the xCELLigence system based on impedance measurement for real-time detection of cell death in a neuronal cell line of immortalized hippocampal neurons (HT-22 cells), neuronal progenitor cells (NPC) and differentiated primary cortical neurons. We found a good correlation between impedance measurements and endpoint viability assays in HT-22 cells and NPC, for detecting proliferation, cell death kinetics and also neuroprotective effects of pharmacological inhibitors of apoptosis. In primary neurons we could not detect dendritic outgrowth during differentiation of the cells. Cell death in primary neurons was detectable by the xCELLigence system, however, the changes in the cell index on the basis of impedance measurements depended to a great extent on the severity of the insult. Cell death induced by ionomycin, e.g. shows as a fast paced process involving a strong cellular disintegration, which allows for impedance-based detection. Cell death accompanied by less pronounced morphological changes like glutamate induced cell death, however, is not well accessible by this approach. In conclusion, our data show that impedance measurement is a convenient and reliable method for the detection of proliferation and kinetics of cell death in neuronal cell lines, whereas this method is less suitable for the assessment of neuronal differentiation and viability of primary neurons.

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Detailed tracking of body and leg movements of a freely walking female cricket during phonotaxis.

Publication Date: 2012 Jan 15 PMID: 21951620
Authors: Petrou, G. - Webb, B.
Journal: J Neurosci Methods

We describe a semi-automated tracking system for insect motion based on commercially available high-speed video cameras and freely available software. We use it to collect detailed three-dimensional kinematic information from female crickets performing free walking phonotaxis towards a calling song stimulus. We mark the insect's joints with small dots of paint and record the movements from underneath with a pair of cameras following the insect as it walks on the transparent floor of an arena. Tracking is done offline, utilizing a kinematic model to constrain the processing. We can obtain the positions and angles of all joints of all legs and six additional body joints, synchronised with stance-swing transitions and the sound pattern, at a 300 Hz frame rate. This data will be used in the further development of models of neural control of phonotaxis.

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High-resolution detection of (1)(3)C multiplets from the conscious mouse brain by ex vivo NMR spectroscopy.

Publication Date: 2012 Jan 15 PMID: 21946227
Authors: Marin-Valencia, I. - Good, L. B. - Ma, Q. - Jeffrey, F. M. - Malloy, C. R. - Pascual, J. M.
Journal: J Neurosci Methods

Glucose readily supplies the brain with the majority of carbon needed to sustain neurotransmitter production and utilization. The rate of brain glucose metabolism can be computed using (13)C nuclear magnetic resonance (NMR) spectroscopy by detecting changes in (13)C contents of products generated by cerebral metabolism. As previously observed, scalar coupling between adjacent (13)C carbons (multiplets) can provide additional information to (13)C contents for the computation of metabolic rates. Most NMR studies have been conducted in large animals (often under anesthesia) because the mass of the target organ is a limiting factor for NMR. Yet, despite the challengingly small size of the mouse brain, NMR studies are highly desirable because the mouse constitutes a common animal model for human neurological disorders. We have developed a method for the ex vivo resolution of NMR multiplets arising from the brain of an awake mouse after the infusion of [1,6-(13)C(2)]glucose. NMR spectra obtained by this method display favorable signal-to-noise ratios. With this infusion protocol, the (13)C multiplets of glutamate, glutamine, GABA and aspartate achieved steady state after 150 min. The method enables the accurate resolution of multiplets over time in the awake mouse brain. We anticipate that this method can be broadly applicable to compute brain fluxes in normal and transgenic mouse models of neurological disorders.

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Study of cerebral gene expression densities using Voronoi analysis.

Publication Date: 2012 Jan 15 PMID: 21946226
Authors: Miazaki, M. - Costa Lda, F.
Journal: J Neurosci Methods

As the available public cerebral gene expression image data increasingly grows, the demand for automated methods to analyze such large amount of data also increases. An important study that can be carried out on these data is related to the spatial relationship between gene expressions. Similar spatial density distribution of expression between genes may indicate they are functionally correlated, thus the identification of these similarities is useful in suggesting directions of investigation to discover gene interactions and their correlated functions. In this paper, we describe the use of a high-throughput methodology based on Voronoi diagrams to automatically analyze and search for possible local spatial density relationships between gene expression images. We tested this method using mouse brain section images from the Allen Mouse Brain Atlas public database. This methodology provided measurements able to characterize the similarity of the density distribution between gene expressions and allowed the visualization of the results through networks and Principal Component Analysis (PCA). These visualizations are useful to analyze the similarity level between gene expression patterns, as well as to compare connection patterns between region networks. Some genes were found to have the same type of function and to be near each other in the PCA visualizations. These results suggest cerebral density correlations between gene expressions that could be further explored.

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Selective, quantitative measurement of releasable synaptic zinc in human autopsy hippocampal brain tissue from Alzheimer's disease patients.

Publication Date: 2012 Jan 15 PMID: 21945000
Authors: Bjorklund, N. L. - Sadagoparamanujam, V. M. - Taglialatela, G.
Journal: J Neurosci Methods

Aberrant central nervous system zinc homeostasis has been reported in Alzheimer's disease (AD). However, there are conflicting reports describing zinc concentration either increased or decreased in the brain of AD patients. Such discrepancies may be due to differences in the brain area examined, zinc detection method, and/or tissue composition. Furthermore, detection and measurement of the releasable zinc pool in autopsy tissue is difficult and usually unreliable. Obtaining an adequate assessment of this releasable zinc pool is of particular significance in AD research in that zinc can coordinate with and stabilize toxic amyloid beta oligomers, which are believed to play a key role in AD neuropathology. In addition, zinc released into the synaptic cleft can interact with the postsynaptic neurons causing altered signaling and synaptic dysfunction, which is a well established event in AD. The method presented here combines two approaches, biochemical fractionation and atomic absorption spectrophotometry, to allow, in addition to extracellular zinc concentration, the reliable and quantitative measurement of zinc specifically localized in synaptic vesicles, which contain the majority of the neuronal releasable zinc. Using this methodology, we found that synaptic vesicle zinc concentrations were increased in AD hippocampi compared to age-matched controls and that this increase in releasable zinc matched increased concentration of zinc in the extracellular space.

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Inference of Granger causal time-dependent influences in noisy multivariate time series.

Publication Date: 2012 Jan 15 PMID: 21944999
Authors: Sommerlade, L. - Thiel, M. - Platt, B. - Plano, A. - Riedel, G. - Grebogi, C. - Timmer, J. - Schelter, B.
Journal: J Neurosci Methods

Inferring Granger-causal interactions between processes promises deeper insights into mechanisms underlying network phenomena, e.g. in the neurosciences where the level of connectivity in neural networks is of particular interest. Renormalized partial directed coherence has been introduced as a means to investigate Granger causality in such multivariate systems. A major challenge in estimating respective coherences is a reliable parameter estimation of vector autoregressive processes. We discuss two shortcomings typical in relevant applications, i.e. non-stationarity of the processes generating the time series and contamination with observational noise. To overcome both, we present a new approach by combining renormalized partial directed coherence with state space modeling. A numerical efficient way to perform both the estimation as well as the statistical inference will be presented.

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In vivo imaging of epileptic activity using 2-NBDG, a fluorescent deoxyglucose analog.

Publication Date: 2012 Jan 15 PMID: 21939688
Authors: Tsytsarev, V. - Maslov, K. I. - Yao, J. - Parameswar, A. R. - Demchenko, A. V. - Wang, L. V.
Journal: J Neurosci Methods

Accurately locating epileptic foci has great importance in advancing the treatment of epilepsy. In this study, epileptic seizures were first induced by intracortical injection of 4-aminopyridine in rats. A fluorescent deoxyglucose substitute, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), was then continuously injected via the tail vein. Brain glucose metabolism was subsequently monitored by fluorescence imaging of 2-NBDG. The initial uptake rate of 2-NBDG at the injection site of 4-aminopyridine significantly exceeded that of the control injection site, which indicated local hypermetabolism induced by seizures. Our results show that 2-NBDG can be used for localizing epileptic foci.

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Wireless multi-channel single unit recording in freely moving and vocalizing primates.

Publication Date: 2012 Jan 15 PMID: 21933683
Authors: Roy, S. - Wang, X.
Journal: J Neurosci Methods

The ability to record well-isolated action potentials from individual neurons in naturally behaving animals is crucial for understanding neural mechanisms underlying natural behaviors. Traditional neurophysiology techniques, however, require the animal to be restrained which often restricts natural behavior. An example is the common marmoset (Callithrix jacchus), a highly vocal New World primate species, used in our laboratory to study the neural correlates of vocal production and sensory feedback. When restrained by traditional neurophysiological techniques marmoset vocal behavior is severely inhibited. Tethered recording systems, while proven effective in rodents pose limitations in arboreal animals such as the marmoset that typically roam in a three-dimensional environment. To overcome these obstacles, we have developed a wireless neural recording technique that is capable of collecting single-unit data from chronically implanted multi-electrodes in freely moving marmosets. A lightweight, low power and low noise wireless transmitter (headstage) is attached to a multi-electrode array placed in the premotor cortex of the marmoset. The wireless headstage is capable of transmitting 15 channels of neural data with signal-to-noise ratio (SNR) comparable to a tethered system. To minimize radio-frequency (RF) and electro-magnetic interference (EMI), the experiments were conducted within a custom designed RF/EMI and acoustically shielded chamber. The individual electrodes of the multi-electrode array were periodically advanced to densely sample the cortical layers. We recorded single-unit data over a period of several months from the frontal cortex of two marmosets. These recordings demonstrate the feasibility of using our wireless recording method to study single neuron activity in freely roaming primates.

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Characterization of [(3)H]CFT binding to the norepinephrine transporter suggests that binding of CFT and nisoxetine is not mutually exclusive.

Publication Date: 2012 Jan 15 PMID: 21933682
Authors: Zhen, J. - Ali, S. - Dutta, A. K. - Reith, M. E.
Journal: J Neurosci Methods

The norepinephrine transporter (NET) is an important target for a wide variety of antidepressants and psychostimulants. Despite its prominence as a drug target, there is only one radioligand in use for NET competitive binding assays, [(3)H]nisoxetine. However, traditional [(3)H]nisoxetine binding protocols often give an underestimation for the affinity of certain classes of NET ligands, particularly cocaine and other tropanes. Here, we explore the feasibility of using the phenyltropane [(3)H]CFT for labeling human NET (hNET) in heterologous cell-based binding studies. Assays were optimized for time and protein content and specific, one-site binding was observed. Potencies of tested NET ligands for inhibition of [(3)H]CFT binding to whole cells (at physiological [Na(+)] and 25 degrees C) were similar to potencies observed in the [(3)H]NE uptake assay. Inhibition constants (K(i)) for binding assays were highly correlated with uptake inhibition constants for all compounds tested (R(2)=0.99, p<0.0001). Cell-free membrane preparations did not display the same pharmacological profile. Under conditions routinely used for measuring [(3)H]nisoxetine binding to membrane preparations (4 degrees C for 3h, [Na(+)] at 295 mM), the potency of nisoxetine and desipramine in inhibiting [(3)H]CFT binding became greater than that measured in a functional assay of [(3)H]NE uptake at physiological [Na(+)]. However, the opposite was true for CFT and cocaine. Interestingly, while investigating [(3)H]CFT as a potential NET radioligand, we uncovered evidence suggesting that CFT and nisoxetine are not mutually exclusive in binding to the NET. Dixon plots of the interaction between nisoxetine and CFT in inhibition of [(3)H]dopamine uptake by the NET indicate that the two compounds can simultaneously bind to the transporter.

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Algorithms for computing spike time distance and point process prototypes with application to feline neuronal responses to acoustic stimuli.

Publication Date: 2012 Jan 15 PMID: 21933681
Authors: Diez, D. M. - Schoenberg, F. P. - Woody, C. D.
Journal: J Neurosci Methods

The spike-time distance metric and the prototype of a collection of point patterns can be used to provide a metric description of repeated observations of point processes. Due to algorithmic limitations in computing spike-time distance, previous work in determining approximations of the prototype has largely been limited to single dimensional or small data sets. We develop new algorithms for each of these methods that are suitable for larger data sets. The first, an incremental matching algorithm, is a method to compute spike-time distance. The second algorithm involves the use of kernel smoothing for prototype construction. These methods readily extend to multiple dimensions and to marked point processes. We use a combination of these approaches to analyze neuronal spike data of cats in different behavioral states and across animals, and we compare our results to previous time series analyses that used averaged frequency histograms. Evidence is found for short latency responses to the acoustic CS that were sensitive to behavioral state.

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A mouse model of human repetitive mild traumatic brain injury.

Publication Date: 2012 Jan 15 PMID: 21930157
Authors: Kane, M. J. - Angoa-Perez, M. - Briggs, D. I. - Viano, D. C. - Kreipke, C. W. - Kuhn, D. M.
Journal: J Neurosci Methods

A novel method for the study of repetitive mild traumatic brain injury (rmTBI) that models the most common form of head injury in humans is presented. Existing animal models of TBI impart focal, severe damage unlike that seen in repeated and mild concussive injuries, and few are configured for repetitive application. Our model is a modification of the Marmarou weight drop method and allows repeated head impacts to lightly anesthetized mice. A key facet of this method is the delivery of an impact to the cranium of an unrestrained subject allowing rapid acceleration of the free-moving head and torso, an essential characteristic known to be important for concussive injury in humans, and a factor that is missing from existing animal models of TBI. Our method does not require scalp incision, emplacement of protective skull helmets or surgery and the procedure can be completed in 1-2 min. Mice spontaneously recover the righting reflex and show no evidence of seizures, paralysis or impaired behavior. Skull fractures and intracranial bleeding are very rare. Minor deficits in motor coordination and locomotor hyperactivity recover over time. Histological analyses reveal mild astrocytic reactivity (increased expression of GFAP) and increased phospho-tau but a lack of blood-brain-barrier disruption, edema and microglial activation. This new animal model is simple and cost-effective and will facilitate characterization of the neurobiological and behavioral consequences of rmTBI. It is also ideal for high throughput screening of potential new therapies for mild concussive injuries as experienced by athletes and military personnel.

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Increased throughput assays of locomotor dysfunction in Drosophila larvae.

Publication Date: 2012 Jan 30 PMID: 21925540
Authors: Sinadinos, C. - Cowan, C. M. - Wyttenbach, A. - Mudher, A.
Journal: J Neurosci Methods

Larval locomotion is a sensitive readout of a range of nervous system deficits in Drosophila, and has been utilised to quantify modulation of the disease phenotype in models of human disease. Single larvae are typically analysed in series using manual quantification of parameters such as contraction rate, or grouped together and studied en-masse. Here, we describe the development of tests for the analysis of several spatially isolated third instar larvae in parallel. We rapidly quantify larval turning rate and velocity during wandering behaviour in a 4 plate assay. In a second test, larvae are recorded as they race along five parallel lanes towards a yeast stimulus. This allows increased throughput analysis of comparative genotypes simultaneously, video archiving, and detection of exacerbation or rescue of defective locomotion in a Drosophila model of tauopathy, as we demonstrate genetically and through delivery of candidate therapeutic chemicals in fly food. The tests are well-suited for rapid comparison of locomotion capability in Drosophila mutants or candidate modulation screens in Drosophila models of human disease.

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Modeling the orientation distribution function by mixtures of angular central Gaussian distributions.

Publication Date: 2012 Jan 15 PMID: 21925539
Authors: Tabelow, K. - Voss, H. U. - Polzehl, J.
Journal: J Neurosci Methods

In this paper we develop a tensor mixture model for diffusion weighted imaging data using an automatic model order selection criterion for the number of tensor components in a voxel. We show that the weighted orientation distribution function for this model can be expanded into a mixture of angular central Gaussian distributions. We investigate properties of this model in extensive simulations and in a high angular resolution scan of a human brain. The results suggest that the model improves imaging of cerebral fiber tracts. In addition, inference on canonical model parameters could potentially provide novel clinical markers of altered white matter. Software to compute the tensor mixture model from diffusion weighted MRI data is made available in the programming language R.

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Orientation selective or not? - Measuring significance of tuning to a circular parameter.

Publication Date: 2012 Jan 15 PMID: 21924292
Authors: Grabska-Barwinska, A. - Ng, B. S. - Jancke, D.
Journal: J Neurosci Methods

Orientation and direction tuning are among the most studied features of the visual system and are routinely measured during experiments to estimate the quality of neuronal responses. However, standard approaches to report orientation selectivity are only narrowly quantitative and strongly depend on the signal quality, while the more sophisticated ones are computationally exhaustive, making them difficult to use during ongoing experiments. We propose a fast and efficient method for reporting the reliability of coding applicable to any circular parameter. Similar to standard deviation in the linear statistics, reproducibility measures trial-to-trial variability of a circular response parameter. Reproducibility is a normalized measure easily transformed to p-values, which provide explicit information about significance of the estimated orientation preference. The proposed approach is applicable to a wide range of signal types. Here, we discuss examples from optical imaging and electrophysiological recordings, and provide a more thorough examination based on tuning curves modeled in silico.

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A Cellular Neural Network methodology for the automated segmentation of multiple sclerosis lesions.

Publication Date: 2012 Jan 15 PMID: 21920384
Authors: Cerasa, A. - Bilotta, E. - Augimeri, A. - Cherubini, A. - Pantano, P. - Zito, G. - Lanza, P. - Valentino, P. - Gioia, M. C. - Quattrone, A.
Journal: J Neurosci Methods

We present a new application based on genetic algorithms (GAs) that evolves a Cellular Neural Network (CNN) capable of automatically determining the lesion load in multiple sclerosis (MS) patients from magnetic resonance imaging (MRI). In particular, it seeks to identify brain areas affected by lesions, whose presence is revealed by areas of higher intensity if compared to healthy tissue. The performance of the CNN algorithm has been quantitatively evaluated by comparing the CNN output with the expert's manual delineation of MS lesions. The CNN algorithm was run on a data set of 11 MS patients; for each one a single dataset of MRI images (matrix resolution of 256x256 pixels) was acquired. Our automated approach gives satisfactory results showing that after the learning process the CNN is capable of detecting MS lesions with different shapes and intensities (mean DICE coefficient=0.64). The system could provide a useful support tool for the evaluation of lesions in MS patients, although it needs to be evolved and developed in the future.

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Response to Westlund's commentary: 'Can conditioned reinforcers and variable-Ratio Schedules make food- and fluid control redundant?'.

Publication Date: 2012 Feb 15 PMID: 21920383
Authors: Prescott, M. J. - Brown, V. J. - Flecknell, P. A. - Garrod, K. - Lemon, R. N. - Parker, A. J. - Ryder, K. - Schultz, W. - Scott, L. - Watson, J. - Whitfield, L.
Journal: J Neurosci Methods

In this short communication, we respond to Westlund's critique of the NC3Rs Working Group report on refinement of the use of food and fluid control as motivational tools for macaques used in behavioural neuroscience research. The suggestions Westlund makes - in particular, the use of conditioned reinforcers and variable ratio schedules - were considered by the Working Group but were not included in the report as specific recommendations. We outline the reasons for this and also address some misunderstandings of our position.

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Adaptive estimation of EEG-rhythms for optimal band identification in BCI.

Publication Date: 2012 Jan 15 PMID: 21911006
Authors: Veluvolu, K. C. - Wang, Y. - Kavuri, S. S.
Journal: J Neurosci Methods

The amplitude of EEG mu-rhythm is large when the subject does not perform or imagine movement and attenuates when the subject either performs or imagines movement. The knowledge of EEG individual frequency components in the time-domain provides useful insight into the classification process. Identification of subject-specific reactive band is crucial for accurate event classification in brain-computer interfaces (BCI). This work develops a simple time-frequency decomposition method for EEG mu rhythm by adaptive modeling. With the time-domain decomposition of the signal, subject-specific reactive band identification method is proposed. Study is conducted on 30 subjects for optimal band selection for four movement classes. Our results show that over 93% the subjects have an optimal band and selection of this band improves the relative power spectral density by 200% with respect to normalized power.

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FACS purification of immunolabeled cell types from adult rat brain.

Publication Date: 2012 Jan 15 PMID: 21911005
Authors: Guez-Barber, D. - Fanous, S. - Harvey, B. K. - Zhang, Y. - Lehrmann, E. - Becker, K. G. - Picciotto, M. R. - Hope, B. T.
Journal: J Neurosci Methods

Molecular analysis of brain tissue is greatly complicated by having many different classes of neurons and glia interspersed throughout the brain. Fluorescence-activated cell sorting (FACS) has been used to purify selected cell types from brain tissue. However, its use has been limited to brain tissue from embryos or transgenic mice with promoter-driven reporter genes. To overcome these limitations, we developed a FACS procedure for dissociating intact cell bodies from adult wild-type rat brains and sorting them using commercially available antibodies against intracellular and extracellular proteins. As an example, we isolated neurons using a NeuN antibody and confirmed their identity using microarray and real time PCR of mRNA from the sorted cells. Our FACS procedure allows rapid, high-throughput, quantitative assays of molecular alterations in identified cell types with widespread applications in neuroscience.

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Can conditioned reinforcers and Variable-Ratio Schedules make food- and fluid control redundant? A comment on the NC3Rs Working Group's report.

Publication Date: 2012 Feb 15 PMID: 21459109
Authors: Westlund, K.
Journal: J Neurosci Methods

This commentary challenges the conclusions of the NC3Rs Working Group's recent special report that food- or fluid control is sometimes necessary to conduct modern neuroscientific investigations in macaque monkeys (Prescott et al., 2010). Given the potential suffering of animals subjected to food- or fluid control, the decision to subject an animal to such practices should be taken hesitantly. That decision hinges on to which extent the animal is willing to be involved in the task. The authors have done a scientific literature search and express expert opinion, but fail to mention two techniques that may greatly influence the animals' motivation to participate in the task and thus reduce the need for food- or fluid control, namely (1) the use of conditioned reinforcers in addition to primary reinforcers; and (2) the use of Variable-Ratio Schedules rather than continuous reinforcement. An ethical and humane approach to animal experimentation suggests that all options should be explored and thoroughly investigated before resorting to methods potentially challenging the animals' welfare.

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