Journal of Neuroscience Methods

High-throughput optogenetic functional magnetic resonance imaging with parallel computations.

Publication Date: 2013 Jun 4 PMID: 23747482
Authors: Fang, Z. - Lee, J. H.
Journal: J Neurosci Methods

Optogenetic functional magnetic resonance imaging (ofMRI) technology enables cell-type specific, temporally precise neuronal control and accurate, in vivo readout of resulting activity across the whole brain. With the ability to precisely control excitation and inhibition parameters, and to accurately record the resulting activity, there is an increased need for a high-throughput method to bring the ofMRI studies to their full potential. In this paper, an advanced system that can allow real-time fMRI with interactive control and analysis in a fraction of the MRI acquisition repetition time (TR) is proposed. With such high processing speed, sufficient time will be available for integration of future developments that can further enhance ofMRI data quality or better streamline the study. We designed and implemented a highly optimized, massively parallel system using graphics processing unit (GPU)s which achieves reconstruction, motion correction, and analysis of 3D volume data in approximately 12.80ms. As a result, with a 750ms TR and 4 interleaf fMRI acquisition, we can now conduct sliding window reconstruction, motion correction, analysis and display in approximately 1.7% of the TR. Therefore, a significant amount of time can now be allocated to integrating advanced but computationally intensive methods that can enable higher image quality and better analysis results all within a TR. Utilizing the proposed high-throughput imaging platform with sliding window reconstruction, we were also able to observe the much-debated initial dips in our ofMRI data. Combined with methods to further improve SNR, the proposed system will enable efficient real-time, interactive, high-throughput ofMRI studies.

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Approaches and tools for modeling signaling pathways and calcium dynamics in neurons.

Publication Date: 2013 Jun 3 PMID: 23743449
Authors: Blackwell, K. T.
Journal: J Neurosci Methods

Signaling pathways are cascades of intracellular biochemical reactions that are activated by transmembrane receptors, and ultimately lead to transcription in the nucleus. In neurons, both calcium permeable synaptic and ionic channels as well as G protein coupled receptors initiate activation of signaling pathway molecules that interact with electrical activity at multiple spatial and time scales. At small temporal and spatial scales, calcium modifies the properties of ionic channels, whereas at larger temporal and spatial scales, various kinases and phosphatases modify the properties of ionic channels, producing phenomena such as synaptic plasticity and homeostatic plasticity. The elongated structure of neuronal dendrites and the organization of multi-protein complexes by anchoring proteins implies that the spatial dimension must be explicit. Therefore, modeling signaling pathways in neurons utilizes algorithms for both diffusion and reactions. The small size of spines coupled with small concentrations of some molecules implies that some reactions occur stochastically. The need for stochastic simulation of many reaction and diffusion events coupled with the multiple temporal and spatial scales makes modeling of signaling pathways a difficult problem. Several different software programs have achieved different aspects of these capabilities. This review explains some of the mathematical formulas used for modeling reactions and diffusion. In addition, it briefly presents the simulators used for modeling reaction-diffusion systems in neurons, together with scientific problems addressed.

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Repeatability of timing of eye-hand coordinated movements across different cognitive tasks.

Publication Date: 2013 May 31 PMID: 23732535
Authors: de Boer, C. - van der Steen, J. - Schol, R. J. - Pel, J. J.
Journal: J Neurosci Methods

Quantification of eye-hand coordinated behaviour is a relatively new tool to study neurodegeneration in humans. Its sensitivity depends on assessment of different behavioural strategies, multiple task testing and repeating tasks within one session. However, a large number of repetition trials will pose a strong burden on subjects. To introduce this method in large scale population studies, it is necessary to determine whether reducing the number of task repetitions, which will lower subject burden, still leads to acceptable measurement accuracy. The objective of this study was to investigate in a test-retest approach the validity and reliability of eye-hand coordination outcome parameters in eight healthy volunteers assessed during a shortened test procedure consisting of eight repetitions of three behavioural tasks: a reflex based tapping task, a planning based tapping task and a memory based tapping task. Eye-hand coordination was quantified in terms of timing (eye and hand latencies), kinematics and accuracy. Eye and hand latencies were found within a normal range (between 150 and 450ms). A paired samples t-test revealed no differences in timing parameters between the first and second measurements. It was concluded that eight trial repetitions are sufficient for quantifying eye-hand coordination in terms of timing, kinematics and accuracy. This approach allows multiple tasks testing of visuomotor behaviour within a reasonable time span of few minutes per task.

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Extracting cortical current dipoles from MEG recordings.

Publication Date: 2013 May 28 PMID: 23727444
Authors: Lots, I. S. - Robinson, S. E. - Abeles, M.
Journal: J Neurosci Methods

A novel way of using synthetic aperture magnetometry to extract local current dipoles is proposed. This method is used to extract the current-dipoles at multiple points in the cortex. It is shown that in this way the correlation between cortical points is lower and falls faster with distance when compared to the original MEG and other methods. Evoked auditory responses are well localized. They show higher signal to noise ratio and are more reproducible then the MEG evoked fields.

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A Comparison of Automated Anatomical-Behavioural Mapping Methods in a Rodent Model of Stroke.

Publication Date: 2013 May 29 PMID: 23727124
Authors: Crum, W. R. - Giampietro, V. P. - Smith, E. J. - Gorenkova, N. - Stroemer, R. P. - Modo, M.
Journal: J Neurosci Methods

Neurological damage, caused by conditions such as stroke, results in a complex pattern of structural change and significant behavioural dysfunctions; the automated analysis of Magnetic Resonance Imaging (MRI) and discovery of structural-behavioural correlates in such disorders remains challenging. Voxel Lesion Symptom Mapping (VLSM) has been used to associate behaviour with lesion location in MRI but requires lesion masks to be defined on each subject and does not exploit the rich structural information in the images. Tensor Based Morphometry (TBM) has been used to perform voxel-wise structural analysis over the whole brain, and can include behaviour as a co-variate; however interpretation may be confounded by a combination of lesion hyper-intensities and subtle structural remodelling away from the lesion. In this paper, we compare and contrast these techniques in a rodent model of stroke (n=58) to assess their efficacy in a challenging pre-clinical application. The results from the automated techniques are compared with manually derived region-of-interest measures of lesion, cortex, striatum, ventricle and hippocampus, and considered against model power calculations. The automated TBM techniques can successfully detect both lesion and non-lesion effects, and are consistent with manual measurements. They do not require manual segmentation to the same extent as VLSM and should be considered part of the toolkit for unbiased analysis of pre-clinical imaging-based studies.

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Local landmark alignment for high-resolution fMRI group studies: Toward a fine cortical investigation of hand movements in human.

Publication Date: 2013 May 31 PMID: 23727047
Authors: Pizzagalli, F. - Auzias, G. - Delon-Martin, C. - Dojat, M.
Journal: J Neurosci Methods

Converging evidence conclusively demonstrates the robust relationship between anatomical landmarks and underlying functional organization in primary cortical regions. In consequence, a precise alignment across subjects of such specific individual landmarks should improve the overlap of the corresponding functional areas and thus the detection of active clusters at the group level. In an effort to define a dedicated processing pipeline for a fine non-invasive exploration of the motor cortex in human, we evaluated four recent non-linear registration methods based on anatomical and functional indexes. We used high-resolution functional MRI data to finely reveal the impact of the registration on the cortical assignment of the detected clusters. Our results first demonstrate that the quality of registration strongly affects the statistical significance and the assignment of activated clusters to specific anatomical regions, here in the primary motor area. Our results also illustrate the bias induced by the chosen reference template on the detected clusters. The analysis of the Jacobian of the deformation field informs us about how each method deforms the anatomical structures and functional maps. The methodology we propose, combining high resolution fMRI and non-linear registration method, allows a robust non-invasive exploration of the motor cortex.

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Establishment of an optimised protocol for a Golgi-electron microscopy method based on a Golgi-Cox staining procedure with a commercial kit.

Publication Date: 2013 May 28 PMID: 23721893
Authors: Koyama, Y. - Nishida, T. - Tohyama, M.
Journal: J Neurosci Methods

The introduction of the FD Rapid GolgiStain Kit has enabled many researchers to easily examine the morphology of neurons. Owing to an improved Golgi-Cox impregnation method, both stable and highly sensitive stained images of neurons can be acquired using this kit. To analyse microstructures such as dendritic spines and growth cones at detail, we attempted to create a useful method combining Golgi-Cox staining and electron microscopy (Golgi-EM method) so that the prepared Kit-stained samples would be appropriate for use in electron microscopy (EM) studies. Results from the present study suggest the importance of the glutaraldehyde fixation step in the Golgi-EM method. Here, we introduce the recommended protocol for the Golgi-EM method, which is available for ultrahigh-voltage electron microscopy and transmission electron microscopy. We are confident that our Golgi-EM method may become a reliable and important method that can contribute to new discoveries in neuronal morphology.

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Quantifying bursting neuron activity from calcium signals using blind deconvolution.

Publication Date: 2013 May 24 PMID: 23711821
Authors: Park, I. J. - Bobkov, Y. V. - Ache, B. W. - Principe, J. C.
Journal: J Neurosci Methods

Advances in calcium imaging have enabled studies of the dynamic activity of both individual neurons and neuronal assemblies. However, challenges, such as unknown nonlinearities in the spike-calcium relationship, noise, and the often relatively low temporal resolution of the calcium signal compared to the time-scale of spike generation, restrict the accurate estimation of action potentials from the calcium signal. Complex neuronal discharge, such as the activity demonstrated by bursting and rhythmically active neurons, represents an even greater challenge for reconstructing spike trains based on calcium signals. We propose a method using blind calcium signal deconvolution based on an information-theoretic approach. This model is meant to maximise the output entropy of a nonlinear filter where the nonlinearity is defined by the cumulative distribution function of the spike signal. We tested our maximum entropy (ME) algorithm using bursting olfactory receptor neurons (bORNs) of the lobster olfactory organ. The advantage of the ME algorithm is that the filter can be trained online based only on the statistics of the spike signal, without any assumptions regarding the unknown transfer function generated by the calcium image obtained from spiking. We show that the ME method is able to more accurately reconstruct the timing of the first and last spikes of a burst compared to other methods and that it improves the temporal precision fivefold compared to direct estimation using calcium imaging landmarks.

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Anti-Abeta antibodies induced by Abeta-HBc virus-like particles prevent Abeta aggregation and protect PC12 cells against toxicity of Abeta

Publication Date: 2013 May 20 PMID: 23701997
Authors: Feng, G. - Wang, W. - Qian, Y. - Jin, H.
Journal: J Neurosci Methods

beta-Amyloid peptide (Abeta) immunization is regarded as the most promising therapy to Alzheimer' s disease. The full length Abeta as antigen might induce meningoencepholontis adverse effect since the middle and C-terminal fragments of Abeta contain T cell epitopes. While N-terminal fragment of Abeta, containing B cell epitope, has weak or no immunogenicity. To improve the immunogenicity, we used HBV core antigen as carrier to make fusion protein containing 2 Abeta1-15. The fusion protein was expressed in Escherichia coli harboring the recombinant plasmid pET/c-2Abeta15-c. Transmission electron microscope (TEM) showed that fusion protein could form virus-like particles (VLPs). After 7-weeks immunization with Abeta-HBc VLPs through subcutaneous injection, the titer of anti-Abeta antibody in sera of BALB/c mice reached up to 105, higher than Abeta peptide immunization. Abeta-HBc VLPs immunization did not elicit Abeta-specific T cell proliferation. The main isotypes of antibody in mice immunized with Abeta-HBc VLPs were IgG1 and IgG2b, while isotype in mice immunized with Abeta1-42 was IgG2a. When the antisera from mice immunized with Abeta-HBc VLPs were co-incubated for 1 week at 37 degrees C with Abeta, fibers of aggregated Abeta was reduced or diminished. The antibodies also prevented PC12 cells from injury by toxicity of Abeta. In conclusion, recombinant c-2Abeta15-c gene can be expressed in E. coli. The expressed protein could form VLPs and has strong immunogenicity. The antisera prevented Abeta fiber formation and protected the PC12 cells against toxicity of Abeta. This study lays the foundation for the experimental study of AD gene engineering vaccine.

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Combining ERD and ERS features to create a system-paced BCI.

Publication Date: 2013 Jun 15 PMID: 23624244
Authors: Thomas, E. - Fruitet, J. - Clerc, M.
Journal: J Neurosci Methods

An important factor in the usability of a brain-computer interface (BCI) is the setup and calibration time required for the interface to perform accurately. Recently, brain-switches based on the beta rebound following motor imagery of a single limb effector have been investigated as basic BCIs due to their good performance with limited electrodes, and brief training session requirements. Here, a BCI is proposed which expands the methodology of brain-switches to design an interface composed of multiple brain-controlled buttons. The algorithm is designed as a system paced interface which can recognise 2 intentional-control tasks and a no-control state based on the activity during and following motor imagery in only 3 electroencephalogram channels. An online experiment was performed over 6 subjects to validate the algorithm, and the results show that a working BCI can be trained from a single calibration session and that the post motor imagery features are both informative and robust over multiple sessions.

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In vivo imaging of brain metabolism activity using a phosphorescent oxygen-sensitive probe.

Publication Date: 2013 Jun 15 PMID: 23624034
Authors: Tsytsarev, V. - Arakawa, H. - Borisov, S. - Pumbo, E. - Erzurumlu, R. S. - Papkovsky, D. B.
Journal: J Neurosci Methods

Several approaches have been adopted for real-time imaging of neural activity in vivo. We tested a new cell-penetrating phosphorescent oxygen-sensitive probe, NanO2-IR, to monitor temporal and spatial dynamics of oxygen metabolism in the neocortex following peripheral sensory stimulation. Probe solution was applied to the surface of anesthetized mouse brain; optical imaging was performed using a MiCAM-02 system. Trains of whisker stimuli were delivered and associated changes in phosphorescent signal were recorded in the contralateral somatosensory ("barrel") cortex. Sensory stimulation led to changes in oxygenation of activated areas of the barrel cortex. The oxygen imaging results were compared to those produced by the voltage-sensitive dye RH-1691. While the signals emitted by the two probes differed in shape and amplitude, they both faithfully indicated specific whisker evoked cortical activity. Thus, NanO2-IR probe can be used as a tool in visualization and real-time analysis of sensory-evoked neural activity in vivo.

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Enhanced loading of Fura-2/AM calcium indicator dye in adult rodent brain slices via a microfluidic oxygenator.

Publication Date: 2013 Jun 15 PMID: 23608309
Authors: Mauleon, G. - Lo, J. F. - Peterson, B. L. - Fall, C. P. - Eddington, D. T.
Journal: J Neurosci Methods

A microfluidic oxygenator is used to deliver constant oxygen to rodent brain slices, enabling the loading of the cell-permeant calcium indicator Fura-2/AM into cells of adult brain slices. When compared to traditional methods, our microfluidic oxygenator improves loading efficiency, measured by the number of loaded cells per unit area, for all tested age groups. Loading in slices from 1-year-old mice was achieved, which has not been possible with current bulk loading methods. This technique significantly expands the age range for which calcium studies are possible without cellular injection. This technique will facilitate opportunities for the study of calcium signaling of aging and long term stress related diseases. Moreover, it should be applicable to other membrane-permeant physiological indicator varieties.

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Photoacoustic and optical coherence tomography of epilepsy with high temporal and spatial resolution and dual optical contrasts.

Publication Date: 2013 Jun 15 PMID: 23603664
Authors: Tsytsarev, V. - Rao, B. - Maslov, K. I. - Li, L. - Wang, L. V.
Journal: J Neurosci Methods

Epilepsy mapping with high spatial and temporal resolution has great significance for both fundamental research on epileptic neurons and the clinical management of epilepsy. In this communication, we demonstrate for the first time in vivo epilepsy mapping with high spatial and temporal resolution and dual optical contrasts in an animal model. Through the variations of a depthresolved optical coherence tomography signal with optical scattering contrast, we observed that epileptic neuron activities modulated the optical refractive index of epileptic neurons and their surrounding tissue. Simultaneously, through neurovasculature coupling mechanisms and optical absorption contrast, we used photoacoustic signals to document the hemodynamic changes of the microvasculature surrounding the epileptic neurons. The epilepsy mapping results were confirmed by a simultaneously recorded electroencephalogram signal during epileptic seizure. Our new epilepsy mapping tool, with high temporal and spatial resolution and dual optical contrasts, may find many applications, such as drug development and epilepsy surgery.

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Sorted averaging improves quality of auditory steady-state responses.

Publication Date: 2013 May 30 PMID: 23603663
Authors: Rahne, T. - Verhey, J. L. - Muhler, R.
Journal: J Neurosci Methods

Increasing signal-to-noise ratio (SNR) is essential for the recording of auditory evoked potentials with electroencephalography (EEG). Several protocols have been proposed to increase the SNR, starting with an averaging of EEG epochs which decreases noise level. Since artifacts decrease the SNR by increasing the noise level, artifact detection and reduction protocols are other important tools to reduce the noise level. The current study focuses on the sorted averaging protocol where the epochs are sort according to their estimated root-mean-square (RMS) amplitude. Calculating an estimated SNR by averaging of the sorted epochs, this process of averaging can be interrupted at the maximum SNR, i.e., at an optimal number of epochs. In contrast to the often used protocol that weighs every epoch by its inverse average root-mean-square amplitude, sorted averaging is a linear operation, i.e., it does not change signal amplitudes. In this study, the sorted averaging is, for the first time, applied to auditory steady-state responses (ASSR) which are evoked by amplitude modulated tones or trains of transient acoustic stimuli. In contrast to other evoked potentials, the ASSR is analyzed in the frequency domain, using the property of auditory system to retain the modulation frequency (or the repetition rate) of the stimulus. ASSR were recorded in 11 subjects with normal hearing. Results of four artifact processing protocols (1) fixed rejection level, (2) adaptive rejection level, (3) weighted averaging and (4) sorted averaging were compared. The results showed a higher normalized SNR with a sorted averaging protocol than with adaptive rejection level and weighted averaging protocols. An advantage of the sorted averaging protocol is that, compared to a fixed-rejection threshold, the ASSR amplitudes were unchanged when the sorted averaging protocol was used, whereas they were significantly reduced by the weighted averaging protocol. The residual noise was also significantly lower for the sorted averaging protocol than for the weighted averaging and adaptive rejection protocols. Thus, the sorted averaging may be a powerful tool to increase the quality of ASSR.

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A rapid fluorescent method to quantify neuronal loss after experimental intracerebral hemorrhage.

Publication Date: 2013 Jun 15 PMID: 23583700
Authors: Chen-Roetling, J. - Lu, X. - Regan, K. A. - Regan, R. F.
Journal: J Neurosci Methods

Neuronal loss in tissue surrounding an intracerebral hemorrhage (ICH) is usually quantified by labor-intensive histological methods that are subject to bias. Fluorescent protein expression has been successfully used as a marker of cell viability in vitro and in retinal studies in vivo, but not in any ICH model to date. The potential of this approach was investigated using transgenic mice that constitutively express the red fluorescent protein variant dTomato in central neurons under the control of the Thy1 promoter. Breeding and growth of these mice were similar to their wild-type counterparts; behavioral phenotyping by digital analysis of home cage video recordings detected no differences. Bright fluorescence was evident in fresh brain samples with minimal background fluorescence, and was reduced in tissue surrounding the hematoma. In order to assess fluorescence loss as an injury marker in a planned study, these mice were crossed with heme oxygenase (HO)-2 knockouts and wild-type controls; striatal hemorrhage was induced by stereotactic injection of collagenase. Fluorescence in hemorrhagic striata was reduced to 86.4+/-3.9%, 62.2+/-5.1%, and 58.3+/-3.0% of contra-lateral on days 1, 4 and 8, respectively, and correlated closely with reduction in striatal cell viability as quantified by MTT assay. HO-2 knockout and wild-type values did not differ significantly. Similar results were observed with stereological cell counts of striatal neurons identified by NeuN immunoreactivity. These results suggest that loss of constitutive dTomato fluorescence is an accurate and efficient marker of neuronal loss in tissue surrounding a striatal hematoma.

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Antibody testing for brain immunohistochemistry: Brain immunolabeling for the cannabinoid CB2 receptor.

Publication Date: 2013 Jun 15 PMID: 23583232
Authors: Baek, J. H. - Darlington, C. L. - Smith, P. F. - Ashton, J. C.
Journal: J Neurosci Methods

The question of whether cannabinoid CB2 receptors are expressed on neurons in the brain and under what circumstances they are expressed is controversial in cannabinoid neuropharmacology. While some studies have reported that CB2 receptors are not detectable on neurons under normal circumstances, other studies have reported abundant neuronal expression. One reason for these apparent discrepancies is the reliance on incompletely validated CB2 receptor antibodies and immunohistochemical procedures. In this study, we demonstrate some of the methodological problems encountered using three different commercial CB2 receptor antibodies. We show that (1) the commonly used antibodies that were confirmed by many of the tests used for antibody validation still failed when examined using the knockout control test; (2) the coherence between the labeling patterns provided by two antibodies for the same protein at different epitopes may be misleading and must be validated using both low- and high-magnification microscopy; and (3) although CB2 receptor antibodies may label neurons in the brain, the protein that the antibodies are labeling is not necessarily CB2. These results showed that great caution needs to be exercised when interpreting the results of brain immunohistochemistry using CB2 receptor antibodies and that, in general, none of the tests for antibody validity that have been proposed, apart from the knockout control test, are reliable.

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SACICA: A sparse approximation coefficient-based ICA model for functional magnetic resonance imaging data analysis.

Publication Date: 2013 May 30 PMID: 23563324
Authors: Wang, N. - Zeng, W. - Chen, L.
Journal: J Neurosci Methods

Independent component analysis (ICA) has been widely used in functional magnetic resonance imaging (fMRI) data to evaluate the functional connectivity, which assumes that the sources of functional networks are statistically independent. Recently, many researchers have demonstrated that sparsity is an effective assumption for fMRI signal separation. In this research, we present a sparse approximation coefficient-based ICA (SACICA) model to analyse fMRI data, which is a promising combination model of sparse features and an ICA technique. The SACICA method consists of three procedures. The wavelet packet decomposition procedure, which decomposes the fMRI data into wavelet tree nodes with different degrees of sparsity, is first. Then, the sparse approximation coefficients set formation procedure, in which an effective Lp norm is proposed to measure the sparse degree of the distinct wavelet tree nodes, is second. The ICA decomposition and reconstruction procedure, which utilises the sparse approximation coefficients set of the fMRI data, is last. The hybrid data experimental results demonstrated that the SACICA method exhibited the stronger spatial source reconstruction ability with respect to the unsmoothed fMRI data and better detection sensitivity of the functional signal on the smoothed fMRI data than the FastICA method. Furthermore, task-related experiments also revealed that SACICA was not only effective in discovering the functional networks but also exhibited a better detection sensitivity of the visual-related functional signal. In addition, the SACICA combined with Fast-FENICA proposed by Wang et al. (2012) was demonstrated to conduct the group analysis effectively on the resting-state data set.

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A novel telemetric system to measure polysomnographic biopotentials in freely moving animals.

Publication Date: 2013 Jun 15 PMID: 23563323
Authors: Zielinski, M. R. - Gerashchenko, L. - Karpova, S. A. - Gerashchenko, D.
Journal: J Neurosci Methods

Mice are by far the most widely used species for scientific research and have been used in many studies involving biopotentials, such as the electroencephalogram (EEG) and electromyogram (EMG) signals monitored for sleep analysis. Unfortunately, current methods for the analysis of these signals involve either tethered systems that are restrictive and heavy for the animal or wireless systems that use transponders that are large relative to the animal and require invasive surgery for implantation; as a result, natural behavior/activity is altered. Here, we propose a novel and inexpensive system for measuring electroencephalographic signals and other biopotentials in mice that allows for natural movement. We also evaluate the new system for the analysis of sleep architecture and EEG power during both spontaneous sleep and the sleep that follows sleep deprivation in mice. Using our new system, vigilance states including non-rapid eye movement sleep (NREMS), rapid eye movement sleep (REMS), and wakefulness, as well as EEG power and NREMS EEG delta power in the 0.5-4Hz range (an indicator of sleep intensity) showed the diurnal rhythms typically found in mice. These values were also similar to values obtained in mice using telemetry transponders. Mice that used the new system also demonstrated enhanced NREMS EEG delta power responses that are typical following sleep deprivation and few signal artifacts. Moreover, similar movement activity counts were found when using the new system compared to a wireless system. This novel system for measuring biopotentials can be used for polysomnography, infusion, microdialysis, and optogenetic studies, reduces artifacts, and allows for a more natural moving environment and a more accurate investigation of biological systems and pharmaceutical development.

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Acute reduction in oxygen tension enhances the induction of neurons from human fibroblasts.

Publication Date: 2013 Jun 15 PMID: 23562599
Authors: Davila, J. - Chanda, S. - Ang, C. E. - Sudhof, T. C. - Wernig, M.
Journal: J Neurosci Methods

We and others have reported the successful conversion of human fibroblasts into functional induced neuronal (iN) cells; however the reprogramming efficiencies were very low. Robust reprogramming methods must be developed before iN cells can be used for translational applications such as disease modeling or transplantation-based therapies. Here, we describe a novel approach in which we significantly enhance iN cell conversion efficiency of human fibroblast cells by reprogramming under hypoxic conditions (5% O2). Fibroblasts were derived under high (21%) or low (5%) oxygen conditions and reprogrammed into iN cells using a combination of the four transcription factors BRN2, ASCL1, MYT1L and NEUROD1. An increase in Map2 immunostaining was only observed when fibroblasts experienced an acute drop in O2 tension upon infection. Interestingly, cells derived and reprogrammed under hypoxic conditions did not produce more iN cells. Approximately 100% of patched cells fired action potentials in low O2 conditions compared to 50% under high O2 growth conditions, confirming the beneficial aspect of reprogramming under low O2. Further characterization showed no significant difference in the intrinsic properties of iN cells reprogrammed in either condition. Surprisingly, the acute drop in oxygen tension did not affect cell proliferation or cell survival and was not synergistic with the blockade of GSK3beta and Smad-mediated pathways. Our results showed that lowering the O2 tension at the initiation of reprogramming is a simple and efficient stratergy to enhance the production of iN cells which will facilitate their use for basic discovery and regenerative medicine.

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A multidimensional approach to an in-depth proteomics analysis of transcriptional regulators in neuroblastoma cells.

Publication Date: 2013 Jun 15 PMID: 23558336
Authors: Li, Q. - Jain, M. R. - Chen, W. - Li, H.
Journal: J Neurosci Methods

The dynamic regulation of transcriptional events is fundamental to many aspects of neuronal cell functions. However, proteomics methods have not been routinely used in global neuroproteomics analyses of transcriptional regulators because they are much less abundant than the "house-keeping" proteins in cells and tissues. Recent improvements in both biochemical preparations of nuclear proteins and detection sensitivities of proteomics technologies have made the global analysis of nuclear transcriptional regulators possible. We report here an optimised neuroproteomic method for the analysis of transcriptional regulators in the nuclear extracts of SHSY-5Y neuroblastoma cells by combining an improved nuclear protein extraction procedure with multidimensional peptide separation approaches. We found that rigorous removal of cytoplasmic proteins and solubilisation of DNA-associated proteins improved the number of nuclear proteins identified. Furthermore, we discovered that multidimensional peptide separations by either strong cation exchange (SCX) chromatography or electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) analysis detected more than 1800 nuclear proteins, which constitutes one of the largest datasets of nuclear proteins reported for a neuronal cell. Thus, in-depth analysis of transcriptional regulators for studying neurological diseases are increasingly feasible.

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Registration of challenging pre-clinical brain images.

Publication Date: 2013 May 30 PMID: 23558335
Authors: Crum, W. R. - Modo, M. - Vernon, A. C. - Barker, G. J. - Williams, S. C.
Journal: J Neurosci Methods

The size and complexity of brain imaging studies in pre-clinical populations are increasing, and automated image analysis pipelines are urgently required. Pre-clinical populations can be subjected to controlled interventions (e.g., targeted lesions), which significantly change the appearance of the brain obtained by imaging. Existing systems for registration (the systematic alignment of scans into a consistent anatomical coordinate system), which assume image similarity to a reference scan, may fail when applied to these images. However, affine registration is a particularly vital pre-processing step for subsequent image analysis which is assumed to be an effective procedure in recent literature describing sophisticated techniques such as manifold learning. Therefore, in this paper, we present an affine registration solution that uses a graphical model of a population to decompose difficult pairwise registrations into a composition of steps using other members of the population. We developed this methodology in the context of a pre-clinical model of stroke in which large, variable hyper-intense lesions significantly impact registration performance. We tested this technique systematically in a simulated human population of brain tumour images before applying it to pre-clinical models of Parkinson's disease and stroke.

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Development of tube tetrodes and a multi-tetrode drive for deep structure electrophysiological recordings in the macaque brain.

Publication Date: 2013 May 30 PMID: 23549063
Authors: Kapoor, V. - Krampe, E. - Klug, A. - Logothetis, N. K. - Panagiotaropoulos, T. I.
Journal: J Neurosci Methods

Understanding the principles that underlie information processing by neuronal networks requires simultaneous recordings from large populations of well isolated single units. Twisted wire tetrodes (TWTs), typically made by winding together four ultrathin wires (diameter: 12-25mum), are ideally suited for such population recordings. They are advantageous over single electrodes; both with respect to quality of isolation as well as the number of single units isolated and have therefore been used extensively for superficial cortical recordings. However, their limited tensile strength poses a difficulty to their use for recordings in deep brain areas. We therefore developed a method to overcome this limitation and utilize tetrodes for electrophysiological recordings in the inferotemporal cortex of rhesus macaque. We fabricated a novel, stiff tetrode called the tube tetrode (TuTe) and developed a multi-tetrode driving system for advancing up to 5 TuTes through a ball and socket chamber to precise locations in the temporal lobe of a rhesus macaque. The signal quality acquired with TuTes was comparable to conventional TWTs and allowed excellent isolation of multiple single units. We describe here a simple method for constructing TuTes, which requires only standard laboratory equipment. Further, our TuTes can be easily adapted to work with other microdrives commonly used for electrophysiological investigation in the macaque brain and produce minimal damage to the cortex along its path because of their ultrathin diameter. The tetrode development described here could allow studying neuronal populations in deep lying brain structures previously difficult to reach with the current technology.

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A simple and fast method for tissue cryohomogenization enabling multifarious molecular extraction.

Publication Date: 2013 Jun 15 PMID: 23541735
Authors: von Ziegler, L. M. - Saab, B. J. - Mansuy, I. M.
Journal: J Neurosci Methods

Research in molecular biology often relies on parallel analysis of nucleic acids, protein and other molecules from a given tissue. When extracted from a single sample however, the quality and quantity of these products can be compromised. One solution is to obtain near-identical samples from multiple animals and dedicate each to a given molecular component, but this approach is not optimal from both an operational and ethical perspective. Thus, we refined the methods for cryohomogenization to allow efficient use of a single experimental sample so that it can easily be divided into fractions for extraction of different molecular components, immediately or after storage. Using western blot, nanodrop UV/V spectrometry, and a bioanalyzer, we show that cryohomogenized hippocampus samples provide high-quality RNA and protein without significant loss in abundance. The method may be particularly advantageous for parallel molecular extraction from brain structures with known hemispheric lateralization, such as the hippocampus, parietal cortex, suprachiasmatic nucleus, and amygdala.

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Susceptibility-based functional brain mapping by 3D deconvolution of an MR-phase activation map.

Publication Date: 2013 May 30 PMID: 23528239
Authors: Chen, Z. - Liu, J. - Calhoun, V. D.
Journal: J Neurosci Methods

The underlying source of T2*-weighted magnetic resonance imaging (T2*MRI) for brain imaging is magnetic susceptibility (denoted by chi). T2*MRI outputs a complex-valued MR image consisting of magnitude and phase information. Recent research has shown that both the magnitude and the phase images are morphologically different from the source chi, primarily due to 3D convolution, and that the source chi can be reconstructed from complex MR images by computed inverse MRI (CIMRI). Thus, we can obtain a 4D chi dataset from a complex 4D MR dataset acquired from a brain functional MRI study by repeating CIMRI to reconstruct 3D chi volumes at each timepoint. Because the reconstructed chi is a more direct representation of neuronal activity than the MR image, we propose a method for chi-based functional brain mapping, which is numerically characterised by a temporal correlation map of chi responses to a stimulant task. Under the linear imaging conditions used for T2*MRI, we show that the chi activation map can be calculated from the MR phase map by CIMRI. We validate our approach using numerical simulations and Gd-phantom experiments. We also analyse real data from a finger-tapping visuomotor experiment and show that the chi-based functional mapping provides additional activation details (in the form of positive and negative correlation patterns) beyond those generated by conventional MR-magnitude-based mapping.

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Complexity in differentiating the expression of truncated or matured forms of MMP-2 and MMP-9 through zymography in rat brain tissues after acute ischaemic stroke.

Publication Date: 2013 May 30 PMID: 23523510
Authors: Alam, M. - Shuaib, A.
Journal: J Neurosci Methods

Matrix metalloproteinases (MMPs) play an important role in the pathogenesis of ischaemic stroke. In particular, the mature forms of MMPs 2 and 9 have similar sizes and share gelatine as a common substrate. Both MMPs are upregulated in ischaemic stroke and play detrimental roles during stroke pathogenesis. Throughout this study, we demonstrated that pro-MMP-2 and pro-MMP-9 from ischaemic rat brain tissue homogenate is detected either through immunoblotting or zymography because of the remarkable size difference between these enzymes (72 versus 95kDa, respectively). However, the mature MMP-2 and MMP-9 cannot be discriminated through zymography because of the almost identical sizes of these forms (66 and 67kDa, respectively). The use of gelatine zymography on ischaemic rat brain tissue homogenate revealed a 65-kDa MMP band, corresponding to the heterogeneous band of mature MMP-2 and/or MMP-9. Furthermore, we also detected mature MMPs of 65kDa generated from both recombinant human MMP-2 and MMP-9. Using a pull down assay in rat brain tissue homogenate with gelatine-agarose beads, we showed increased activities for both the pro and mature forms of MMP-2 and MMP-9. However, we could not determine the origin of the respective mature MMPs from the heterogeneous band. Thus, in this study, we demonstrated that the identification and quantification of mature MMP-2 and MMP-9 could not be achieved using zymography alone. Therefore, the development of a reliable technique to identify and measure the respective MMPs is needed to test new stroke therapies targeting MMP-2 and MMP-9.

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Pig lumbar spine anatomy and imaging-guided lateral lumbar puncture: A new large animal model for intrathecal drug delivery.

Publication Date: 2013 May 30 PMID: 23518340
Authors: Pleticha, J. - Maus, T. P. - Jeng-Singh, C. - Marsh, M. P. - Al-Saiegh, F. - Christner, J. A. - Lee, K. H. - Beutler, A. S.
Journal: J Neurosci Methods

Intrathecal (IT) administration is an important route of drug delivery, and its modelling in a large animal species is of critical value. Although domestic swine is the preferred species for preclinical pharmacology, no minimally invasive method has been established to deliver agents into the IT space. While a "blind" lumbar puncture (LP) can sample cerebrospinal fluid (CSF), it is unreliable for drug delivery in pigs. Using computed tomography (CT), we determined the underlying anatomical reasons for this irregularity. The pig spinal cord was visualised terminating at the S2-S3 level. The lumbar region contained only small amounts of CSF found in the lateral recess. Additional anatomical constraints included ossification of the midline ligaments, overlapping lamina with small interlaminar spaces, and a large bulk of epidural adipose tissue. Accommodating the the pig CT anatomy, we developed a lateral LP (LLP) injection technique that employs advanced planning of the needle path and monitoring of the IT injection progress. The key features of the LLP procedure involved choosing a vertebral level without overlapping lamina or spinal ligament ossification, a needle trajectory crossing the midline, and entering the IT space in its lateral recess. Effective IT delivery was validated by the injection of contrast media to obtain a CT myelogram. LLP represents a safe and reliable method to deliver agents to the lumbar pig IT space, which can be implemented in a straightforward way by any laboratory with access to CT equipment. Therefore, LLP is an attractive large animal model for preclinical studies of IT therapies.

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A magnetic resonance imaging-safe method for the study of human eyeblink conditioning.

Publication Date: 2013 May 30 PMID: 23500969
Authors: Kent, J. S. - Michael Bailey, D. - Vollmer, J. M. - Newman, S. D. - Bolbecker, A. R. - O'Donnell, B. F. - Hetrick, W. P.
Journal: J Neurosci Methods

Eyeblink conditioning (EBC) is a widely used translational probe of cerebellar function in both humans and non-human animals. Decades of animal research have identified the cerebellum as critical for EBC. While there is evidence for the involvement of the cerebellum in human EBC, the neural circuitry of EBC in healthy humans has yet to be fully elucidated. The purpose of this study was to design and validate a highly customisable system for EBC stimulus presentation and response recording using infrared (IR) reflectance suitable for use in magnetic resonance imaging (MRI) environments; in this way, the neural activity of EBC could be investigated using fMRI in humans. Four participants underwent delay EBC and simultaneous fMRI. The results indicate (1) a high signal-to-noise ratio in the IR reflectance data that effectively quantifies the eyeblink morphology and timing and (2) evidence of conditioning in the fMRI environment. The quality of the data, the feasibility of conducting EBC experiments in the fMRI environment, and the customisability of the current system to fit a variety of EBC experimental design parameters are discussed.

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Repeated measurement of the attention components of patients with multiple sclerosis using the Attention Network Test-Interaction (ANT-I): Stability, isolability, robustness, and reliability.

Publication Date: 2013 May 30 PMID: 23473799
Authors: Ishigami, Y. - Fisk, J. D. - Wojtowicz, M. - Klein, R. M.
Journal: J Neurosci Methods

The Attention Network Test (ANT) assesses the networks of attention (alerting, orienting, and executive control). The utility of measuring attention network performances with the repeated administration of the ANT in clinical populations has not yet been explored. In the present study, we administered a variant of the ANT (ANT-I) to 11 multiple sclerosis (MS) patients and 11 controls over six monthly sessions to examine the stability, isolability, robustness, and reliability of the ANT-I. Participants responded through button presses to indicate the direction of a target arrow presented either above or below a fixation cross. The target was accompanied by distracting arrows, pointing either in the same or opposite direction of the target arrow. Target arrows were preceded in some trials by alerting auditory signals and/or by uninformative visual cues. Network scores were calculated using orthogonal subtractions of performance under selected conditions. The MS patients were slower than the controls, though group differences in network performance were rare. Even after five test sessions, the network scores remained stable, despite some practice effects that were the largest under conditions that tested the executive network. The reliabilities of the orienting and executive network effects were good in both groups and increased across sessions, especially with the MS patients. The alerting network was the least reliable, especially for MS patients, but it also became more reliable across sessions. The results suggest that the ANT-I can be used to measure the integrity of attention networks for MS patients in applications that require repeated testing.

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