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Targeted single-neuron infection with rabies virus for transneuronal multisynaptic tracing

Targeted single-neuron infection with rabies virus for transneuronal multisynaptic tracing | Neuroscience_technics | Scoop.it

The transynaptic and retrograde spread of rabies virus make it an efficient and robust transneuronal tracer, capable of revealing connectivity patterns of multisynaptic, neuronal circuits with great detail. Current techniques begin by infecting many neurons simultaneously, from which higher-order neurons are then labeled sequentially in time. Here we report on a method that can initially infect a single neuron-of-choice, allowing for greater precision and specificity of labeled circuits. - by Nguyen TD et al., Journal of Neuroscience Methods , Vol. 209(2), 15 August 2012, Pages 367–370

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Calcium channel auxiliary α2δ and β subunits: trafficking and one step beyond

Calcium channel auxiliary α2δ and β subunits: trafficking and one step beyond | Neuroscience_technics | Scoop.it

The voltage-gated calcium channel α2δ and β subunits are traditionally considered to be auxiliary subunits that enhance channel trafficking, increase the expression of functional calcium channels at the plasma membrane and influence the channels' biophysical properties. Accumulating evidence indicates that these subunits may also have roles in the nervous system that are not directly linked to calcium channel function. For example, β subunits may act as transcriptional regulators, and certain α2δ subunits may function in synaptogenesis. The aim of this Review is to examine both the classic and novel roles for these auxiliary subunits in voltage-gated calcium channel function and beyond. - Dolphin AC in Nature Reviews Neuroscience 13, 542-555 (August 2012)

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Quantitative single-cell ion-channel gene expression profiling through an improved qRT-PCR technique combined with whole cell patch clamp

Quantitative single-cell ion-channel gene expression profiling through an improved qRT-PCR technique combined with whole cell patch clamp | Neuroscience_technics | Scoop.it

Cellular excitability originates from a concerted action of different ion channels. The genomic diversity of ion channels (over 100 different genes) underlies the functional diversity of neurons in the central nervous system (CNS) and even within a specific type of neurons large differences in channel expression have been observed. Patch-clamp is a powerful technique to study the electrophysiology of excitability at the single cell level, allowing exploration of cell-to-cell variability. Only a few attempts have been made to link electrophysiological profiling to mRNA transcript levels and most suffered from experimental noise precluding conclusive quantitative correlations. Here we describe a refinement to the technique that combines patch-clamp analysis with quantitative real-time (qRT) PCR at the single cell level. Hereto the expression of a housekeeping gene was used to normalize for cell-to-cell variability in mRNA isolation and the subsequent processing steps for performing qRT-PCR. However, the mRNA yield from a single cell was insufficient for performing a valid qRT-PCR assay; this was resolved by including a RNA amplification step. The technique was validated on a stable Ltk− cell line expressing the Kv2.1 channel and on embryonic dorsal root ganglion (DRG) cells probing for the expression of Kv2.1. Current density and transcript quantity displayed a clear correlation when the qRT-PCR assay was done in twofold and the data normalized to the transcript level of the housekeeping gene GAPD. Without this normalization no significant correlation was obtained. This improved technique should prove very valuable for studying the molecular background of diversity in cellular excitability - Veys K. et al., Journal of Neuroscience Methods 209(1), 30 July 2012

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High-resolution optical control of spatiotemporal neuronal activity patterns in zebrafish using a digital micromirror device : Nature Protocols : Nature Publishing Group

High-resolution optical control of spatiotemporal neuronal activity patterns in zebrafish using a digital micromirror device : Nature Protocols : Nature Publishing Group | Neuroscience_technics | Scoop.it

How to build your own DMD-based photostimulation device: free access paper including Matlab code for DMD control

[Abstract]Optogenetic approaches allow the manipulation of neuronal activity patterns in space and time by light, particularly in small animals such as zebrafish. However, most techniques cannot control neuronal activity independently at different locations. Here we describe equipment and provide a protocol for single-photon patterned optical stimulation of neurons using a digital micromirror device (DMD). This method can create arbitrary spatiotemporal light patterns with spatial and temporal resolutions in the micrometer and submillisecond range, respectively. Different options to integrate a DMD into a multiphoton microscope are presented and compared. We also describe an ex vivo preparation of the adult zebrafish head that greatly facilitates optogenetic and other experiments. After assembly, the initial alignment takes about one day and the zebrafish preparation takes <30 min. The method has previously been used to activate channelrhodopsin-2 and manipulate oscillatory synchrony among spatially distributed neurons in the zebrafish olfactory bulb. It can be adapted easily to a wide range of other species, optogenetic probes and scientific applications. - Nature Protocols 7, 1410–1425 (2012)

 

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Tetrode recordings in the cerebellar cortex

Tetrode recordings in the cerebellar cortex | Neuroscience_technics | Scoop.it

Multi-unit recordings with tetrodes have been used in brain studies for many years, but surprisingly, scarcely in the cerebellum. The cerebellum is subdivided in multiple small functional zones. Understanding the proper features of the cerebellar computations requires a characterization of neuronal activity within each area. By allowing simultaneous recordings of neighboring cells, tetrodes provide a helpful technique to study the dynamics of the cerebellar local networks. Here, we discuss experimental configurations to optimize such recordings and demonstrate their use in the different layers of the cerebellar cortex. We show that tetrodes can also be used to perform simultaneous recordings from neighboring units in freely moving rats using a custom-made drive, thus permitting studies of cerebellar network dynamics in a large variety of behavioral conditions. - Journal of Physiology-Paris

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Concepts and Methods for the Study of Axonal Regeneration in the CNS

Concepts and Methods for the Study of Axonal Regeneration in the CNS | Neuroscience_technics | Scoop.it

Progress in the field of axonal regeneration research has been like the process of axonal growth itself: there is steady progress toward reaching the target, but there are episodes of mistargeting, misguidance along false routes, and connections that must later be withdrawn. This primer will address issues in the study of axonal growth after central nervous system injury in an attempt to provide guidance toward the goal of progress in the field. We address definitions of axonal growth, sprouting and regeneration after injury, and the research tools to assess growth. - Neuron 74(5), 2012

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Automated multi-slice extracellular and patch-clamp experiments using the WinLTP data acquisition system with automated perfusion control

Automated multi-slice extracellular and patch-clamp experiments using the WinLTP data acquisition system with automated perfusion control | Neuroscience_technics | Scoop.it

Highlights
► Almost complete automation of synaptic plasticity and pharmacology experiments is achieved by adding automated perfusion to stimulation/acquisition protocol scripting. ► For multi-slice experiments, automated perfusion control is helpful when there is a need to change many solutions on many separately perfused slices. ► For single cell patch-clamp experiments, automated perfusion control is helpful when many solutions have to be rapidly changed with accurate timing. ► For slice and single cell, single-line automated perfusion, up to 16 different solutions can be delivered. ► For single cell, triple-line automated perfusion with stepper control, up to 48 different solutions can be delivered. - Journal of Neuroscience Methods 207(2), 2012

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In vivo patch-clamp recording from locus coeruleus neurones in the rat brainstem

In vivo patch-clamp recording from locus coeruleus neurones in the rat brainstem | Neuroscience_technics | Scoop.it

Locus coeruleus (LC) neurones extend noradrenergic projections throughout the neuroaxis and are involved in homeostatic functions such as pain modulation, arousal and cardio-respiratory control. To address the cellular mechanisms underlying pain modulation we have developed a patch-clamp recording technique from LC neurones in anaesthetized rats. These recordings showed LC discharge in vivo to be driven by both spontaneous membrane potential oscillations and CNQX-sensitive EPSCs opposed by bicuculine-sensitive IPSCs. Hindlimb pinch evoked a biphasic action potential response underpinned by a slow monophasic excitatory current. This approach allows detailed characterisation of the synaptic and integrative mechanisms of LC responses to naturalistic stimulation. - J Physiol 590, 2225-2231, 2012

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Robots that reveal the inner workings of brain cells

Robots that reveal the inner workings of brain cells | Neuroscience_technics | Scoop.it

New method offers automated way to record electrical activity inside neurons in the living brain. - MIT News Office (May 7, 2012)

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A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing : Nature Neuroscience : Nature Publishing Group

A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing : Nature Neuroscience : Nature Publishing Group | Neuroscience_technics | Scoop.it

This study describes the generation of knock-in mouse lines that express optogenetic activators or silencers in a CRE recombinase-dependent manner, and demonstrates the reliability and utility of these tools with in vivo and ex vivo light-induced... NatureNeuroscience 15, 2012

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Progress in Biophysics and Molecular Biology - Voltage clamp methods for the study of membrane currents and SR Ca2+ release in adult skeletal muscle fibres

Progress in Biophysics and Molecular Biology - Voltage clamp methods for the study of membrane currents and SR Ca2+ release in adult skeletal muscle fibres | Neuroscience_technics | Scoop.it

Skeletal muscle excitation–contraction (E–C)1 coupling is a process composed of multiple sequential stages, by which an action potential triggers sarcoplasmic reticulum (SR)2 Ca2+ release and subsequent contractile activation. The various steps in the E–C coupling process in skeletal muscle can be studied using different techniques. The simultaneous recordings of sarcolemmal electrical signals and the accompanying elevation in myoplasmic Ca2+, due to depolarization-initiated SR Ca2+ release in skeletal muscle fibres, have been useful to obtain a better understanding of muscle function. In studying the origin and mechanism of voltage dependency of E–C coupling a variety of different techniques have been used to control the voltage in adult skeletal fibres. Pioneering work in muscles isolated from amphibians or crustaceans used microelectrodes or ‘high resistance gap’ techniques to manipulate the voltage in the muscle fibres. The development of the patch clamp technique and its variant, the whole-cell clamp configuration that facilitates the manipulation of the intracellular environment, allowed the use of the voltage clamp techniques in different cell types, including skeletal muscle fibres. The aim of this article is to present an historical perspective of the voltage clamp methods used to study skeletal muscle E–C coupling as well as to describe the current status of using the whole-cell patch clamp technique in studies in which the electrical and Ca2+ signalling properties of mouse skeletal muscle membranes are being investigated. - Progress Biophysics & Mol Biol 108(3) 2012

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ScienceDirect.com - Journal of Neuroscience Methods - The use of macroelectrodes in recording cellular spiking activity

ScienceDirect.com - Journal of Neuroscience Methods - The use of macroelectrodes in recording cellular spiking activity | Neuroscience_technics | Scoop.it

Microelectrode recording (MER) is an important navigational and investigational tool, specifically with regard to deep brain stimulation (DBS) surgery. MER is often utilized when targeting the subthalamic nucleus (STN) and other deep brain nuclei in the management of Parkinson's disease (PD), tremor, dystonia and other emerging applications. Microelectrodes are used to detect and measure cellular spiking activity while macroelectrodes are considered more suitable for measuring the collective sum of slow potentials from multiple cells near the electrode, the local field potential (LFP). Precisely how the characteristics of an electrode affect the data recorded is still unclear. Technical idiosyncrasies of some surgical cases allowed serendipitous data collection from a 250 to 6000 Hz bandpassed macroelectrode recording during DBS implantation for PD. Simultaneous recording from both a microelectrode and macroelectrode were compared along the same surgical trajectory. Audio, normalized root mean square of the recorded signal, and power spectrograms were used to analyze the data. The analyses demonstrate similar results in detecting cellular spiking activity when recording with macroelectrodes compared with microelectrodes. This has important implications for the standardization of recording electrophysiological data as well as for the development of next generation closed-loop deep brain stimulation systems. - Journal of Neuroscience Methods 206(1), 2012

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Light sheet microscopy of living or cleared specimens Current Opinion in Neurobiology

Light sheet microscopy of living or cleared specimens Current Opinion in Neurobiology | Neuroscience_technics | Scoop.it

Light sheet microscopy is a versatile imaging technique with a unique combination of capabilities. It provides high imaging speed, high signal-to-noise ratio and low levels of photobleaching and phototoxic effects. These properties are crucial in a wide range of applications in the life sciences, from live imaging of fast dynamic processes in single cells to long-term observation of developmental dynamics in entire large organisms. When combined with tissue clearing methods, light sheet microscopy furthermore allows rapid imaging of large specimens with excellent coverage and high spatial resolution. Even samples up to the size of entire mammalian brains can be efficiently recorded and quantitatively analyzed.

Here, we provide an overview of the history of light sheet microscopy, review the development of tissue clearing methods, and discuss recent technical breakthroughs that have the potential to influence the future direction of the field. - Curr Op Neurobiology 22(11), 2012

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An amplified promoter system for targeted expression of calcium indicator proteins in the cerebellar cortex

An amplified promoter system for targeted expression of calcium indicator proteins in the cerebellar cortex | Neuroscience_technics | Scoop.it

Recording of identified neuronal network activity using genetically encoded calcium indicators (GECIs) requires labeling that is cell type-specific and bright enough for the detection of functional signals. However, specificity and strong expression are often not achievable using the same promoter. Here we present a combinatorial approach for targeted expression and single-cell-level quantification in which a weak promoter is used to drive trans-amplification under a strong general promoter. We demonstrated this approach using recombinant adeno-associated viruses (rAAVs) to deliver the sequence of the GECI D3cpv in the mouse cerebellar cortex. (...) Kuhn B et al. in Frontiers in Neural Circuits 6:49., 31 July 2012

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Imaging Neural Circuit Dynamics with a Voltage-Sensitive Fluorescent Protein

Imaging Neural Circuit Dynamics with a Voltage-Sensitive Fluorescent Protein | Neuroscience_technics | Scoop.it

Population signals from neuronal ensembles in cortex during behaviour are commonly measured with EEG, LFP and voltage-sensitive dyes. A genetically encoded voltage indicator would be useful for detection of such signals in specific cell types. Here, we describe how his goal can be achieved with Butterfly, a voltage-sensitive fluorescent protein (VSFP) with a subthreshold detection range and enhancements designed for the voltage imaging from single neurons to brain in vivo. VSFP-Butterfly showed reliable membrane targeting, maximum response gain around standard neuronal resting membrane potential, fast kinetics for single cell synaptic responses, and a high signal/noise ratio. Butterfly reports EPSPs in cortical neurons, whisker-evoked responses in barrel cortex, 25 Hz gamma oscillations in hippocampal slices, 2-12 Hz slow waves during brain state modulation in vivo. Our findings demonstrate that cell class specific voltage imaging is practical with VSFP-Butterfly, and expand the genetic toolbox for the detection of neuronal population dynamics. - Akemann W et al., Journal of Neurophysiology 18, 2012

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Optogenetic manipulation of neural circuits and behavior in Drosophila larvae

Optogenetics is a powerful tool that enables the spatiotemporal control of neuronal activity and circuits in behaving animals. Here, we describe our protocol for optical activation of neurons in Drosophila larvae. As an example, we discuss the use of optogenetics to activate larval nociceptors and nociception behaviors in the third-larval instar. We have previously shown that, using spatially defined GAL4 drivers and potent UAS (upstream activation sequence)-channelrhodopsin-2∷YFP transgenic strains developed in our laboratory, it is possible to manipulate neuronal populations in response to illumination by blue light and to test whether the activation of defined neural circuits is sufficient to shape behaviors of interest. Although we have only used the protocol described here in larval stages, the procedure can be adapted to study neurons in adult flies—with the caveat that blue light may not sufficiently penetrate the adult cuticle to stimulate neurons deep in the brain. This procedure takes 1 week to culture optogenetic flies and ∼1 h per group for the behavioral assays. - Nature Protocols 7, 1470–1478 (2012)

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Making neurophysiological data analysis reproducible: Why and how?

Making neurophysiological data analysis reproducible: Why and how? | Neuroscience_technics | Scoop.it

Highlights
► The “Reproducible Research”/“Reproducible Data Analysis” paradigm is presented and the relevant literature is reviewed. ► Two open source software making a daily implementation of the paradigm easy, R/Sweave and the org-mode of the editor emacs, are presented next. ► A toy example is used to illustrate the implementation of both tools. - J. Physiol-Paris 106(3-4), 2012

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Automated whole-cell patch-clamp electrophysiology of neurons in vivo

Automated whole-cell patch-clamp electrophysiology of neurons in vivo | Neuroscience_technics | Scoop.it

A robot, algorithm and software for automated in vivo intracellular electrophysiology are reported that can automatically perform whole-cell patch clamping in the living mouse brain with quality comparable to that for a trained human experimenter. -  Nature Methods 9, 2012

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A window into the synapse

A window into the synapse | Neuroscience_technics | Scoop.it

Visualizing the trafficking of synaptic proteins can help illuminate the functions of these critical neuronal components. However, a synapse's tiny dimensions and molecularly dense nature have made this endeavor difficult. Tanaka and Hirano tackled this problem by making cultured neurons synapse directly onto a coverslip and then monitoring the trafficking of fluorescently labeled receptors via total internal reflection fluorescence microscopy. The authors coated glass surfaces with the synaptic adhesion protein neurexin, which is known to induce postsynaptic differentiation in cells, and then plated rat hippocampal neurons onto them. The neurons bound to the neurexin molecules and formed postsynaptic-like structures. Tanaka and Hirano used this preparation to visualize the recruitment of fluorescently labeled glutamate receptors to the 'pseudo-synapses'. - Nature Methods 9, 533 (2012) about Tanaka, H. & Hirano, T. Cell Reports 1, 291–298 (2012). http://www.nature.com/nmeth/journal/v9/n6/full/nmeth.2055.html

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The origin of extracellular fields and currents — EEG, ECoG, LFP and spikes

The origin of extracellular fields and currents — EEG, ECoG, LFP and spikes | Neuroscience_technics | Scoop.it

[Review] Neuronal activity in the brain gives rise to transmembrane currents that can be measured in the extracellular medium. Although the major contributor of the extracellular signal is the synaptic transmembrane current, other sources — including Na+ and Ca2+ spikes, ionic fluxes through voltage- and ligand-gated channels, and intrinsic membrane oscillations — can substantially shape the extracellular field. High-density recordings of field activity in animals and subdural grid recordings in humans, combined with recently developed data processing tools and computational modelling, can provide insight into the cooperative behaviour of neurons, their average synaptic input and their spiking output, and can increase our understanding of how these processes contribute to the extracellular signal. - Nature Reviews Neuroscience 13, 2012

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Brain Research Bulletin, special issue: Genetically Engineered Mouse Models of Neurologic Diseases

Brain Research Bulletin, special issue: Genetically Engineered Mouse Models of Neurologic Diseases | Neuroscience_technics | Scoop.it

Mouse for Alzheimer's and Parkinson's diseases, spinocerebellar ataxias, diffuse gliomas... - Brain Research Bulletin | Vol 88, Iss 1, Pgs 1-80, (1 May, 2012)

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SigMate: A Matlab-based automated tool for extracellular neuronal signal processing and analysis

SigMate: A Matlab-based automated tool for extracellular neuronal signal processing and analysis | Neuroscience_technics | Scoop.it

Highlights
► SigMate is a comprehensive neuronal signal processing and analysis package. ► Can process and analyze multichannel and multisource neuronal signals. ► Multipurpose package independent of neuronal signal acquisition systems. ► Friendly graphical user interfaces (GUIs) based processing and analysis. ► Open platform for neuronal signal analysis tools.

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A 'nano' era for electrophysiology

A 'nano' era for electrophysiology | Neuroscience_technics | Scoop.it

Researchers in three independent laboratories develop nanoscale devices for network-level electrophysiology. 

 

http://www.nature.com/nmeth/journal/v9/n4/full/nmeth.1961.html

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Rapid optical control of nociception with an ion-channel photoswitch

Rapid optical control of nociception with an ion-channel photoswitch | Neuroscience_technics | Scoop.it

Local anesthetics effectively suppress pain sensation, but most of these compounds act nonselectively, inhibiting activity of all neurons. Moreover, their actions abate slowly, preventing precise spatial and temporal control of nociception. We developed a photoisomerizable molecule, quaternary ammonium–azobenzene–quaternary ammonium (QAQ), that enables rapid and selective optical control of nociception. QAQ is membrane-impermeant and has no effect on most cells, but it infiltrates pain-sensing neurons through endogenous ion channels that are activated by noxious stimuli, primarily TRPV1. After QAQ accumulates intracellularly, it blocks voltage-gated ion channels in the trans form but not the cis form. QAQ enables reversible optical silencing of mouse nociceptive neuron firing without exogenous gene expression and can serve as a light-sensitive analgesic in rats in vivo. Because intracellular QAQ accumulation is a consequence of nociceptive ion-channel activity, QAQ-mediated photosensitization is a platform for understanding signaling mechanisms in acute and chronic pain. - Nature Methods

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Optogenetic investigation of neural circuits underlying brain disease in animal models

[Review] Optogenetic tools have provided a new way to establish causal relationships between brain activity and behaviour in health and disease. Although no animal model captures human disease precisely, behaviours that recapitulate disease symptoms may be elicited and modulated by optogenetic methods, including behaviours that are relevant to anxiety, fear, depression, addiction, autism and parkinsonism. The rapid proliferation of optogenetic reagents together with the swift advancement of strategies for implementation has created new opportunities for causal and precise dissection of the circuits underlying brain diseases in animal models.

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