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Neuroscience_technics
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Sparse and combinatorial neuron labelling Current Opinion in Neurobiology

Sparse and combinatorial neuron labelling Current Opinion in Neurobiology | Neuroscience_technics | Scoop.it

Sparse, random labelling of individual cells is a key approach to study brain circuit organisation and development. An array of methods based on genetic engineering now complements older methods such as Golgi staining, facilitating analysis while providing higher information content. Increasingly refined expression strategies based on transcriptional modulators and site-specific recombinases are used to distribute markers or combinations of markers within specific neuronal subsets. Several trends are emerging: first, increasing labelling density with multiplexed markers to allow more cells to be reliably distinguished; second, using labels to report lineage relationships among defined cells in addition to anatomy; third, coupling cell labelling with genetic manipulations that reveal or perturb cell function. These strategies offer new opportunities for characterizing the fine scale architecture of neuronal circuits, and understanding lineage and functional relations among their cellular components in normal or experimental situations.

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Measuring the Firing Rate of High-Resistance Neurons with Cell-Attached Recording

Measuring the Firing Rate of High-Resistance Neurons with Cell-Attached Recording | Neuroscience_technics | Scoop.it

Cell-attached recording is extensively used to study the firing rate of mammalian neurons, but potential limitations of the method have not been investigated in detail. Here we perform cell-attached recording of molecular layer interneurons in cerebellar slices from rats and mice, and we study how experimental conditions influence the measured firing rate. We find that this rate depends on time in cell-attached mode, on pipette potential, and on pipette ionic composition. In the first minute after sealing, action currents are variable in shape and size, presumably reflecting membrane instability. The firing rate remains approximately constant during the first 4 min after sealing and gradually increases afterward. Making the pipette potential more positive leads to an increase in the firing rate, with a steeper dependence on voltage if the pipette solution contains K+ as the main cation than if it contains Na+. Ca2+ imaging experiments show that establishing a cell-attached recording can result in an increased somatic Ca2+ concentration, reflecting an increased firing rate linked to an increase in the pipette–cell conductance. Pipette effects on cell firing are traced to a combination of passive electrical coupling, opening of voltage- and Ca2+-sensitive K+ channels (BK channels) after action potentials, and random activation of voltage-insensitive, presumably mechanosensitive, cationic channels. We conclude that, unless experimental conditions are optimized, cell-attached recordings in small neurons may report erroneous firing rates.

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Genetically encoded neural activity indicators Current Opinion in Neurobiology

Genetically encoded neural activity indicators Current Opinion in Neurobiology | Neuroscience_technics | Scoop.it

Recording activity from identified populations of neurons is a central goal of neuroscience. Changes in membrane depolarization, particularly action potentials, are the most important features of neural physiology to extract, although ions, neurotransmitters, neuromodulators, second messengers, and the activation state of specific proteins are also crucial. Modern fluorescence microscopy provides the basis for such activity mapping, through multi-photon imaging and other optical schemes. Probes remain the rate-limiting step for progress in this field: they should be bright and photostable, and ideally come in multiple colors. Only protein-based reagents permit chronic imaging from genetically specified cells. Here we review recent progress in the design, optimization and deployment of genetically encoded indicators for calcium ions (a proxy for action potentials), membrane potential, and neurotransmitters. We highlight seminal experiments, and present an outlook for future progress. 

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Chronic in vivo imaging in the mouse spinal cord using an implanted chamber

Chronic in vivo imaging in the mouse spinal cord using an implanted chamber | Neuroscience_technics | Scoop.it
An imaging chamber implanted over the mouse spinal cord enables long-term longitudinal two-photon microscopy of cellular dynamics in normal or pathological conditions.
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Aberration-free three-dimensional multiphoton imaging of neuronal activity at kHz rates

Aberration-free three-dimensional multiphoton imaging of neuronal activity at kHz rates | Neuroscience_technics | Scoop.it

Multiphoton microscopy is a powerful tool in neuroscience, promising to deliver important data on the spatiotemporal activity within individual neurons as well as in networks of neurons. A major limitation of current technologies is the relatively slow scan rates along the z direction compared to the kHz rates obtainable in the x and y directions. Here, we describe a custom-built microscope system based on an architecture that allows kHz scan rates over hundreds of microns in all three dimensions without introducing aberration. We further demonstrate how this high-speed 3D multiphoton imaging system can be used to study neuronal activity at millisecond resolution at the subcellular as well as the population level. (PNAS February 21, 2012)

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Genetically encoded probes for optical imaging of brain electrical activity

Genetically encoded probes for optical imaging of brain electrical activity | Neuroscience_technics | Scoop.it

The combination of optical imaging methods with targeted expression of protein-based fluorescent probes constitutes a powerful approach for functional analysis of selected cell populations within intact neuronal circuitries. Herein, we lay out the conceptual motivation for optogenetic recording of brain electrical activity using genetically encoded voltage-sensitive fluorescent proteins (VSFPs), describe how the current generation of VSFPs has evolved, and demonstrate how VSFPs report membrane voltage signals in isolated cells, brain slices, and living animals. We conclude with a critical appraisal of VSFPs for voltage recording and highlight promising applications of this emerging methodology for bridging cellular and intact systems biology. (Progress in Brain Research vol. 196, 2012)

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Optogenetic excitation of neurons with channelrhodopsins: Light instrumentation, expression systems, and channelrhodopsin variants Progress in Brain

Optogenetic excitation of neurons with channelrhodopsins: Light instrumentation, expression systems, and channelrhodopsin variants Progress in Brain | Neuroscience_technics | Scoop.it

[Review] Classically, temporally precise excitation of membrane potential in neurons within intact tissue can be achieved by direct electrical stimulation or indirect electrical stimulation induced by changing magnetic fields. Both of these approaches have a predetermined selectivity based on the biophysical properties of the nervous tissue and membrane in the region of the stimulation. A recent advance in selective excitation of neurons is the “optogenetic” approach utilizing channelrhodopsins (ChRs). By expressing the light-responsive ChR in neurons using cell-type selective promoters or other methods, specific neurons can be depolarized by light in a temporally precise manner with millisecond resolution even if their membrane biophysical properties are less favorable for electrical stimulation. In addition, ChRs can be used to depolarize nonneuronal cells in the nervous tissue, and to sustain depolarization over a prolonged period of time, both of which cannot be achieved with electrical or magnetic stimulations.

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Functional profiling of neurons through cellular neuropharmacology

Functional profiling of neurons through cellular neuropharmacology | Neuroscience_technics | Scoop.it

We describe a functional profiling strategy to identify and characterize subtypes of neurons present in a peripheral ganglion, which should be extendable to neurons in the CNS. In this study, dissociated dorsal-root ganglion neurons from mice were exposed to various pharmacological agents (challenge compounds), while at the same time the individual responses of >100 neurons were simultaneously monitored by calcium imaging. Each challenge compound elicited responses in only a subset of dorsal-root ganglion neurons. Two general types of challenge compounds were used: agonists of receptors (ionotropic and metabotropic) that alter cytoplasmic calcium concentration (receptor–agonist challenges) and compounds that affect voltage-gated ion channels (membrane–potential challenges). Notably, among the latter are K-channel antagonists, which elicited unexpectedly diverse types of calcium responses in different cells (i.e., phenotypes). We used various challenge compounds to identify several putative neuronal subtypes on the basis of their shared and/or divergent functional, phenotypic profiles. Our results indicate that multiple receptor–agonist and membrane–potential challenges may be applied to a neuronal population to identify, characterize, and discriminate among neuronal subtypes. This experimental approach can uncover constellations of plasma membrane macromolecules that are functionally coupled to confer a specific phenotypic profile on each neuronal subtype. This experimental platform has the potential to bridge a gap between systems and molecular neuroscience with a cellular-focused neuropharmacology, ultimately leading to the identification and functional characterization of all neuronal subtypes at a given locus in the nervous system.

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The gain in brain: novel imaging techniques and multiplexed proteomic imaging of brain tissue ultrastructure

The gain in brain: novel imaging techniques and multiplexed proteomic imaging of brain tissue ultrastructure | Neuroscience_technics | Scoop.it

The rapid accumulation of neuroproteomics data in recent years has prompted the emergence of novel antibody-based imaging methods that aim to understand the anatomical and functional context of the multitude of identified proteins. The pioneering field of ultrastructural multiplexed proteomic imaging now includes a number of high resolution methods, such as array tomography, stimulated emission depletion microscopy, stochastic optical reconstruction microscopy and automated transmission electron microscopy, which allow a detailed molecular characterization of individual synapses and subsynaptic structures within brain tissues for the first time. While all of these methods still face considerable limitations, a combined complementary approach building on the respective strengths of each method is possible and will enable fascinating research into the proteomic diversity of the nervous system. - Curr. Op. Neurobiology 22(1), 2012

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Optical super-resolution microscopy in neurobiology 10.1016/j.conb.2011.10.014 : Current Opinion in Neurobiology | ScienceDirect.com

Optical super-resolution microscopy in neurobiology 10.1016/j.conb.2011.10.014 : Current Opinion in Neurobiology | ScienceDirect.com | Neuroscience_technics | Scoop.it

Understanding the highly plastic nature of neurons requires the dynamic visualization of their molecular and cellular organization in a native context. However, due to the limited resolution of standard light microscopy, many of the structural specializations of neurons cannot be resolved. A recent revolution in light microscopy has given rise to several super-resolution light microscopy methods yielding 2–10-fold higher resolution than conventional microscopy. We here describe the principles behind these techniques as well as their application to the analysis of the molecular architecture of the synapse. Furthermore, we discuss the potential for continued development of super-resolution microscopy as necessary for live imaging of neuronal structure and function in the brain. - Curr. Op. Neurobiology 22(1), 2012

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Towards reliable spike-train recordings from thousands of neurons with multielectrodes

Towards reliable spike-train recordings from thousands of neurons with multielectrodes | Neuroscience_technics | Scoop.it

The new generation of silicon-based multielectrodes comprising hundreds or more electrode contacts offers unprecedented possibilities for simultaneous recordings of spike trains from thousands of neurons. Such data will not only be invaluable for finding out how neural networks in the brain work, but will likely be important also for neural prosthesis applications. This opportunity can only be realized if efficient, accurate and validated methods for automatic spike sorting are provided. In this review we describe some of the challenges that must be met to achieve this goal, and in particular argue for the critical need of realistic model data to be used as ground truth in the validation of spike-sorting algorithms. Cur Op in Neurobiology 22(1), 2012

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Serial two-photon tomography for automated ex vivo mouse brain imaging

Serial two-photon tomography for automated ex vivo mouse brain imaging | Neuroscience_technics | Scoop.it

Here we describe an automated method, named serial two-photon (STP) tomography, that achieves high-throughput fluorescence imaging of mouse brains by integrating two-photon microscopy and tissue sectioning. STP tomography generates high-resolution datasets that are free of distortions and can be readily warped in three dimensions, for example, for comparing multiple anatomical tracings. This method opens the door to routine systematic studies of neuroanatomy in mouse models of human brain disorders. Nature Methods

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Neural activity imaging with genetically encoded calcium indicators

Neural activity imaging with genetically encoded calcium indicators | Neuroscience_technics | Scoop.it

Genetically encoded calcium indicators (GECIs), together with modern microscopy, allow repeated activity measurement, in real time and with cellular resolution, of defined cellular populations. Recent efforts in protein engineering have yielded several high-quality GECIs that facilitate new applications in neuroscience. Here, we summarize recent progress in GECI design, optimization, and characterization, and provide guidelines for selecting the appropriate GECI for a given biological application. We focus on the unique challenges associated with imaging in behaving animals. (Progress in Brain Research, vol. 196, 2012)

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Genetically encoded molecular tools for light-driven silencing of targeted neurons

Genetically encoded molecular tools for light-driven silencing of targeted neurons | Neuroscience_technics | Scoop.it

The ability to silence, in a temporally precise fashion, the electrical activity of specific neurons embedded within intact brain tissue, is important for understanding the role that those neurons play in behaviors, brain disorders, and neural computations. “Optogenetic” silencers, genetically encoded molecules that, when expressed in targeted cells within neural networks, enable their electrical activity to be quieted in response to pulses of light, are enabling these kinds of causal circuit analyses studies. Two major classes of optogenetic silencer are in broad use in species ranging from worm to monkey: light-driven inward chloride pumps, or halorhodopsins, and light-driven outward proton pumps, such as archaerhodopsins and fungal light-driven proton pumps. Both classes of molecule, when expressed in neurons via viral or other transgenic means, enable the targeted neurons to be hyperpolarized by light. We here review the current status of these sets of molecules, and discuss how they are being discovered and engineered. We also discuss their expression properties, ionic properties, spectral characteristics, and kinetics. Such tools may not only find many uses in the quieting of electrical activity for basic science studies but may also, in the future, find clinical uses for their ability to safely and transiently shut down cellular electrical activity in a precise fashion. (Progress in Brain Research, vol. 196, 2012)

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Estimation of the time course of neurotransmitter release at central synapses from the first latency of postsynaptic currents

Estimation of the time course of neurotransmitter release at central synapses from the first latency of postsynaptic currents | Neuroscience_technics | Scoop.it

Measurement of the release time course (RTC) and of the quantal content is important for quantifying synaptic precision and understanding the molecular basis of the release process at central synapses. In theory, the RTC can be determined directly from the histogram of first latencies of quantal events only if a maximum of one vesicle is released per trial, but at most synapses multiple vesicles are released. Traditionally, first latency histograms have been corrected for multiple releases using a simple correction, derived by Barrett and Stevens (BS; 1972b) for quantifying release at the neuromuscular junction. This correction has also been used to quantify release at central synapses. We show, by combining an analytical approach and numerical simulations of stochastic quantal release, that the BS correction gives a biased estimate for RTC and quantal content. The bias increases with release probability, and is therefore particularly problematic for central synapses. We show that this is due to assuming infinite availability of releasable vesicles and we derive a formula for estimating the RTC from first latencies without this assumption. The resulting ‘binomial correction’ requires knowledge of the maximum number of quanta that can be released following an action potential (N), which can be estimated with variance-mean analysis. We show with simulations that estimating RTC and quantal content from first latencies using the binomial correction is robust in the presence of noise and when release probability is non-uniform. We also provide an alternative method for estimating RTC from the first latencies when N cannot be determined.

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Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes : Nature Methods : Nature Publishing Group

Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes : Nature Methods : Nature Publishing Group | Neuroscience_technics | Scoop.it
The understanding of brain computations requires methods that read out neural activity on different spatial and temporal scales. Following signal propagation and integration across a neuron and recording the concerted activity of hundreds of neurons pose distinct challenges, and the design of imaging systems has been mostly focused on tackling one of the two operations. We developed a high-resolution, acousto-optic two-photon microscope with continuous three-dimensional (3D) trajectory and random-access scanning modes that reaches near-cubic-millimeter scan range and can be adapted to imaging different spatial scales. We performed 3D calcium imaging of action potential backpropagation and dendritic spike forward propagation at sub-millisecond temporal resolution in mouse brain slices. We also performed volumetric random-access scanning calcium imaging of spontaneous and visual stimulation–evoked activity in hundreds of neurons of the mouse visual cortex in vivo. These experiments demonstrate the subcellular and network-scale imaging capabilities of our system.
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