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All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins

All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins | Neuroscience_technics | Scoop.it

A combination of a sensitive blue light-gated channelrhodopsin actuator and red-shifted Arch-based voltage sensors allows all-optical electrophysiology without cross-talk in cultured neurons or brain slices. (...) - by Hochbaum DR et al., Nature Methods 11, 825–833, 22 June 2014

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Single-cell axotomy of cultured hippocampal neurons integrated in neuronal circuits

Single-cell axotomy of cultured hippocampal neurons integrated in neuronal circuits | Neuroscience_technics | Scoop.it

An understanding of the molecular mechanisms of axon regeneration after injury is key for the development of potential therapies. Single-cell axotomy of dissociated neurons enables the study of the intrinsic regenerative capacities of injured axons. This protocol describes how to perform single-cell axotomy on dissociated hippocampal neurons containing synapses. Furthermore, to axotomize hippocampal neurons integrated in neuronal circuits, we describe how to set up coculture with a few fluorescently labeled neurons. This approach allows axotomy of single cells in a complex neuronal network and the observation of morphological and molecular changes during axon regeneration. Thus, single-cell axotomy of mature neurons is a valuable tool for gaining insights into cell intrinsic axon regeneration and the plasticity of neuronal polarity of mature neurons. Dissociation of the hippocampus and plating of hippocampal neurons takes ∼2 h. Neurons are then left to grow for 2 weeks, during which time they integrate into neuronal circuits. Subsequent axotomy takes 10 min per neuron and further imaging takes 10 min per neuron. - by Gomis-Rüth S et al., Nature Protocols  9, 1028–1037 (2014) 

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Encoded multisite two-photon microscopy

Encoded multisite two-photon microscopy | Neuroscience_technics | Scoop.it

The advent of scanning two-photon microscopy (2PM) has created a fertile new avenue for noninvasive investigation of brain activity in depth. One principal weakness of this method, however, lies with the limit of scanning speed, which makes optical interrogation of action potential-like activity in a neuronal network problematic. Encoded multisite two-photon microscopy (eMS2PM), a scanless method that allows simultaneous imaging of multiple targets in depth with high temporal resolution, addresses this drawback. eMS2PM uses a liquid crystal spatial light modulator to split a high-power femto-laser beam into multiple subbeams. To distinguish them, a digital micromirror device encodes each subbeam with a specific binary amplitude modulation sequence. Fluorescence signals from all independently targeted sites are then collected simultaneously onto a single photodetector and site-specifically decoded. We demonstrate that eMS2PM can be used to image spike-like voltage transients in cultured cells and fluorescence transients (calcium signals in neurons and red blood cells in capillaries from the cortex) in depth in vivo. These results establish eMS2PM as a unique method for simultaneous acquisition of neuronal network activity. (...) - by Ducros M et al., PNAS August 6, 2013 vol. 110 no. 32 13138-13143

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Selective and regulated gene expression in murine Purkinje cells by in utero electroporation

Selective and regulated gene expression in murine Purkinje cells by in utero electroporation | Neuroscience_technics | Scoop.it

A new in utero electroporation (IUE) protocol has been developed to deliver genes preferentially into cerebellar Purkinje cells. IUE did not alter the physiological characteristics or normal synaptic plasticity of Purkinje cells. IUE allowed selective, effective, and temporally regulated expression of multiple foreign genes in Purkinje cells in vivo. (...) by Nishiyama J et al.European Journal of Neuroscience, Volume 36Issue 7pages 2867–2876October 2012

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[Protocols] In vitro imaging of primary neural cell culture from Drosophila

[Protocols] In vitro imaging of primary neural cell culture from Drosophila | Neuroscience_technics | Scoop.it

Cell culture systems are widely used for molecular, genetic and biochemical studies. Primary cell cultures of animal tissues offer the advantage that specific cell types can be studied in vitro outside of their normal environment. We provide a detailed protocol for generating primary neural cell cultures derived from larval brains of Drosophila melanogaster. The developing larval brain contains stem cells such as neural precursors and intermediate neural progenitors, as well as fully differentiated and functional neurons and glia cells. We describe how to analyze these cell types in vitro by immunofluorescent staining and scanning confocal microscopy. Cell type–specific fluorescent reporter lines and genetically encoded calcium sensors allow the monitoring of developmental, cellular processes and neuronal activity in living cells in vitro. The protocol provides a basis for functional studies of wild-type or genetically manipulated primary neural cells in culture, both in fixed and living samples. The entire procedure takes ∼3 weeks. - by Egger B et al., Nature Protocols 8, 958–965 (2013)

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Whole brain cellular-level activity mapping in a second

Whole brain cellular-level activity mapping in a second | Neuroscience_technics | Scoop.it

It is now possible to map the activity of nearly all the neurons in a vertebrate brain at cellular resolution. What does this mean for neuroscience research and projects like the Brain Activity Map proposal?

In an Article that just went live in Nature Methods, Misha Ahrens and Philipp Keller from HHMI’s Janelia Farm Research Campus used high-speed light sheet microscopy to image the activity of 80% of the neurons in the brain of a fish larva at speeds of a whole brain every 1.3 seconds. This represents—to our knowledge—the first technology that achieves whole brain imaging of a vertebrate brain at cellular resolution with speeds that approximate neural activity patterns and behavior. (...) - by erika pastrana, Nature Methods18 Mar 2013

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Whole-brain functional imaging at cellular resolution using light-sheet microscopy by Misha B Ahrens Philipp J Keller in Nature Methods (2013) doi:10.1038/nmeth.2434


See the movie : http://ht.ly/jk9iL

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High-fidelity optical reporting of neuronal electrical activity with an ultrafast fluorescent voltage sensor

High-fidelity optical reporting of neuronal electrical activity with an ultrafast fluorescent voltage sensor | Neuroscience_technics | Scoop.it

In this technical report, St-Pierre and colleagues introduce a new genetically encoded voltage sensor called Accelerated Sensor of Action Potentials 1 (ASAP1), which consists of a circularly permuted GFP inserted in the extracellular voltage-sensing domain of a phosphatase. ASAP1 surpasses existing sensors in reliably detecting single action potentials and tracking subthreshold potentials and high-frequency spike trains. (...) -  by St-Pierre F. et al., Nature Neuroscience 17, 884–889 (2014)

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Nanoscale-Targeted Patch-Clamp Recordings of Functional Presynaptic Ion Channels

Nanoscale-Targeted Patch-Clamp Recordings of Functional Presynaptic Ion Channels | Neuroscience_technics | Scoop.it

Direct electrical access to presynaptic ion channels has hitherto been limited to large specialized terminals such as the calyx of Held or hippocampal mossy fiber bouton. The electrophysiology and ion-channel complement of far more abundant small synaptic terminals (≤1 μm) remain poorly understood. Here we report a method based on superresolution scanning ion conductance imaging of small synapses in culture at approximately 100–150 nm 3D resolution, which allows presynaptic patch-clamp recordings in all four configurations (cell-attached, inside-out, outside-out, and whole-cell). Using this technique, we report presynaptic recordings of K+, Na+, Cl, and Ca2+ channels. This semiautomated approach allows direct investigation of the distribution and properties of presynaptic ion channels at small central synapses. - by Novak P. et al., Neuron Volume 79, Issue 6, 18 September 2013, Pages 1067–1077

Julien Hering, PhD's insight:

This semiautomated method seems to be robust and accesible to low experienced scientists in electrophysiology. The nanoscale resolution opens a window on the physiology of small presynaptic boutons and the ion channels distribution and properties in these neuronal terminals. - Article in open access !

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A new nonscanning confocal microscopy module for functional voltage-sensitive dye and Ca2+ imaging of neuronal circuit activity

Recent advances in fluorescent confocal microscopy and voltage-sensitive and Ca2+ dyes have vastly improved our ability to image neuronal circuits. However, existing confocal systems are not fast enough or too noisy for many live-cell functional imaging studies. Here, we describe and demonstrate the function of a novel, nonscanning confocal microscopy module. The optics, which are designed to fit the standard camera port of the Olympus BX51WI epifluorescent microscope, achieve a high signal-to-noise ratio (SNR) at high temporal resolution, making this configuration ideal for functional imaging of neuronal activities such as the voltage-sensitive dye (VSD) imaging. The optics employ fixed 100- × 100-pinhole arrays at the back focal plane (optical conjugation plane), above the tube lens of a usual upright microscope. The excitation light travels through these pinholes, and the fluorescence signal, emitted from subject, passes through corresponding pinholes before exciting the photodiodes of the imager: a 100- × 100-pixel metal-oxide semiconductor (MOS)-type pixel imager with each pixel corresponding to a single 100- × 100-μm photodiode. This design eliminated the need for a scanning device; therefore, acquisition rate of the imager (maximum rate of 10 kHz) is the only factor limiting acquisition speed. We tested the application of the system for VSD and Ca2+ imaging of evoked neuronal responses on electrical stimuli in rat hippocampal slices. The results indicate that, at least for these applications, the new microscope maintains a high SNR at image acquisition rates of ≤0.3 ms per frame. (...) - by Tominaga T & Tominaga Y, Journal Of Neurophysiologie July 15, 2013 vol. 110 no. 2 553-561

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Viral transduction of the neonatal brain delivers controllable genetic mosaicism for visualising and manipulating neuronal circuits in vivo

Viral transduction of the neonatal brain delivers controllable genetic mosaicism for visualising and manipulating neuronal circuits in vivo | Neuroscience_technics | Scoop.it

Freehand injection of recombinant AAV into the neonatal mouse brain offers a fast and easy way to attain widespread genetic manipulation of neurons throughout the brain. Rapid onset and year-long persistence of viral expression permits study of both critical periods and aging. Viral titer can be used to control mosaicism, and multiple viruses can be co-injected for bigenic expression. The technique’s simplicity and the availability of viral reagents should facilitate a range of experiments. (...) - by Ji-Yoen Kim et al.European Journal of Neuroscience, Volume 37Issue 8pages 1203–1220April 2013

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See-through brains clarify connections

See-through brains clarify connections | Neuroscience_technics | Scoop.it

Technique to make tissue transparent offers three-dimensional view of neural networks.


A chemical treatment that turns whole organs transparent offers a big boost to the field of ‘connectomics’ — the push to map the brain’s fiendishly complicated wiring. Scientists could use the technique to view large networks of neurons with unprecedented ease and accuracy. The technology also opens up new research avenues for old brains that were saved from patients and healthy donors. (...) - by Helen Shen, Nature News, 10 April 2013


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Catherine McAnally's curator insight, October 14, 12:56 PM

Amazing technology!

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Genetically encoded optical indicators for the analysis of neuronal circuits

[Review] In a departure from previous top-down or bottom-up strategies used to understand neuronal circuits, many forward-looking research programs now place the circuit itself at their centre. This has led to an emphasis on the dissection and elucidation of neuronal circuit elements and mechanisms, and on studies that ask how these circuits generate behavioural outputs. This movement towards circuit-centric strategies is progressing rapidly as a result of technological advances that combine genetic manipulation with light-based methods. The core tools of these new approaches are genetically encoded optical indicators and actuators that enable non-destructive interrogation and manipulation of neuronal circuits in behaving animals with cellular-level precision. This Review examines genetically encoded reporters of neuronal function and assesses their value for circuit-oriented neuroscientific investigations. (...) - by Thomas KnöpfelNature Reviews Neuroscience 13, 687-700 (October 2012)

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