Neuroscience_technics
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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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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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Seeing the forest tree by tree: super-resolution light microscopy meets the neurosciences

Seeing the forest tree by tree: super-resolution light microscopy meets the neurosciences | Neuroscience_technics | Scoop.it

Light microscopy can be applied in vivo and can sample large tissue volumes, features crucial for the study of single neurons and neural circuits. However, light microscopy per se is diffraction-limited in resolution, and the substructure of core signaling compartments of neuronal circuits—axons, presynaptic active zones, postsynaptic densities and dendritic spines—can be only insufficiently characterized by standard light microscopy. Recently, several forms of super-resolution light microscopy breaking the diffraction-imposed resolution limit have started to allow highly resolved, dynamic imaging in the cell-biologically highly relevant 10–100 nanometer range ('mesoscale'). New, sometimes surprising answers concerning how protein mobility and protein architectures shape neuronal communication have already emerged. Here we start by briefly introducing super-resolution microscopy techniques, before we describe their use in the analysis of neuronal compartments. We conclude with long-term prospects for super-resolution light microscopy in the molecular and cellular neurosciences.(...) - by Marta Maglione & Stephan J SigristNature Neuroscience 16, 790–797, 25 June 2013

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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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Single Action Potentials and Subthreshold Electrical Events Imaged in Neurons with a Fluorescent Protein Voltage Probe

Single Action Potentials and Subthreshold Electrical Events Imaged in Neurons with a Fluorescent Protein Voltage Probe | Neuroscience_technics | Scoop.it

Monitoring neuronal electrical activity using fluorescent protein-based voltage sensors has been limited by small response magnitudes and slow kinetics of existing probes. Here we report the development of a fluorescent protein voltage sensor, named ArcLight, and derivative probes that exhibit large changes in fluorescence intensity in response to voltage changes. ArcLight consists of the voltage-sensing domain of Ciona intestinalis voltage-sensitive phosphatase and super ecliptic pHluorin that carries the point mutation A227D. The fluorescence intensity of ArcLight A242 decreases by 35% in response to a 100mV depolarization when measured in HEK293 cells, which is more than five times larger than the signals from previously reported fluorescent protein voltage sensors. We show that the combination of signal size and response speed of these new probes allows the reliable detection of single action potentials and excitatory potentials in individual neurons and dendrites. - by Jin L et al., Neuron 75(5-6),  September 2012, Pages 779–785

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Conversion of Channelrhodopsin into a Light-Gated Chloride Channel

The field of optogenetics uses channelrhodopsins (ChRs) for light-induced neuronal activation. However, optimized tools for cellular inhibition at moderate light levels are lacking. We found that replacement of E90 in the central gate of ChR with positively charged residues produces chloride-conducting ChRs (ChloCs) with only negligible cation conductance. Molecular dynamics modeling unveiled that a high-affinity Cl–-binding site had been generated near the gate. Stabilizing the open state dramatically increased the operational light sensitivity of expressing cells (slow ChloC). In CA1 pyramidal cells, ChloCs completely inhibited action potentials triggered by depolarizing current injections or synaptic stimulation. Thus, by inverting the charge of the selectivity filter, we have created a class of directly light-gated anion channels that can be used to block neuronal output in a fully reversible fashion. - by Wietek J. et al., Science 25 April 2014: Vol. 344 no. 6182 pp. 409-412

Julien Hering, PhD's insight:
Silencing Neurons with Light 

Neural networks control the activity of living individuals as central processing units control the functions of modern computers. In a neuronal circuit, information is transmitted through neurons in the form of an action potential, which is the electric potential difference between the inside and the outside of a neuron. Ion channel proteins in the neuronal membrane act as molecular devices that create and regulate action potentials. A technology called optogenetics  allows neuronal circuits to be manipulated by a combination of optics and genetically targeted incorporation of microbial retinal binding proteins, called opsins, into neurons. On pages 409 and 420 of this Science issue, Wietek et al. use structure-based molecular engineering to invert the charge selectivity of different opsins, channelrhodopsins from algae, resulting in much improved neuron silencers for use in optogenetics.

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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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Targeting neurons and photons for optogenetics

Targeting neurons and photons for optogenetics | Neuroscience_technics | Scoop.it

activity in genetically or functionally defined neurons with millisecond precision. Harnessing the full potential of optogenetic tools, however, requires light to be targeted to the right neurons at the right time. Here we discuss some barriers and potential solutions to this problem. We review methods for targeting the expression of light-activatable molecules to specific cell types, under genetic, viral or activity-dependent control. Next we explore new ways to target light to individual neurons to allow their precise activation and inactivation. These techniques provide a precision in the temporal and spatial activation of neurons that was not achievable in previous experiments. In combination with simultaneous recording and imaging techniques, these strategies will allow us to mimic the natural activity patterns of neurons in vivo, enabling previously impossible 'dream experiments'. (...) - by Packer AM et al., Nature Neuroscience 16, 805–815 (2013)

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Towards Neuronal Organoids: A Method for Long-Term Culturing of High-Density Hippocampal Neurons [openaccess]

Towards Neuronal Organoids: A Method for Long-Term Culturing of High-Density Hippocampal Neurons [openaccess] | Neuroscience_technics | Scoop.it

One of the goals in neuroscience is to obtain tractable laboratory cultures that closely recapitulate in vivo systems while still providing ease of use in the lab. Because neurons can exist in the body over a lifetime, long-term culture systems are necessary so as to closely mimic the physiological conditions under laboratory culture conditions. Ideally, such a neuronal organoid culture would contain multiple cell types, be highly differentiated, and have a high density of interconnected cells. However, before these types of cultures can be created, certain problems associated with long-term neuronal culturing must be addressed. We sought to develop a new protocol which may further prolong the duration and integrity of E18 rat hippocampal cultures. We have developed a protocol that allows for culturing of E18 hippocampal neurons at high densities for more than 120 days. These cultured hippocampal neurons are (i) well differentiated with high numbers of synapses, (ii) anchored securely to their substrate, (iii) have high levels of functional connectivity, and (iv) form dense multi-layered cellular networks. We propose that our culture methodology is likely to be effective for multiple neuronal subtypes–particularly those that can be grown in Neurobasal/B27 media. This methodology presents new avenues for long-term functional studies in neurons. - by Todd GK et al., PLoS ONE 8(4): e58996. doi:10.1371/journal.pone.0058996

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Biolistic delivery of voltage-sensitive dyes for fast recording of membrane potential changes in individual neurons in rat brain slices

Biolistic delivery of voltage-sensitive dyes for fast recording of membrane potential changes in individual neurons in rat brain slices | Neuroscience_technics | Scoop.it

[Abstract] Optical recording of membrane potential changes with fast voltage-sensitive dyes (VSDs) in neurons is one of the very few available methods for studying the generation and propagation of electrical signals to the distant compartments of excitable cells. The more lipophilic is the VSD, the better signal-to-noise ratio of the optical signal can be achieved. At present there are no effective ways to deliver water-insoluble dyes into the membranes of live cells. Here, we report a possibility to stain individual live neurons with highly lipophilic VSDs in acute brain slices using biolistic delivery. We tested four ANEP-based VSDs with different lipophilic properties and showed their ability to stain single neurons in a slice area of up to 150 μm in diameter after being delivered by a biolistic apparatus. In the slices of neocortex and hippocampus, the two most lipophilic dyes, di-8-ANEPPS and di-12-ANEPPQ, showed cell-specific loading and Golgi-like staining patterns with minimal background fluorescence. Simultaneous patch-clamp and optical recording of biolistically stained neurons demonstrated a good match of optical and electrical signals both for spontaneous APs (action potentials) and stimulus-evoked events. Our results demonstrate the high efficiency of a fast and targeted method of biolistic delivery of lipophilic VSDs for optical signals recording from mammalian neurons in vitro. - by Nikolay Aseyev et al., Journal of Neuroscience Methods, Volume 212, Issue 1, 15 January 2013, Pages 17–27

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