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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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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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The OpenPicoAmp : an open-source planar lipid bilayer amplifier for hands-on learning of neuroscience

The OpenPicoAmp : an open-source planar lipid bilayer amplifier for hands-on learning of neuroscience | Neuroscience_technics | Scoop.it

Neuroscience education can be promoted by the availability of low cost and engaging teaching materials. To address this issue, we developed an open-source lipid bilayer amplifier, the OpenPicoAmp, which is appropriate for use in introductory courses in biophysics or neurosciences dealing with the electrical properties of the cell membrane. The amplifier is designed using the common lithographic printed circuit board fabrication process and off-the-shelf electronic components. In addition, we propose a specific design for experimental chambers allowing the insertion of a commercially available polytetrafluoroethylene film. This experimental setup can be used in simple experiments in which students monitor the bilayer formation by capacitance measurement and record unitary currents produced by ionic channels like gramicidin A. Used in combination with a low-cost data acquisition board this system provides a complete solution for hands-on lessons, therefore improving the effectiveness in teaching basic neurosciences or biophysics. - by Shlyonsky V et al., arXiv:1403.7439 2014 

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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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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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Method for stationarity-segmentation of spike train data with application to the Pearson cross-correlation

Correlations among neurons are supposed to play an important role in computation and information coding in the nervous system. Empirically, functional interactions between neurons are most commonly assessed by cross-correlation functions. Recent studies have suggested that pairwise correlations may indeed be sufficient to capture most of the information present in neural interactions. Many applications of correlation functions, however, implicitly tend to assume that the underlying processes are stationary. This assumption will usually fail for real neurons recorded in vivo since their activity during behavioral tasks is heavily influenced by stimulus-, movement-, or cognition-related processes as well as by more general processes like slow oscillations or changes in state of alertness. To address the problem of nonstationarity, we introduce a method for assessing stationarity empirically and then “slicing” spike trains into stationary segments according to the statistical definition of weak-sense stationarity. We examine pairwise Pearson cross-correlations (PCCs) under both stationary and nonstationary conditions and identify another source of covariance that can be differentiated from the covariance of the spike times and emerges as a consequence of residual nonstationarities after the slicing process: the covariance of the firing rates defined on each segment. Based on this, a correction of the PCC is introduced that accounts for the effect of segmentation. We probe these methods both on simulated data sets and on in vivo recordings from the prefrontal cortex of behaving rats. Rather than for removing nonstationarities, the present method may also be used for detecting significant events in spike trains. - by Quiroga-Lombard CS et al., Journal of Neurophysiology July 15, 2013 vol. 110 no. 2 562-572

Julien Hering, PhD's insight:

Very technical issue but very useful for researchers who are performing in vivo electrophysiology recordings!

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Contamination of current-clamp measurement of neuron capacitance by voltage-dependent phenomena

Measuring neuron capacitance is important for morphological description, conductance characterization, and neuron modeling. One method to estimate capacitance is to inject current pulses into a neuron and fit the resulting changes in membrane potential with multiple exponentials; if the neuron is purely passive, the amplitude and time constant of the slowest exponential give neuron capacitance (Major G, Evans JD, Jack JJ. Biophys J 65: 423–449, 1993). Golowasch et al. (Golowasch J, Thomas G, Taylor AL, Patel A, Pineda A, Khalil C, Nadim F. J Neurophysiol 102: 2161–2175, 2009) have shown that this is the best method for measuring the capacitance of nonisopotential (i.e., most) neurons. However, prior work has not tested for, or examined how much error would be introduced by, slow voltage-dependent phenomena possibly present at the membrane potentials typically used in such work. We investigated this issue in lobster (Panulirus interruptus) stomatogastric neurons by performing current clamp-based capacitance measurements at multiple membrane potentials. A slow, voltage-dependent phenomenon consistent with residual voltage-dependent conductances was present at all tested membrane potentials (−95 to −35 mV). This phenomenon was the slowest component of the neuron's voltage response, and failure to recognize and exclude it would lead to capacitance overestimates of several hundredfold. Most methods of estimating capacitance depend on the absence of voltage-dependent phenomena. Our demonstration that such phenomena make nonnegligible contributions to neuron responses even at well-hyperpolarized membrane potentials highlights the critical importance of checking for such phenomena in all work measuring neuron capacitance. We show here how to identify such phenomena and minimize their contaminating influence. - by White WE et al., Journal of Neurophysiologie, July 1, 2013 vol. 110 no. 1 257-268

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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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Opportunities and challenges of pluripotent stem cell neurodegenerative disease models

Opportunities and challenges of pluripotent stem cell neurodegenerative disease models | Neuroscience_technics | Scoop.it

Human neurodegenerative disorders are among the most difficult to study. In particular, the inability to readily obtain the faulty cell types most relevant to these diseases has impeded progress for decades. Recent advances in pluripotent stem cell technology now grant access to substantial quantities of disease-pertinent neurons both with and without predisposing mutations. While this suite of technologies has revolutionized the field of 'in vitro disease modeling', great care must be taken in their deployment if robust, durable discoveries are to be made. Here we review what we perceive to be several of the stumbling blocks in the use of stem cells for the study of neurological disease and offer strategies to overcome them. (...) - by Jackson Sandoe Kevin Eggan, Nature Neuroscience 16, 780–789, 25 June 2013



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Localization of single-cell current sources based on extracellular potential patterns: the spike CSD method

Localization of single-cell current sources based on extracellular potential patterns: the spike CSD method | Neuroscience_technics | Scoop.it

A new, spike CSD (sCSD) method has been developed to reveal CSD distribution of single cells during action potential generation, based on the inverse solution of the Poisson-equation. Simulations showed, that the sCSD method reconstructed the original CSD more precisely than the traditional CSD. Applying our method to spikes, measured in cat A1 cortex with a 16 channel linear probe in vivo, the cell-electrode distances were estimated and the spatio-temporal CSD distributions were reconstructed. (...) - by Zoltán Somogyvári, Dorottya Cserpán, István Ulbert and Péter Érdi, European Journal of Neuroscience, Volume 36Issue 10pages 3299–3313November 2012

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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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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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[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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Ion Channels "Methods and Protocols"

Ion Channels "Methods and Protocols" | Neuroscience_technics | Scoop.it

Every cell in our body contains a great variety and number of permeability pathways for various organic and inorganic ions, water, metabolites, nutrients, and signaling molecules. Maintenance and precise control of gating within these pathways are fundamental principles of life as these underlie basic cellular functions such as communication, contractility, and
metabolism. This book focuses on the strategies, approaches, methods, and protocols for studying a large family of proteins that form ionic channels in the plasma membrane and intracellular membranes of cells. (...) - SpringerEditorsNikita Gamper

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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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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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Idealizing Ion Channel Recordings by a Jump Segmentation Multiresolution Filter

Based on a combination of jump segmentation and statistical multiresolution analysis for dependent data, a new approach called J-SMURF to idealize ion channel recordings has been developed. It is model-free in the sense that no a-priori assumptions about the channel's characteristics have to be made; it thus complements existing methods which assume a model for the channel's dynamics, like hidden Markov models. The method accounts for the effect of an analog filter being applied before the data analysis, which results in colored noise, by adapting existing muliresolution statistics to this situation. J-SMURF's ability to denoise the signal without missing events even when the signal-to-noise ratio is low is demonstrated on simulations as well as on ion current traces obtained from gramicidin A channels reconstituted into solvent-free planar membranes. When analyzing a newly synthesized acylated system of a fatty acid modified gramicidin channel, we are able to give statistical evidence for unknown gating characteristics such as subgating. - by Hotz T et al., NanoBioscience, IEEE Transactions on  (Volume:12 ,  Issue: 4 )

Julien Hering, PhD's insight:

Great improvement of single channels electrophysiology recordings.

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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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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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A chamber for the perfusion of in vitro tissue with multiple solutions

There are currently no practical systems that allow extended regions (>5 mm2) of a tissue slice in vitro to be exposed, in isolation, to changes in ionic conditions or to pharmacological manipulation. Previous work has only achieved this at the expense of access to the tissue for recording electrodes. Here, we present a chamber that allows a tissue slice to be maintained in multiple solutions, at physiological temperatures, and preserves the ability to record from the slice. We demonstrate the effectiveness of the tissue bath with respect to minimizing the mixing of the solutions, maintaining the viability of the tissue, and preserving the ability to record from the slice simultaneously. - by Thomas MG et al.Journal of Neurophysiology July 1, 2013 vol. 110 no. 1 269-277

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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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Researchers Perform Fastest Measurements Ever Made of Ion Channel Proteins

Researchers Perform Fastest Measurements Ever Made of Ion Channel Proteins | Neuroscience_technics | Scoop.it

The miniaturization of electronics continues to create unprecedented capabilities in computer and communications applications, enabling handheld wireless devices with tremendous computing performance operating on battery power. This same miniaturization of electronic systems is also creating new opportunities in biotechnology and biophysics.

A team of researchers at Columbia Engineering has used miniaturized electronics to measure the activity of individual ion-channel proteins with temporal resolution as fine as one microsecond, producing the fastest recordings of single ion channels ever performed. Ion channels are biomolecules that allow charged atoms to flow in and out of cells, and they are an important work-horse in cell signaling, sensing, and energetics. They are also being explored for nanopore sequencing applications. As the “transistors” of living systems, they are the target of many drugs, and the ability to perform such fast measurements of these proteins will lead to new understanding of their functions.

The researchers have designed a custom integrated circuit to perform these measurements, in which an artificial cell membrane and ion channel are attached directly to the surface of the amplifier chip. The results are described in a paper published online May 1, 2013, in Nano Letters.


The Fu Foundation School of Engineering & Applied Science - Columbia University, May 20, 2103

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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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A strategy to capture and characterize the synaptic transcriptome

Here we describe a strategy designed to identify RNAs that are actively transported to synapses during learning. Our approach is based on the characterization of RNA transport complexes carried by molecular motor kinesin. Using this strategy in Aplysia, we have identified 5,657 unique sequences consisting of both coding and noncoding RNAs from the CNS. Several of these RNAs have key roles in the maintenance of synaptic function and growth. One of these RNAs, myosin heavy chain, is critical in presynaptic sensory neurons for the establishment of long-term facilitation, but not for its persistence. - by Puthanveettil SV et al., PNAS, vol.110 no 18, 7464-7469, 2013

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Small-scale isolation of synaptic vesicles from mammalian brain : Nature Protocols : Nature Publishing Group

Small-scale isolation of synaptic vesicles from mammalian brain : Nature Protocols : Nature Publishing Group | Neuroscience_technics | Scoop.it

Synaptic vesicles (SVs) are essential organelles that participate in the release of neurotransmitters from a neuron. Biochemical analysis of purified SVs was instrumental in the identification of proteins involved in exocytotic membrane fusion and neurotransmitter uptake. Although numerous protocols have been published detailing the isolation of SVs from the brain, those that give the highest-purity vesicles often have low yields. Here we describe a protocol for the small-scale isolation of SVs from mouse and rat brain. The procedure relies on standard fractionation techniques, including differential centrifugation, rate-zonal centrifugation and size-exclusion chromatography, but it has been optimized for minimal vesicle loss while maintaining a high degree of purity. The protocol can be completed in less than 1 d and allows the recovery of ∼150 μg of vesicle protein from a single mouse brain, thus allowing vesicle isolation from transgenic mice. (...) - by Ahmed S et al., Nature Protocols 8, 998–1009 (2013)

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Power failure: why small sample size undermines the reliability of neuroscience

A study with low statistical power has a reduced chance of detecting a true effect, but it is less well appreciated that low power also reduces the likelihood that a statistically significant result reflects a true effect. Here, we show that the average statistical power of studies in the neurosciences is very low. The consequences of this include overestimates of effect size and low reproducibility of results. There are also ethical dimensions to this problem, as unreliable research is inefficient and wasteful. Improving reproducibility in neuroscience is a key priority and requires attention to well-established but often ignored methodological principles. - By Button KS et al., Nature Reviews Neuroscience 14365-376 (May 2013)

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