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[Review] Highlights: - Optogenetic tools allow selective activation/silencing of specific neuronal populations.
- Inhibitory opsins have been shown to efficiently reduce epileptiform activity in brain slices.
- New strategies for controlling seizures could be explored using optogenetics.
- Technically challenging optimisation of optogenetic approaches.
- Some uncertainty in predicting outcomes of optogenetic interventions on excitability of complex neuronal networks. (...) - by Kokaia M et al., Neuropharmacology, Volume 69, June 2013, Pages 89–95
- We present Neurient, an algorithm developed to trace dense fluorescent neurite images.
- Neurient allows unsupervised image processing, with optional tunable parameters.
- Method: find seed points, index into a directed lookup table, trace neurite segments.
- Trace coordinates and orientations can be used to evaluate neurite alignment.
- The Neurient code is open source and freely available for use and customization.
by Mitchel JA et al., Journal of Neuroscience Methods, Volume 214, Issue 2, 15 April 2013, Pages 210–222
The era of Big Neuroscience has arrived.
In late January, The Human Brain Project—an attempt to create a computer simulation of the brain at every scale from the nano nano to the macro biotic—announced that it had successfully arranged a billion Euro funding package for a 10-year run. And then on Feb. 18, an article in The New York Times took the wraps off a plan to spend perhaps billions of dollars for an effort to record large collections of brain cells and figure out what exactly they are doing. (...) - by Gary Stix, Nature, 28 February 2013
[Abstract] A microfluidic chip for culturing neurons and spatially isolating axons from somas is presented for use with visually guided whole-cell electrophysiological measurements. A modular design consisting of detachable and re-sealable layers is used to satisfy the requirements of both long-term neuron culturing as well as electrophysiological measurements. Whole cell patch clamp recordings indicate functional viability of neurons with isolated axons. Fluidic isolation was used to achieve asymmetric lentiviral infection of neurons on a single side reservoir. Neurons were asymmetrically infected with lentiviruses expressing the light-activated cationic channel channelrhodopsin-2. Light-evoked excitatory postsynaptic responses were detected by whole cell recordings of neurons on the uninfected side showing functional synaptic connectivity between the two isolated but axonally connected sides of the device. - by Jokinen V. et al., Journal of Neuroscience Methods, Volume 212, Issue 2, 30 January 2013, Pages 276–282
We describe an intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) with signal-to-noise ratio and kinetics appropriate for in vivo imaging. We engineered iGluSnFR in vitro to maximize its fluorescence change, and we validated its utility for visualizing glutamate release by neurons and astrocytes in increasingly intact neurological systems. In hippocampal culture, iGluSnFR detected single field stimulus–evoked glutamate release events. In pyramidal neurons in acute brain slices, glutamate uncaging at single spines showed that iGluSnFR responds robustly and specifically to glutamate in situ, and responses correlate with voltage changes. In mouse retina, iGluSnFR-expressing neurons showed intact light-evoked excitatory currents, and the sensor revealed tonic glutamate signaling in response to light stimuli. In worms, glutamate signals preceded and predicted postsynaptic calcium transients. In zebrafish, iGluSnFR revealed spatial organization of direction-selective synaptic activity in the optic tectum. Finally, in mouse forelimb motor cortex, iGluSnFR expression in layer V pyramidal neurons revealed task-dependent single-spine activity during running. - by Marvin JS et al., Nature Methods 10, 162–170 (2013) doi:10.1038/nmeth.2333
A new way to link artificial arms and hands to the nervous system could allow the brain to control prostheses as smoothly as if they were natural limbs (...) - By D. Kacy Cullen and Douglas H. Smith, Scientific American, January 14, 2013
by Henneberger C Rusakov DA, Nature Protocols 7, 2171–2179 (2012)
[Abstract] In a single-electrode current-clamp recording, the measured potential includes both the response of the membrane and that of the measuring electrode. The electrode response is traditionally removed using bridge balance, where the response of an ideal resistor representing the electrode is subtracted from the measurement. Because the electrode is not an ideal resistor, this procedure produces capacitive transients in response to fast or discontinuous currents. More sophisticated methods exist, but they all require a preliminary calibration phase, to estimate the properties of the electrode. If these properties change after calibration, the measurements are corrupted. We propose a compensation method that does not require preliminary calibration. Measurements are compensated offline by fitting a model of the neuron and electrode to the trace and subtracting the predicted electrode response. The error criterion is designed to avoid the distortion of compensated traces by spikes. The technique allows electrode properties to be tracked over time and can be extended to arbitrary models of electrode and neuron. We demonstrate the method using biophysical models and whole cell recordings in cortical and brain-stem neurons. - by Rossant C et al., Journal of Neurophysiology, November 1, 2012 vol. 108 no. 9 2629-2639
Spontaneous postsynaptic currents (PSCs) provide key information about the mechanisms of synaptic transmission and the activity modes of neuronal networks. However, detecting spontaneous PSCs in vitro and in vivo has been challenging, because of the small amplitude, the variable kinetics, and the undefined time of generation of these events. Here, we describe a, to our knowledge, new method for detecting spontaneous synaptic events by deconvolution, using a template that approximates the average time course of spontaneous PSCs. A recorded PSC trace is deconvolved from the template, resulting in a series of delta-like functions. The maxima of these delta-like events are reliably detected, revealing the precise onset times of the spontaneous PSCs. Among all detection methods, the deconvolution-based method has a unique temporal resolution, allowing the detection of individual events in high-frequency bursts. Furthermore, the deconvolution-based method has a high amplitude resolution, because deconvolution can substantially increase the signal/noise ratio. When tested against previously published methods using experimental data, the deconvolution-based method was superior for spontaneous PSCs recorded in vivo. Using the high-resolution deconvolution-based detection algorithm, we show that the frequency of spontaneous excitatory postsynaptic currents in dentate gyrus granule cells is 4.5 times higher in vivo than in vitro. (...) - by Pernía-Andrade AJ et al., Biophysical Journal, Volume 103, Issue 7, 3 October 2012, Pages 1429–1439
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
In this post I would like to start an online discussion about a very interesting recent PNAS paper: Tracking a complete voltage-sensor cycle with metal-ion bridges by Henrion et al. I know that other people in the voltage-gated cation channel field are very interested in this paper and it also relates to many of the topics I have been discussing in my posts. I thought it would be a good idea to discuss it and a journal club type format encourages contributions from anyone who is interested. So read the paper and join in the discussion! (...) - by M Letts on LettsScience, August 26, 2012
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
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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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
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 Methods, 18 Mar 2013
Many cellular structures and organelles are too small to be properly resolved by conventional light microscopy. This is particularly true for dendritic spines and glial processes, which are very small, dynamic, and embedded in dense tissue, making it difficult to image them under realistic experimental conditions. Two-photon microscopy is currently the method of choice for imaging in thick living tissue preparations, both in acute brain slices and in vivo. However, the spatial resolution of a two-photon microscope, which is limited to ∼350 nm by the diffraction of light, is not sufficient for resolving many important details of neural morphology, such as the width of spine necks or thin glial processes. Recently developed superresolution approaches, such as stimulated emission depletion microscopy, have set new standards of optical resolution in imaging living tissue. However, the important goal of superresolution imaging with significant subdiffraction resolution has not yet been accomplished in acute brain slices. To overcome this limitation, we have developed a new microscope based on two-photon excitation and pulsed stimulated emission depletion microscopy, which provides unprecedented spatial resolution and excellent experimental access in acute brain slices using a long-working distance objective. The new microscope improves on the spatial resolution of a regular two-photon microscope by a factor of four to six, and it is compatible with time-lapse and simultaneous two-color superresolution imaging in living cells. We demonstrate the potential of this nanoscopy approach for brain slice physiology by imaging the morphology of dendritic spines and microglial cells well below the surface of acute brain slices. (...) - by Bethge P. et al., Biophysical Journal, Volume 104, Issue 4, 778-785, 19 February 2013
Highlights - The optopatcher: a new holder for simultaneous patch-clamp recording and light stimulation.
- We used the optopatcher for in vivo cortical patch-clamp recording and optogenetic activation.
- The holder can be used in multiple platforms whenever a glass pipette is used.
by Katz Y. et al., Journal of Neuroscience Methods, 28 January 2013 (In Press)
[Abstract] We recently introduced fluorescent false neurotransmitters (FFNs) as optical tracers that enable the visualization of neurotransmitter release at individual presynaptic terminals. Here, we describe a pH-responsive FFN probe, FFN102, which as a polar dopamine transporter substrate selectively labels dopamine cell bodies and dendrites in ventral midbrain and dopaminergic synaptic terminals in dorsal striatum. FFN102 exhibits greater fluorescence emission in neutral than acidic environments, and thus affords a means to optically measure evoked release of synaptic vesicle content into the extracellular space. Simultaneously, FFN102 allows the measurement of individual synaptic terminal activity by following fluorescence loss upon stimulation. Thus, FFN102 enables not only the identification of dopamine cells and their processes in brain tissue, but also the optical measurement of functional parameters including dopamine transporter activity and dopamine release at the level of individual synapses. As such, the development of FFN102 demonstrates that, by bringing together organic chemistry and neuroscience, molecular entities can be generated that match the endogenous transmitters in selectivity and distribution, allowing for the study of both the microanatomy and functional plasticity of the normal and diseased nervous system. - by Rodriguez PC et al., PNAS vol. 110 no. 3, 870–875
Northwestern researchers have developed a novel push-pen patch clamp electrode system that integrates a linear hydraulic actuator in the pipette holder. The actuator moves the metal Ag/AgCl electrode within the pipette to a position where it protrudes from the pipette orifice. This mechanism has multiple benefits in conventional whole-cell experiments. For example, it lowers the series resistance since the resistivity of the electrode is less than that of the pipette solution. The reduced series resistance permits the recording of higher bandwidth signals. Further, the push-pen operation serves as a physical structure to help remove the commonly found cellular debris clog in the pipette tip by pushing it out and clearing it. Lastly, the push-pen operation also reduces the leakage of cytosol into the pipette which results in the ability to conduct longer experiments. (...) - flintbox , Dec 14, 2012
[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
Recording simultaneously from essentially all of the relevant neurons in a local circuit is crucial to understand how they collectively represent information. Here we show that the combination of a large, dense multielectrode array and a novel, mostly automated spike-sorting algorithm allowed us to record simultaneously from a highly overlapping population of >200 ganglion cells in the salamander retina. By combining these methods with labeling and imaging, we showed that up to 95% of the ganglion cells over the area of the array were recorded. By measuring the coverage of visual space by the receptive fields of the recorded cells, we concluded that our technique captured a neural population that forms an essentially complete representation of a region of visual space. This completeness allowed us to determine the spatial layout of different cell types as well as identify a novel group of ganglion cells that responded reliably to a set of naturalistic and artificial stimuli but had no measurable receptive field. Thus, our method allows unprecedented access to the complete neural representation of visual information, a crucial step for the understanding of population coding in sensory systems. - by Marre O. et al., The Journal of Neuroscience, 24 October 2012, 32(43): 14859-14873
[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 Knöpfel, Nature Reviews Neuroscience 13, 687-700 (October 2012)
Stem Cells: Rapid neuronal differentiation
The differentiation of human pluripotent stem cells (hPSCs) into neurons in the culture dish is a long, drawn-out process, typically requiring a month or more. This is much longer than is needed for the equivalent process in the mouse system in vitro… - Chambers, S.M. et al., Nat. Biotechnol. 30, 715–720 (2012) via Nature Methods 9, 866, 30 August 2012
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
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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