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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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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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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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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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A microfluidic chip for axonal isolation and electrophysiological measurements

A microfluidic chip for axonal isolation and electrophysiological measurements | Neuroscience_technics | Scoop.it

[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 MethodsVolume 212, Issue 2, 30 January 2013, Pages 276–282

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[Protocol] Monitoring local synaptic activity with astrocytic patch pipettes

[Protocol] Monitoring local synaptic activity with astrocytic patch pipettes | Neuroscience_technics | Scoop.it
by Henneberger C Rusakov DA, Nature Protocols 7, 2171–2179 (2012)
Julien Hering, PhD's insight:

[Abstract] Rapid signal exchange between astroglia and neurons has emerged as a key player in neural communication in the brain. To understand the mechanisms involved, it is often important to have access to individual astrocytes while monitoring the activity of nearby synapses. Achieving this with standard electrophysiological tools is not always feasible. The protocol presented here enables the monitoring of synaptic activity using whole-cell current-clamp recordings from a local astrocyte. This approach takes advantage of the fact that the low input resistance of electrically passive astroglia allows extracellular currents to pass through the astrocytic membrane with relatively little attenuation. Once the slice preparation is ready, it takes ∼30 min to several hours to implement this protocol, depending on the experimental design, which is similar to other patch-clamp techniques. The technique presented here can be used to directly access the intracellular medium of individual astrocytes while examining synapses functioning in their immediate proximity.

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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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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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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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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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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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Novel "Push-Pen" Design for Patch-Clamp Electrode

Novel "Push-Pen" Design for Patch-Clamp Electrode | Neuroscience_technics | Scoop.it
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
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A calibration-free electrode compensation method

A calibration-free electrode compensation method | Neuroscience_technics | Scoop.it

[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

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