Neuroscience_technics
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Neuroscience_technics
Neuroscience requires a wide range of technologies
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Scooped by Julien Hering, PhD
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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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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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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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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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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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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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A Deconvolution-Based Method with High Sensitivity and Temporal Resolution for Detection of Spontaneous Synaptic Currents In Vitro and In Vivo

A Deconvolution-Based Method with High Sensitivity and Temporal Resolution for Detection of Spontaneous Synaptic Currents In Vitro and In Vivo | Neuroscience_technics | Scoop.it

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

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