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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 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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An optimized fluorescent probe for visualizing glutamate neurotransmission

An optimized fluorescent probe for visualizing glutamate neurotransmission | Neuroscience_technics | Scoop.it

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 

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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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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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Fluorescent dopamine tracer resolves individual dopaminergic synapses and their activity in the brain

Fluorescent dopamine tracer resolves individual dopaminergic synapses and their activity in the brain | Neuroscience_technics | Scoop.it

[AbstractWe 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, 870875


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