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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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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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Optopatcher—An electrode holder for simultaneous intracellular patch-clamp recording and optical manipulation

Optopatcher—An electrode holder for simultaneous intracellular patch-clamp recording and optical manipulation | Neuroscience_technics | Scoop.it
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)

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Single Action Potentials and Subthreshold Electrical Events Imaged in Neurons with a Fluorescent Protein Voltage Probe

Single Action Potentials and Subthreshold Electrical Events Imaged in Neurons with a Fluorescent Protein Voltage Probe | Neuroscience_technics | Scoop.it

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

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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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An optogenetic approach in epilepsy

An optogenetic approach in epilepsy | Neuroscience_technics | Scoop.it

[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., NeuropharmacologyVolume 69, June 2013, Pages 89–95
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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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