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Multiple-color optical activation, silencing, and desynchronization of neural activity, with single-spike temporal resolution.

Han X, Boyden ES - PLoS ONE (2007)

Bottom Line: The quest to determine how precise neural activity patterns mediate computation, behavior, and pathology would be greatly aided by a set of tools for reliably activating and inactivating genetically targeted neurons, in a temporally precise and rapidly reversible fashion.Having earlier adapted a light-activated cation channel, channelrhodopsin-2 (ChR2), for allowing neurons to be stimulated by blue light, we searched for a complementary tool that would enable optical neuronal inhibition, driven by light of a second color.The Halo/ChR2 system thus constitutes a powerful toolbox for multichannel photoinhibition and photostimulation of virally or transgenically targeted neural circuits without need for exogenous chemicals, enabling systematic analysis and engineering of the brain, and quantitative bioengineering of excitable cells.

View Article: PubMed Central - PubMed

Affiliation: Stanford University School of Medicine, Stanford, California, United States of America.

ABSTRACT
The quest to determine how precise neural activity patterns mediate computation, behavior, and pathology would be greatly aided by a set of tools for reliably activating and inactivating genetically targeted neurons, in a temporally precise and rapidly reversible fashion. Having earlier adapted a light-activated cation channel, channelrhodopsin-2 (ChR2), for allowing neurons to be stimulated by blue light, we searched for a complementary tool that would enable optical neuronal inhibition, driven by light of a second color. Here we report that targeting the codon-optimized form of the light-driven chloride pump halorhodopsin from the archaebacterium Natronomas pharaonis (hereafter abbreviated Halo) to genetically-specified neurons enables them to be silenced reliably, and reversibly, by millisecond-timescale pulses of yellow light. We show that trains of yellow and blue light pulses can drive high-fidelity sequences of hyperpolarizations and depolarizations in neurons simultaneously expressing yellow light-driven Halo and blue light-driven ChR2, allowing for the first time manipulations of neural synchrony without perturbation of other parameters such as spiking rates. The Halo/ChR2 system thus constitutes a powerful toolbox for multichannel photoinhibition and photostimulation of virally or transgenically targeted neural circuits without need for exogenous chemicals, enabling systematic analysis and engineering of the brain, and quantitative bioengineering of excitable cells.

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Halo-mediated silencing of neuronal spiking.(A) Light-driven spike blockade, demonstrated for a representative hippocampal neuron. Top (“I-injection”), neuronal firing of 20 spikes at 5 Hz, induced by pulsed somatic current injection (∼300 pA, 4 ms). Middle (“Light”), membrane hyperpolarization induced by two periods of yellow light, timed so as to be capable of blocking spikes 7–11 and spike 17, out of the train of 20 spikes. Bottom (“I-injection±Light”), yellow light drives Halo to block neuron spiking (note absence of spikes 7–11 and of spike 17), while leaving spikes elicited during periods of darkness largely intact. (B) Population data (n = 6 neurons) for light-driven, Halo-mediated spike blockade, showing high spike probability during periods of darkness (spikes 1–6, 12–16, and 18–20), and low spike probability during periods of yellow light illumination (spikes 7–11 and spike 17). Error bars (S.E.M.) are smaller than the points plotted.
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pone-0000299-g004: Halo-mediated silencing of neuronal spiking.(A) Light-driven spike blockade, demonstrated for a representative hippocampal neuron. Top (“I-injection”), neuronal firing of 20 spikes at 5 Hz, induced by pulsed somatic current injection (∼300 pA, 4 ms). Middle (“Light”), membrane hyperpolarization induced by two periods of yellow light, timed so as to be capable of blocking spikes 7–11 and spike 17, out of the train of 20 spikes. Bottom (“I-injection±Light”), yellow light drives Halo to block neuron spiking (note absence of spikes 7–11 and of spike 17), while leaving spikes elicited during periods of darkness largely intact. (B) Population data (n = 6 neurons) for light-driven, Halo-mediated spike blockade, showing high spike probability during periods of darkness (spikes 1–6, 12–16, and 18–20), and low spike probability during periods of yellow light illumination (spikes 7–11 and spike 17). Error bars (S.E.M.) are smaller than the points plotted.

Mentions: We next analyzed the ability of Halo to enable rapidly-inducible and rapidly-reversible silencing of neural activity. In patch clamped neurons, we delivered trains of intracellularly-injected somatic currents (∼300 pA, lasting 4 ms each), causing the neurons to fire 20 action potentials at 5 Hz with a 100% success rate (Fig. 4A, “I-injection”). We then scheduled yellow light pulses to occur at certain phases within the somatic current-injection protocol–specifically, during the times when spikes 7 through 11, and spike 17, would normally be driven (Fig. 4A, “Light”). Finally, we presented the light pulses and the somatic current injections simultaneously (Fig. 4A, “I-injection±light”, three trials shown), and discovered that strikingly, spiking was blocked precisely during the periods of yellow light exposure, and at no other times. Most remarkably, the rapid onset and offset kinetics of Halo allowed the deletion of even single spikes–specifically, the second yellow light pulse, timed to silence just spike 17, was able to eliminate spike 17 without affecting neighboring spikes 16 or 18 at all. We repeated this experiment five times, on each of n = 6 neurons (Fig. 4B). Across all these experiments, the second pulse of yellow light reduced the probability of firing spike 17 to 3.3%, whereas neighboring spikes 16 and 18 fired 96.7% of the time; this latter success probability was not significantly different from the success rate of the first spike in the train, before any light exposure at all (χ2 = 1.02, p>0.30). In total, during periods when the yellow light was off, somatic current pulses elicited spikes 98.7% of the time, whereas during periods when the yellow light was on, somatic current pulses elicited spikes only 1.2% of the time. The temporal precision of Halo in silencing spikes therefore offers a novel method of creating ultratransient, precise, and effective inhibition of activity in genetically-specified neurons.


Multiple-color optical activation, silencing, and desynchronization of neural activity, with single-spike temporal resolution.

Han X, Boyden ES - PLoS ONE (2007)

Halo-mediated silencing of neuronal spiking.(A) Light-driven spike blockade, demonstrated for a representative hippocampal neuron. Top (“I-injection”), neuronal firing of 20 spikes at 5 Hz, induced by pulsed somatic current injection (∼300 pA, 4 ms). Middle (“Light”), membrane hyperpolarization induced by two periods of yellow light, timed so as to be capable of blocking spikes 7–11 and spike 17, out of the train of 20 spikes. Bottom (“I-injection±Light”), yellow light drives Halo to block neuron spiking (note absence of spikes 7–11 and of spike 17), while leaving spikes elicited during periods of darkness largely intact. (B) Population data (n = 6 neurons) for light-driven, Halo-mediated spike blockade, showing high spike probability during periods of darkness (spikes 1–6, 12–16, and 18–20), and low spike probability during periods of yellow light illumination (spikes 7–11 and spike 17). Error bars (S.E.M.) are smaller than the points plotted.
© Copyright Policy
Related In: Results  -  Collection

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pone-0000299-g004: Halo-mediated silencing of neuronal spiking.(A) Light-driven spike blockade, demonstrated for a representative hippocampal neuron. Top (“I-injection”), neuronal firing of 20 spikes at 5 Hz, induced by pulsed somatic current injection (∼300 pA, 4 ms). Middle (“Light”), membrane hyperpolarization induced by two periods of yellow light, timed so as to be capable of blocking spikes 7–11 and spike 17, out of the train of 20 spikes. Bottom (“I-injection±Light”), yellow light drives Halo to block neuron spiking (note absence of spikes 7–11 and of spike 17), while leaving spikes elicited during periods of darkness largely intact. (B) Population data (n = 6 neurons) for light-driven, Halo-mediated spike blockade, showing high spike probability during periods of darkness (spikes 1–6, 12–16, and 18–20), and low spike probability during periods of yellow light illumination (spikes 7–11 and spike 17). Error bars (S.E.M.) are smaller than the points plotted.
Mentions: We next analyzed the ability of Halo to enable rapidly-inducible and rapidly-reversible silencing of neural activity. In patch clamped neurons, we delivered trains of intracellularly-injected somatic currents (∼300 pA, lasting 4 ms each), causing the neurons to fire 20 action potentials at 5 Hz with a 100% success rate (Fig. 4A, “I-injection”). We then scheduled yellow light pulses to occur at certain phases within the somatic current-injection protocol–specifically, during the times when spikes 7 through 11, and spike 17, would normally be driven (Fig. 4A, “Light”). Finally, we presented the light pulses and the somatic current injections simultaneously (Fig. 4A, “I-injection±light”, three trials shown), and discovered that strikingly, spiking was blocked precisely during the periods of yellow light exposure, and at no other times. Most remarkably, the rapid onset and offset kinetics of Halo allowed the deletion of even single spikes–specifically, the second yellow light pulse, timed to silence just spike 17, was able to eliminate spike 17 without affecting neighboring spikes 16 or 18 at all. We repeated this experiment five times, on each of n = 6 neurons (Fig. 4B). Across all these experiments, the second pulse of yellow light reduced the probability of firing spike 17 to 3.3%, whereas neighboring spikes 16 and 18 fired 96.7% of the time; this latter success probability was not significantly different from the success rate of the first spike in the train, before any light exposure at all (χ2 = 1.02, p>0.30). In total, during periods when the yellow light was off, somatic current pulses elicited spikes 98.7% of the time, whereas during periods when the yellow light was on, somatic current pulses elicited spikes only 1.2% of the time. The temporal precision of Halo in silencing spikes therefore offers a novel method of creating ultratransient, precise, and effective inhibition of activity in genetically-specified neurons.

Bottom Line: The quest to determine how precise neural activity patterns mediate computation, behavior, and pathology would be greatly aided by a set of tools for reliably activating and inactivating genetically targeted neurons, in a temporally precise and rapidly reversible fashion.Having earlier adapted a light-activated cation channel, channelrhodopsin-2 (ChR2), for allowing neurons to be stimulated by blue light, we searched for a complementary tool that would enable optical neuronal inhibition, driven by light of a second color.The Halo/ChR2 system thus constitutes a powerful toolbox for multichannel photoinhibition and photostimulation of virally or transgenically targeted neural circuits without need for exogenous chemicals, enabling systematic analysis and engineering of the brain, and quantitative bioengineering of excitable cells.

View Article: PubMed Central - PubMed

Affiliation: Stanford University School of Medicine, Stanford, California, United States of America.

ABSTRACT
The quest to determine how precise neural activity patterns mediate computation, behavior, and pathology would be greatly aided by a set of tools for reliably activating and inactivating genetically targeted neurons, in a temporally precise and rapidly reversible fashion. Having earlier adapted a light-activated cation channel, channelrhodopsin-2 (ChR2), for allowing neurons to be stimulated by blue light, we searched for a complementary tool that would enable optical neuronal inhibition, driven by light of a second color. Here we report that targeting the codon-optimized form of the light-driven chloride pump halorhodopsin from the archaebacterium Natronomas pharaonis (hereafter abbreviated Halo) to genetically-specified neurons enables them to be silenced reliably, and reversibly, by millisecond-timescale pulses of yellow light. We show that trains of yellow and blue light pulses can drive high-fidelity sequences of hyperpolarizations and depolarizations in neurons simultaneously expressing yellow light-driven Halo and blue light-driven ChR2, allowing for the first time manipulations of neural synchrony without perturbation of other parameters such as spiking rates. The Halo/ChR2 system thus constitutes a powerful toolbox for multichannel photoinhibition and photostimulation of virally or transgenically targeted neural circuits without need for exogenous chemicals, enabling systematic analysis and engineering of the brain, and quantitative bioengineering of excitable cells.

Show MeSH
Related in: MedlinePlus