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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 naturalistic trains of inhibitory events.(A) Three voltage traces of a representative current-clamped hippocampal neuron, exposed to a Poisson train of yellow light pulses. Each light pulse lasts 10 ms, and the Poisson train has a mean inter-pulse interval of λ = 100 ms. (B) Voltage traces of three different representative current-clamped neurons exposed to the same Poisson train of light pulses (λ = 100 ms). (C) Properties of hyperpolarization events elicited by Poisson trains with inter-pulse interval λ = 100 ms (i, ii) and λ = 200 ms (iii, iv), plotted versus onset time of each light pulse. Plots (i) and (iii) show the peak amplitude of each hyperpolarization event (black symbols), as well as the across-trials standard deviation of these amplitude values across ten trials (gray symbols). Plots (ii) and (iv) show the latency between the onset time of the light pulse and the time of the hyperpolarization peak (black symbols), as well as the across-trials standard deviation of these timing values across ten trials (gray symbols). In this panel, plotted points are across-neuron mean±S.E.M. (n = 5 neurons). (D) Comparison of the peak hyperpolarization (i) and the time-to-peak (ii) data between the beginning (first5) and end (last5) of each Poisson train, for the n = 5 neurons described in Fig. 3C. In (i): for each neuron, the average of the first 5 or last 5 hyperpolarization peaks (black) or the across-trials standard deviation of these amplitude values (gray) was first computed, then the across-neuron mean±S.E.M. was plotted. In (ii): for each neuron, the average of the first 5 or last 5 times-to-peak (black) or the across-trials standard deviation of these times-to-peak (gray) were first computed, then the across-neuron mean±S.E.M. was plotted. For (ii), the gray bars were stacked on top of the black ones for ease of visualization.
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pone-0000299-g003: Halo-mediated naturalistic trains of inhibitory events.(A) Three voltage traces of a representative current-clamped hippocampal neuron, exposed to a Poisson train of yellow light pulses. Each light pulse lasts 10 ms, and the Poisson train has a mean inter-pulse interval of λ = 100 ms. (B) Voltage traces of three different representative current-clamped neurons exposed to the same Poisson train of light pulses (λ = 100 ms). (C) Properties of hyperpolarization events elicited by Poisson trains with inter-pulse interval λ = 100 ms (i, ii) and λ = 200 ms (iii, iv), plotted versus onset time of each light pulse. Plots (i) and (iii) show the peak amplitude of each hyperpolarization event (black symbols), as well as the across-trials standard deviation of these amplitude values across ten trials (gray symbols). Plots (ii) and (iv) show the latency between the onset time of the light pulse and the time of the hyperpolarization peak (black symbols), as well as the across-trials standard deviation of these timing values across ten trials (gray symbols). In this panel, plotted points are across-neuron mean±S.E.M. (n = 5 neurons). (D) Comparison of the peak hyperpolarization (i) and the time-to-peak (ii) data between the beginning (first5) and end (last5) of each Poisson train, for the n = 5 neurons described in Fig. 3C. In (i): for each neuron, the average of the first 5 or last 5 hyperpolarization peaks (black) or the across-trials standard deviation of these amplitude values (gray) was first computed, then the across-neuron mean±S.E.M. was plotted. In (ii): for each neuron, the average of the first 5 or last 5 times-to-peak (black) or the across-trials standard deviation of these times-to-peak (gray) were first computed, then the across-neuron mean±S.E.M. was plotted. For (ii), the gray bars were stacked on top of the black ones for ease of visualization.

Mentions: We next probed whether the fast response times of Halo could support naturalistic sequences of hyperpolarization events, in response to trains of brief pulses of yellow light. Figure 3A shows three traces of hyperpolarization events elicited in a single neuron, resulting from repeatedly playing back a Poisson train (mean inter-pulse interval, λ = 100 ms, 59 pulses), of 10 ms-duration yellow light pulses, to illustratively simulate stochastic inhibitory (e.g., GABAergic) synaptic input. Figure 3B shows three such hyperpolarization traces, taken from different neurons. The variability of such trains was remarkably low in many regards–across ten repeated trials in a single cell, across multiple cells (n = 5 neurons), and over time throughout a sustained train of 59 pulses (Fig. 3C, 3D). Notably, we found that for hyperpolarizations elicited by 10 ms-duration light pulses during a λ = 100 ms Poisson train, the mean amplitude was −4.56 mV (averaged across trials and neurons), but the trial-to-trial standard deviation of this amplitude was only 400 µV (averaged across neurons, Fig 3Ci and Fig. 3Di, left side). The trial-to-trial jitter of the time the hyperpolarization took to reach its peak value was also small, 1.27 ms (averaged across neurons, Fig. 3Cii and Fig. 3Dii, left side). The neuron-to-neuron variability of amplitude and timing was somewhat larger than the trial-to-trial variability, with standard deviations of 1.45 mV and 1.78 ms, respectively, but these values nevertheless demonstrated that precise inhibitory control of a population of neurons could take place with millivolt and millisecond resolution. Finally, we quantitatively examined the through-train sustainability of light-elicited voltage changes, by comparing the amplitude mean and amplitude variability, and timing variability, between the hyperpolarization events elicited by the first five light pulses in the 59-pulse train, and the last five light pulses in the train (Figs. 3Di and 3Dii, left side). No difference was seen for any of these statistics, compared between the events elicited at the beginning vs. end of the train (p>0.10 for all measures, t-test). Identical conclusions held for the λ = 200 ms Poisson train with 46 pulses (Figs. 3Ciii and 3Civ, and Figs. 3Di and 3Dii, right side). The high temporal and amplitude fidelity of Halo-mediated hyperpolarizations suggests that Halo may be an ideal tool for simulating many forms of synaptic inhibition.


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

Han X, Boyden ES - PLoS ONE (2007)

Halo-mediated naturalistic trains of inhibitory events.(A) Three voltage traces of a representative current-clamped hippocampal neuron, exposed to a Poisson train of yellow light pulses. Each light pulse lasts 10 ms, and the Poisson train has a mean inter-pulse interval of λ = 100 ms. (B) Voltage traces of three different representative current-clamped neurons exposed to the same Poisson train of light pulses (λ = 100 ms). (C) Properties of hyperpolarization events elicited by Poisson trains with inter-pulse interval λ = 100 ms (i, ii) and λ = 200 ms (iii, iv), plotted versus onset time of each light pulse. Plots (i) and (iii) show the peak amplitude of each hyperpolarization event (black symbols), as well as the across-trials standard deviation of these amplitude values across ten trials (gray symbols). Plots (ii) and (iv) show the latency between the onset time of the light pulse and the time of the hyperpolarization peak (black symbols), as well as the across-trials standard deviation of these timing values across ten trials (gray symbols). In this panel, plotted points are across-neuron mean±S.E.M. (n = 5 neurons). (D) Comparison of the peak hyperpolarization (i) and the time-to-peak (ii) data between the beginning (first5) and end (last5) of each Poisson train, for the n = 5 neurons described in Fig. 3C. In (i): for each neuron, the average of the first 5 or last 5 hyperpolarization peaks (black) or the across-trials standard deviation of these amplitude values (gray) was first computed, then the across-neuron mean±S.E.M. was plotted. In (ii): for each neuron, the average of the first 5 or last 5 times-to-peak (black) or the across-trials standard deviation of these times-to-peak (gray) were first computed, then the across-neuron mean±S.E.M. was plotted. For (ii), the gray bars were stacked on top of the black ones for ease of visualization.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1808431&req=5

pone-0000299-g003: Halo-mediated naturalistic trains of inhibitory events.(A) Three voltage traces of a representative current-clamped hippocampal neuron, exposed to a Poisson train of yellow light pulses. Each light pulse lasts 10 ms, and the Poisson train has a mean inter-pulse interval of λ = 100 ms. (B) Voltage traces of three different representative current-clamped neurons exposed to the same Poisson train of light pulses (λ = 100 ms). (C) Properties of hyperpolarization events elicited by Poisson trains with inter-pulse interval λ = 100 ms (i, ii) and λ = 200 ms (iii, iv), plotted versus onset time of each light pulse. Plots (i) and (iii) show the peak amplitude of each hyperpolarization event (black symbols), as well as the across-trials standard deviation of these amplitude values across ten trials (gray symbols). Plots (ii) and (iv) show the latency between the onset time of the light pulse and the time of the hyperpolarization peak (black symbols), as well as the across-trials standard deviation of these timing values across ten trials (gray symbols). In this panel, plotted points are across-neuron mean±S.E.M. (n = 5 neurons). (D) Comparison of the peak hyperpolarization (i) and the time-to-peak (ii) data between the beginning (first5) and end (last5) of each Poisson train, for the n = 5 neurons described in Fig. 3C. In (i): for each neuron, the average of the first 5 or last 5 hyperpolarization peaks (black) or the across-trials standard deviation of these amplitude values (gray) was first computed, then the across-neuron mean±S.E.M. was plotted. In (ii): for each neuron, the average of the first 5 or last 5 times-to-peak (black) or the across-trials standard deviation of these times-to-peak (gray) were first computed, then the across-neuron mean±S.E.M. was plotted. For (ii), the gray bars were stacked on top of the black ones for ease of visualization.
Mentions: We next probed whether the fast response times of Halo could support naturalistic sequences of hyperpolarization events, in response to trains of brief pulses of yellow light. Figure 3A shows three traces of hyperpolarization events elicited in a single neuron, resulting from repeatedly playing back a Poisson train (mean inter-pulse interval, λ = 100 ms, 59 pulses), of 10 ms-duration yellow light pulses, to illustratively simulate stochastic inhibitory (e.g., GABAergic) synaptic input. Figure 3B shows three such hyperpolarization traces, taken from different neurons. The variability of such trains was remarkably low in many regards–across ten repeated trials in a single cell, across multiple cells (n = 5 neurons), and over time throughout a sustained train of 59 pulses (Fig. 3C, 3D). Notably, we found that for hyperpolarizations elicited by 10 ms-duration light pulses during a λ = 100 ms Poisson train, the mean amplitude was −4.56 mV (averaged across trials and neurons), but the trial-to-trial standard deviation of this amplitude was only 400 µV (averaged across neurons, Fig 3Ci and Fig. 3Di, left side). The trial-to-trial jitter of the time the hyperpolarization took to reach its peak value was also small, 1.27 ms (averaged across neurons, Fig. 3Cii and Fig. 3Dii, left side). The neuron-to-neuron variability of amplitude and timing was somewhat larger than the trial-to-trial variability, with standard deviations of 1.45 mV and 1.78 ms, respectively, but these values nevertheless demonstrated that precise inhibitory control of a population of neurons could take place with millivolt and millisecond resolution. Finally, we quantitatively examined the through-train sustainability of light-elicited voltage changes, by comparing the amplitude mean and amplitude variability, and timing variability, between the hyperpolarization events elicited by the first five light pulses in the 59-pulse train, and the last five light pulses in the train (Figs. 3Di and 3Dii, left side). No difference was seen for any of these statistics, compared between the events elicited at the beginning vs. end of the train (p>0.10 for all measures, t-test). Identical conclusions held for the λ = 200 ms Poisson train with 46 pulses (Figs. 3Ciii and 3Civ, and Figs. 3Di and 3Dii, right side). The high temporal and amplitude fidelity of Halo-mediated hyperpolarizations suggests that Halo may be an ideal tool for simulating many forms of synaptic inhibition.

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