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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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Safety of Halo in cultured hippocampal neurons.(A) Membrane resting potential of Halo-expressing vs. wild-type neurons (n = 19 each). (B) Membrane resistance of Halo-expressing vs. wild-type neurons. (C) Fraction of Halo-expressing (n = 22) vs. wild-type (n = 308) cultured neurons whose nuclei stained positive for the membrane-integrity assessing DNA stain ethidium homodimer-1.
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pone-0000299-g002: Safety of Halo in cultured hippocampal neurons.(A) Membrane resting potential of Halo-expressing vs. wild-type neurons (n = 19 each). (B) Membrane resistance of Halo-expressing vs. wild-type neurons. (C) Fraction of Halo-expressing (n = 22) vs. wild-type (n = 308) cultured neurons whose nuclei stained positive for the membrane-integrity assessing DNA stain ethidium homodimer-1.

Mentions: Since it is important to evaluate whether a new technology has unanticipated side effects, such as altering basal cell physiology, or increasing the propensity for cell death, we conducted several control experiments. First, we characterized the basal state of Halo-expressing neurons electrophysiologically when no light was present. When measured in darkness, there was no difference in the resting potentials of neurons expressing Halo, and those of neighboring wild-type neurons (p>0.20, t-test; n = 19 Halo-expressing cells, n = 19 wild-type cells; Fig. 2A). Similarly, membrane resistance was not significantly different between the Halo-expressing cells and the wild-type cells (p>0.70; Fig. 2B). This result suggests that basal neural activity would be little affected by the presence of Halo in the absence of light. As an independent assay for unanticipated effects on cell health, we assayed whether Halo expression for one week in cultured hippocampal neurons could lead to apoptosis, using the membrane-impermeant DNA stain ethidium homodimer-1 to detect any cell membrane breakdown that would accompany apoptotic cell death [36]. We found no difference in cell death between Halo-expressing and control wild-type neurons: 16/308 (5.2%) control neurons counted, and 1/22 (4.5%) Halo-expressing neurons counted, were labeled by ethidium homodimer-1, indicating that Halo was not toxic over the timecourse of a week of expression (χ2 = 0.02, p>0.85; Fig. 2C). Thus, along multiple axes, Halo proved to be well-tolerated by mammalian neurons, perhaps as expected given its structural similarity to the well-tolerated photostimulation protein Channelrhodopsin-2 [18], [28], [29].


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

Han X, Boyden ES - PLoS ONE (2007)

Safety of Halo in cultured hippocampal neurons.(A) Membrane resting potential of Halo-expressing vs. wild-type neurons (n = 19 each). (B) Membrane resistance of Halo-expressing vs. wild-type neurons. (C) Fraction of Halo-expressing (n = 22) vs. wild-type (n = 308) cultured neurons whose nuclei stained positive for the membrane-integrity assessing DNA stain ethidium homodimer-1.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1808431&req=5

pone-0000299-g002: Safety of Halo in cultured hippocampal neurons.(A) Membrane resting potential of Halo-expressing vs. wild-type neurons (n = 19 each). (B) Membrane resistance of Halo-expressing vs. wild-type neurons. (C) Fraction of Halo-expressing (n = 22) vs. wild-type (n = 308) cultured neurons whose nuclei stained positive for the membrane-integrity assessing DNA stain ethidium homodimer-1.
Mentions: Since it is important to evaluate whether a new technology has unanticipated side effects, such as altering basal cell physiology, or increasing the propensity for cell death, we conducted several control experiments. First, we characterized the basal state of Halo-expressing neurons electrophysiologically when no light was present. When measured in darkness, there was no difference in the resting potentials of neurons expressing Halo, and those of neighboring wild-type neurons (p>0.20, t-test; n = 19 Halo-expressing cells, n = 19 wild-type cells; Fig. 2A). Similarly, membrane resistance was not significantly different between the Halo-expressing cells and the wild-type cells (p>0.70; Fig. 2B). This result suggests that basal neural activity would be little affected by the presence of Halo in the absence of light. As an independent assay for unanticipated effects on cell health, we assayed whether Halo expression for one week in cultured hippocampal neurons could lead to apoptosis, using the membrane-impermeant DNA stain ethidium homodimer-1 to detect any cell membrane breakdown that would accompany apoptotic cell death [36]. We found no difference in cell death between Halo-expressing and control wild-type neurons: 16/308 (5.2%) control neurons counted, and 1/22 (4.5%) Halo-expressing neurons counted, were labeled by ethidium homodimer-1, indicating that Halo was not toxic over the timecourse of a week of expression (χ2 = 0.02, p>0.85; Fig. 2C). Thus, along multiple axes, Halo proved to be well-tolerated by mammalian neurons, perhaps as expected given its structural similarity to the well-tolerated photostimulation protein Channelrhodopsin-2 [18], [28], [29].

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