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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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Related in: MedlinePlus

Millisecond-timescale Halo-mediated neuronal hyperpolarization, elicited by pulses of yellow light.(A) A representative cultured hippocampal neuron expressing mammalian codon-optimized N. pharaonis halorhodopsin (abbreviated Halo) fused to GFP, under the CaMKII promoter. Scale bar, 20 µm. (B) Neuronal currents elicited by optical activation of Halo. Left, representative outward currents elicited by two 1-second pulses of yellow (560±27.5 nm) light (∼10 mW/mm2) in a voltage-clamped neuron held at −70mV. Right, population data for n = 22 neurons. In this and subsequent figures, gray bars represent mean ± standard deviation unless otherwise indicated. Yellow bars in this and subsequent figures represent the period of yellow light exposure. (C) Kinetic properties of yellow light-elicited, Halo-mediated currents from voltage-clamped neurons. (i), 15–85% current onset time; (ii), 85–15% offset time. For each measurement, data is presented from neurons held at −70 mV, −30 mV and+10 mV (left to right). In this panel, gray bars represent mean ± standard error of the mean (S.E.M.). (D) Neuronal hyperpolarizations elicited by optical activation of Halo. Left, representative membrane voltage hyperpolarizations elicited by two 1-second pulses of yellow light, in a current-clamped neuron held at resting membrane potential. Right, population data for n = 19 neurons. (E) Kinetic properties of yellow light-elicited, Halo-mediated hyperpolarizations from current-clamped neurons, including both 15–85% voltage change onset time and 85–15% offset time.
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pone-0000299-g001: Millisecond-timescale Halo-mediated neuronal hyperpolarization, elicited by pulses of yellow light.(A) A representative cultured hippocampal neuron expressing mammalian codon-optimized N. pharaonis halorhodopsin (abbreviated Halo) fused to GFP, under the CaMKII promoter. Scale bar, 20 µm. (B) Neuronal currents elicited by optical activation of Halo. Left, representative outward currents elicited by two 1-second pulses of yellow (560±27.5 nm) light (∼10 mW/mm2) in a voltage-clamped neuron held at −70mV. Right, population data for n = 22 neurons. In this and subsequent figures, gray bars represent mean ± standard deviation unless otherwise indicated. Yellow bars in this and subsequent figures represent the period of yellow light exposure. (C) Kinetic properties of yellow light-elicited, Halo-mediated currents from voltage-clamped neurons. (i), 15–85% current onset time; (ii), 85–15% offset time. For each measurement, data is presented from neurons held at −70 mV, −30 mV and+10 mV (left to right). In this panel, gray bars represent mean ± standard error of the mean (S.E.M.). (D) Neuronal hyperpolarizations elicited by optical activation of Halo. Left, representative membrane voltage hyperpolarizations elicited by two 1-second pulses of yellow light, in a current-clamped neuron held at resting membrane potential. Right, population data for n = 19 neurons. (E) Kinetic properties of yellow light-elicited, Halo-mediated hyperpolarizations from current-clamped neurons, including both 15–85% voltage change onset time and 85–15% offset time.

Mentions: We first created a fusion protein comprising the mammalian codon-optimized form of N. pharaonis halorhodopsin (Halo), with EGFP added in-frame at the C-terminus for ease of visualization (see Methods for details). When expressed in cultured hippocampal neurons using the CaMKII promoter, which targets excitatory neurons of the forebrain [35], Halo-EGFP fluoresced brightly, and appeared evenly distributed around the neuron (Fig. 1A). When exposed to ∼10 mW/mm2 yellow light (from a xenon lamp, filtered by a standard Texas red excitation filter from Chroma, bandpass 560±27.5 nm), voltage-clamped hippocampal neurons expressing Halo experienced outward currents with rapid onset, stable steady-state amplitude, and abrupt shut-off after cessation of illumination. No supplementation of culture or recording media with the essential halorhodopsin cofactor all-trans retinal was necessary for strong currents to be elicited, consistent with prior work that demonstrated high enough levels of all-trans retinal in mammalian culture and brain to enable type I opsin functionality [18], [28]. Light pulses elicited pulse amplitudes of 88.7±32.8 pA (mean±st. dev.; n = 22 neurons; Fig. 1B). Repeating a 1-second pulse of yellow light twice, spaced by 1 second of darkness, resulted in similar pulse amplitudes each time (p>0.50, paired t-test), although during each light pulse, a slight perceptible decay was visible (analyzed later in the manuscript). This relatively stable current amplitude is consistent with what is known about the halorhodopsin photocycle, which can fully complete within tens of milliseconds [33]. The light-elicited current amplitude did not vary significantly with holding voltage when assayed at-70 mV, −30 mV, and+10 mV (F = 0.004, p>0.95, ANOVA with factor of holding voltage), nor did any measured kinetic parameters vary across this voltage range, such as onset or offset times of current pulses (F<0.6, p>0.55 for all comparisons, ANOVA; Fig. 1C). The onset and offset times of elicited currents were strikingly rapid, ∼10–15 ms at all holding voltages tested (Fig. 1Ci, 1Cii). When held in current clamp, hippocampal neurons underwent peak hyperpolarizations of 32.9±14.4 mV (n = 19 neurons) in response to pulses of yellow light, with no difference between the peak hyperpolarizations achieved by two 1-second pulses separated by a 1-second pause (p>0.85, paired t-test; Fig. 1D). Furthermore, as expected from the current-clamp experiments, these large voltage changes were quite rapid, with onset and offset times of 68±57 and 73±39 ms respectively for these large voltage swings (Fig. 1E). Thus, Halo was capable of reliably mediating hyperpolarizations of large magnitude, with fast onset and offset times at the beginning and end of light exposure.


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

Han X, Boyden ES - PLoS ONE (2007)

Millisecond-timescale Halo-mediated neuronal hyperpolarization, elicited by pulses of yellow light.(A) A representative cultured hippocampal neuron expressing mammalian codon-optimized N. pharaonis halorhodopsin (abbreviated Halo) fused to GFP, under the CaMKII promoter. Scale bar, 20 µm. (B) Neuronal currents elicited by optical activation of Halo. Left, representative outward currents elicited by two 1-second pulses of yellow (560±27.5 nm) light (∼10 mW/mm2) in a voltage-clamped neuron held at −70mV. Right, population data for n = 22 neurons. In this and subsequent figures, gray bars represent mean ± standard deviation unless otherwise indicated. Yellow bars in this and subsequent figures represent the period of yellow light exposure. (C) Kinetic properties of yellow light-elicited, Halo-mediated currents from voltage-clamped neurons. (i), 15–85% current onset time; (ii), 85–15% offset time. For each measurement, data is presented from neurons held at −70 mV, −30 mV and+10 mV (left to right). In this panel, gray bars represent mean ± standard error of the mean (S.E.M.). (D) Neuronal hyperpolarizations elicited by optical activation of Halo. Left, representative membrane voltage hyperpolarizations elicited by two 1-second pulses of yellow light, in a current-clamped neuron held at resting membrane potential. Right, population data for n = 19 neurons. (E) Kinetic properties of yellow light-elicited, Halo-mediated hyperpolarizations from current-clamped neurons, including both 15–85% voltage change onset time and 85–15% offset time.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0000299-g001: Millisecond-timescale Halo-mediated neuronal hyperpolarization, elicited by pulses of yellow light.(A) A representative cultured hippocampal neuron expressing mammalian codon-optimized N. pharaonis halorhodopsin (abbreviated Halo) fused to GFP, under the CaMKII promoter. Scale bar, 20 µm. (B) Neuronal currents elicited by optical activation of Halo. Left, representative outward currents elicited by two 1-second pulses of yellow (560±27.5 nm) light (∼10 mW/mm2) in a voltage-clamped neuron held at −70mV. Right, population data for n = 22 neurons. In this and subsequent figures, gray bars represent mean ± standard deviation unless otherwise indicated. Yellow bars in this and subsequent figures represent the period of yellow light exposure. (C) Kinetic properties of yellow light-elicited, Halo-mediated currents from voltage-clamped neurons. (i), 15–85% current onset time; (ii), 85–15% offset time. For each measurement, data is presented from neurons held at −70 mV, −30 mV and+10 mV (left to right). In this panel, gray bars represent mean ± standard error of the mean (S.E.M.). (D) Neuronal hyperpolarizations elicited by optical activation of Halo. Left, representative membrane voltage hyperpolarizations elicited by two 1-second pulses of yellow light, in a current-clamped neuron held at resting membrane potential. Right, population data for n = 19 neurons. (E) Kinetic properties of yellow light-elicited, Halo-mediated hyperpolarizations from current-clamped neurons, including both 15–85% voltage change onset time and 85–15% offset time.
Mentions: We first created a fusion protein comprising the mammalian codon-optimized form of N. pharaonis halorhodopsin (Halo), with EGFP added in-frame at the C-terminus for ease of visualization (see Methods for details). When expressed in cultured hippocampal neurons using the CaMKII promoter, which targets excitatory neurons of the forebrain [35], Halo-EGFP fluoresced brightly, and appeared evenly distributed around the neuron (Fig. 1A). When exposed to ∼10 mW/mm2 yellow light (from a xenon lamp, filtered by a standard Texas red excitation filter from Chroma, bandpass 560±27.5 nm), voltage-clamped hippocampal neurons expressing Halo experienced outward currents with rapid onset, stable steady-state amplitude, and abrupt shut-off after cessation of illumination. No supplementation of culture or recording media with the essential halorhodopsin cofactor all-trans retinal was necessary for strong currents to be elicited, consistent with prior work that demonstrated high enough levels of all-trans retinal in mammalian culture and brain to enable type I opsin functionality [18], [28]. Light pulses elicited pulse amplitudes of 88.7±32.8 pA (mean±st. dev.; n = 22 neurons; Fig. 1B). Repeating a 1-second pulse of yellow light twice, spaced by 1 second of darkness, resulted in similar pulse amplitudes each time (p>0.50, paired t-test), although during each light pulse, a slight perceptible decay was visible (analyzed later in the manuscript). This relatively stable current amplitude is consistent with what is known about the halorhodopsin photocycle, which can fully complete within tens of milliseconds [33]. The light-elicited current amplitude did not vary significantly with holding voltage when assayed at-70 mV, −30 mV, and+10 mV (F = 0.004, p>0.95, ANOVA with factor of holding voltage), nor did any measured kinetic parameters vary across this voltage range, such as onset or offset times of current pulses (F<0.6, p>0.55 for all comparisons, ANOVA; Fig. 1C). The onset and offset times of elicited currents were strikingly rapid, ∼10–15 ms at all holding voltages tested (Fig. 1Ci, 1Cii). When held in current clamp, hippocampal neurons underwent peak hyperpolarizations of 32.9±14.4 mV (n = 19 neurons) in response to pulses of yellow light, with no difference between the peak hyperpolarizations achieved by two 1-second pulses separated by a 1-second pause (p>0.85, paired t-test; Fig. 1D). Furthermore, as expected from the current-clamp experiments, these large voltage changes were quite rapid, with onset and offset times of 68±57 and 73±39 ms respectively for these large voltage swings (Fig. 1E). Thus, Halo was capable of reliably mediating hyperpolarizations of large magnitude, with fast onset and offset times at the beginning and end of light exposure.

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