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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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Blue light facilitates optimal Halo function.(A) (i) Timecourse of Halo-mediated hyperpolarizations in a representative current-clamped hippocampal neuron during 15 seconds of continuous yellow light, followed by four 1-second test pulses of yellow light (one every 30 seconds, starting 10 seconds after the end of the first 15-second period of yellow light). (ii) Timecourse of Halo-mediated hyperpolarization for the same cell exhibited in (i), but when Halo function is facilitated by a 400-ms pulse of blue light in between the 15-second period of yellow light and the first 1-second test pulse. (B) Population data for blue-light facilitation of Halo recovery (n = 8 neurons). Plotted are the hyperpolarizations elicited by the four 1-second test pulses of yellow light, normalized to the peak hyperpolarization induced by the original 15-second yellow light pulse. Dots represent mean±S.E.M. Black dots represent experiments when no blue light pulse was delivered (as in Fig. 5Ai.). Open blue dots represent experiments when 400 ms of blue light was delivered to facilitate recovery (as in Fig. 5Aii.).
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pone-0000299-g005: Blue light facilitates optimal Halo function.(A) (i) Timecourse of Halo-mediated hyperpolarizations in a representative current-clamped hippocampal neuron during 15 seconds of continuous yellow light, followed by four 1-second test pulses of yellow light (one every 30 seconds, starting 10 seconds after the end of the first 15-second period of yellow light). (ii) Timecourse of Halo-mediated hyperpolarization for the same cell exhibited in (i), but when Halo function is facilitated by a 400-ms pulse of blue light in between the 15-second period of yellow light and the first 1-second test pulse. (B) Population data for blue-light facilitation of Halo recovery (n = 8 neurons). Plotted are the hyperpolarizations elicited by the four 1-second test pulses of yellow light, normalized to the peak hyperpolarization induced by the original 15-second yellow light pulse. Dots represent mean±S.E.M. Black dots represent experiments when no blue light pulse was delivered (as in Fig. 5Ai.). Open blue dots represent experiments when 400 ms of blue light was delivered to facilitate recovery (as in Fig. 5Aii.).

Mentions: Halo-mediated currents and hyperpolarizations appeared to decay slightly during 1-second long pulses of yellow light (Fig. 1B, D), raising the question of how Halo might perform during longer-duration neural silencing experiments. We exposed Halo-expressing neurons to continuous yellow light, and found that the hyperpolarization decayed with a time constant of approximately 16.8±10.4 seconds (n = 8 neurons; representative trace shown in Fig. 5Ai). We tested for recovery of Halo function after 15 seconds of yellow light, with repeated 1-second test pulses of yellow light delivered every 30 seconds, and found that even after four such rest periods, the hyperpolarization peak amplitude remained down by 33% from its original peak amplitude, suggesting that the Halo protein had entered an inactive state (Fig. 5Ai and Fig. 5B, black dots). Earlier studies on halorhodopsin from another archaebacterium, Halobacterium halobium, found evidence for a similar type of rundown, due to accumulation of an inactive 13-cis retinal form of the halorhodopsin molecule, which required seconds to recover to the original state [37]. However, these earlier studies also found that brief periods of blue light could facilitate rapid recovery of H. halobium halorhodopsin to the active form, by assisting in the re-isomerization of 13-cis retinal to the all-trans form. For Halo, we found that a brief pulse of moderate-intensity blue light (∼10 mW/mm2 400 ms-long), delivered through the GFP excitation filter) could completely restore Halo to its active state (Fig. 5Aii), recovering the hyperpolarizations elicited by the test pulses of yellow light to their original amplitude (p>0.05 for each test pulse, paired t-test; n = 8; Fig. 5B, open blue dots). Thus, despite the existence of rundown during long-lasting exposure of Halo-expressing neurons to yellow light, brief periods of blue light exposure will facilitate the optimal performance of Halo during long periods of neural inhibition in vivo.


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

Han X, Boyden ES - PLoS ONE (2007)

Blue light facilitates optimal Halo function.(A) (i) Timecourse of Halo-mediated hyperpolarizations in a representative current-clamped hippocampal neuron during 15 seconds of continuous yellow light, followed by four 1-second test pulses of yellow light (one every 30 seconds, starting 10 seconds after the end of the first 15-second period of yellow light). (ii) Timecourse of Halo-mediated hyperpolarization for the same cell exhibited in (i), but when Halo function is facilitated by a 400-ms pulse of blue light in between the 15-second period of yellow light and the first 1-second test pulse. (B) Population data for blue-light facilitation of Halo recovery (n = 8 neurons). Plotted are the hyperpolarizations elicited by the four 1-second test pulses of yellow light, normalized to the peak hyperpolarization induced by the original 15-second yellow light pulse. Dots represent mean±S.E.M. Black dots represent experiments when no blue light pulse was delivered (as in Fig. 5Ai.). Open blue dots represent experiments when 400 ms of blue light was delivered to facilitate recovery (as in Fig. 5Aii.).
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pone-0000299-g005: Blue light facilitates optimal Halo function.(A) (i) Timecourse of Halo-mediated hyperpolarizations in a representative current-clamped hippocampal neuron during 15 seconds of continuous yellow light, followed by four 1-second test pulses of yellow light (one every 30 seconds, starting 10 seconds after the end of the first 15-second period of yellow light). (ii) Timecourse of Halo-mediated hyperpolarization for the same cell exhibited in (i), but when Halo function is facilitated by a 400-ms pulse of blue light in between the 15-second period of yellow light and the first 1-second test pulse. (B) Population data for blue-light facilitation of Halo recovery (n = 8 neurons). Plotted are the hyperpolarizations elicited by the four 1-second test pulses of yellow light, normalized to the peak hyperpolarization induced by the original 15-second yellow light pulse. Dots represent mean±S.E.M. Black dots represent experiments when no blue light pulse was delivered (as in Fig. 5Ai.). Open blue dots represent experiments when 400 ms of blue light was delivered to facilitate recovery (as in Fig. 5Aii.).
Mentions: Halo-mediated currents and hyperpolarizations appeared to decay slightly during 1-second long pulses of yellow light (Fig. 1B, D), raising the question of how Halo might perform during longer-duration neural silencing experiments. We exposed Halo-expressing neurons to continuous yellow light, and found that the hyperpolarization decayed with a time constant of approximately 16.8±10.4 seconds (n = 8 neurons; representative trace shown in Fig. 5Ai). We tested for recovery of Halo function after 15 seconds of yellow light, with repeated 1-second test pulses of yellow light delivered every 30 seconds, and found that even after four such rest periods, the hyperpolarization peak amplitude remained down by 33% from its original peak amplitude, suggesting that the Halo protein had entered an inactive state (Fig. 5Ai and Fig. 5B, black dots). Earlier studies on halorhodopsin from another archaebacterium, Halobacterium halobium, found evidence for a similar type of rundown, due to accumulation of an inactive 13-cis retinal form of the halorhodopsin molecule, which required seconds to recover to the original state [37]. However, these earlier studies also found that brief periods of blue light could facilitate rapid recovery of H. halobium halorhodopsin to the active form, by assisting in the re-isomerization of 13-cis retinal to the all-trans form. For Halo, we found that a brief pulse of moderate-intensity blue light (∼10 mW/mm2 400 ms-long), delivered through the GFP excitation filter) could completely restore Halo to its active state (Fig. 5Aii), recovering the hyperpolarizations elicited by the test pulses of yellow light to their original amplitude (p>0.05 for each test pulse, paired t-test; n = 8; Fig. 5B, open blue dots). Thus, despite the existence of rundown during long-lasting exposure of Halo-expressing neurons to yellow light, brief periods of blue light exposure will facilitate the optimal performance of Halo during long periods of neural inhibition in vivo.

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