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Multimodal fast optical interrogation of neural circuitry

Abstract

Our understanding of the cellular implementation of systems-level neural processes like action, thought and emotion has been limited by the availability of tools to interrogate specific classes of neural cells within intact, living brain tissue. Here we identify and develop an archaeal light-driven chloride pump (NpHR) from Natronomonas pharaonis for temporally precise optical inhibition of neural activity. NpHR allows either knockout of single action potentials, or sustained blockade of spiking. NpHR is compatible with ChR2, the previous optical excitation technology we have described, in that the two opposing probes operate at similar light powers but with well-separated action spectra. NpHR, like ChR2, functions in mammals without exogenous cofactors, and the two probes can be integrated with calcium imaging in mammalian brain tissue for bidirectional optical modulation and readout of neural activity. Likewise, NpHR and ChR2 can be targeted together to Caenorhabditis elegans muscle and cholinergic motor neurons to control locomotion bidirectionally. NpHR and ChR2 form a complete system for multimodal, high-speed, genetically targeted, all-optical interrogation of living neural circuits.

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Figure 1: Electrophysiological properties of NpHR in oocytes and hippocampal neurons.
Figure 2: Combining NpHR with ChR2 for noninvasive optical control.
Figure 3: NpHR mediates tunable neuronal inhibition over a range of timescales.
Figure 4: Temporal precision of NpHR-mediated inhibition.
Figure 5: Bidirectional optical neural control and in vivo implementation.
Figure 6: Bidirectional optical control of C. elegans.

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Acknowledgements

We thank J. Huguenard for discussions; L. Meltzer and H.-C. Tsai for assistance with confocal imaging; R. Airan for statistical assistance; V. Gradinaru for help with calcium imaging; M. Engelhard, J. Rand, D. Oesterhelt, R. Abele, and B. Bauer for plasmids; and K. Zehl and E. Grabski for expert technical assistance. F.Z. is supported by a fellowship from the NIH. L.-P.W. is supported by a fellowship from the California Institute of Regenerative Medicine. F.Z. and L.-P.W. are co-first authors. E.B. and G.N. are supported by grants from the Max-Planck-Society and by the Deutsche Forschungsgemeinschaft. A.G. is supported by grants from the Hessisches Ministerium für Wissenschaft und Kunst, and by the Deutsche Forschungsgemeinschaft. K.D. is supported by NARSAD, APIRE and the Snyder, Culpeper, Coulter, Klingenstein, Whitehall, McKnight, and Albert Yu and Mary Bechmann Foundations, as well as by NIMH, NIDA, and the NIH Director’s Pioneer Award Program.

The GenBank accession number is EF474018 for the ‘mammalianized’ NpHR sequence and EF474017 for the ‘mammalianized’ ChR2(1-315) sequence.

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Supplementary information

Supplementary Figures and Table

This file contains Supplementary Figures S1-S3 with Legends and Supplementary Table 1. (PDF 5605 kb)

Supplementary Movie 1

This file contains Supplementary Movie 1 which shows swimming of a transgenic C. elegans expressing NpHR (transgene zxEx29) in muscles is instantaneously, and repeatedly, inhibited by photoactivation of HR (duration of illumination is indicated by appearance of a yellow dot). (MOV 496 kb)

Supplementary Movie 2

This file contains Supplementary Movie 2 which shows swimming of a transgenic C. elegans expressing NpHR in cholinergic motoneurons (transgene zxEx33) is instantaneously inhibited by photoactivation of NpHR (duration of illumination is indicated by appearance of a yellow dot). (MOV 2516 kb)

Supplementary Movie 3

This file contains Supplementary Movie 3 which shows transgenic C. elegans expressing NpHR-ECFP in muscles (transgene zxEx30). Movement is rapidly inhibited (3x) by photoactivation of HR, and the body relaxes and dilates. Duration of illumination episodes is indicated by appearance of a yellow dot. (MOV 899 kb)

Supplementary Movie 4

This file contains Supplementary Movie 4 which shows one transgenic C. elegans expressing NpHR in muscles (transgene zxEx29), and one non-transgenic control animal. Movement of the transgenic animal (on the left) is rapidly inhibited by photoactivation of HR (illumination indicated by a yellow dot), while the non-transgenic animal does not respond. FILE: suppl movie 5.wmv (MOV 1066 kb)

Supplementary Movie 5

This file contains Supplementary Movie 5 which shows transgenic C. elegans expressing NpHR in cholinergic motoneurons (transgene zxEx33). Movement is rapidly inhibited by photoactivation of HR, and the body relaxes and dilates. Duration of illumination is indicated by appearance of a yellow dot. (MOV 1471 kb)

Supplementary Movie 6

This file contains Supplementary Movie 6 which shows co-expression and -activation of ChR2(H134R)-EYFP and NpHR in cholinergic motoneurons of transgenic C. elegans (transgene zxEx34). The animal is illuminated with blue light (for ChR2 activation, indicated by a cyan dot), causing contractions, then, while ChR2 is still photoactivated, NpHR is photoactivated by yellow light (yellow dot), causing significant body relaxation. When NpHR activation ends, the animal contracts again (ChR2 still activated), and finally, when ChR2 activation ends, the animal relaxes to the initial body length. (MOV 891 kb)

Supplementary Movie 7

This file contains Supplementary Movie 7 which shows co-expression and rapidly alternating activation of ChR2(H134R)-EYFP and NpHR in muscles of transgenic C. elegans (transgene zxEx32). The animal is illuminated with alternating blue light (for ChR2 activation, indicated by a cyan dot), causing contractions, and yellow light (for NpHR activation, indicated by a yellow dot), causing significant body relaxation. (MOV 1757 kb)

Supplementary Video Streaming

(HTML 3524 kb)

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Zhang, F., Wang, LP., Brauner, M. et al. Multimodal fast optical interrogation of neural circuitry. Nature 446, 633–639 (2007). https://doi.org/10.1038/nature05744

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