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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.

References

  1. Deisseroth, K. et al. Next-generation optical technologies for illuminating genetically targeted brain circuits. J. Neurosci. 26, 10380–10386 (2006)

    Article  CAS  Google Scholar 

  2. Boyden, E. S., Zhang, F., Bamberg, E., Nagel, G. & Deisseroth, K. Millisecond-timescale, genetically targeted optical control of neural activity. Nature Neurosci. 8, 1263–1268 (2005)

    Article  CAS  Google Scholar 

  3. Zhang, F., Wang, L. P., Boyden, E. S. & Deisseroth, K. Channelrhodopsin-2 and optical control of excitable cells. Nature Methods 3, 785–792 (2006)

    Article  CAS  Google Scholar 

  4. Nagel, G. et al. Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc. Natl Acad. Sci. USA 100, 13940–13945 (2003)

    Article  ADS  CAS  Google Scholar 

  5. Li, X. et al. Fast noninvasive activation and inhibition of neural and network activity by vertebrate rhodopsin and green algae channelrhodopsin. Proc. Natl Acad. Sci. USA 102, 17816–17821 (2005)

    Article  ADS  CAS  Google Scholar 

  6. Nagel, G. et al. Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses. Curr. Biol. 15, 2279–2284 (2005)

    Article  MathSciNet  CAS  Google Scholar 

  7. Ishizuka, T., Kakuda, M., Araki, R. & Yawo, H. Kinetic evaluation of photosensitivity in genetically engineered neurons expressing green algae light-gated channels. Neurosci. Res. 54, 85–94 (2006)

    Article  CAS  Google Scholar 

  8. Bi, A. et al. Ectopic expression of a microbial-type rhodopsin restores visual responses in mice with photoreceptor degeneration. Neuron 50, 23–33 (2006)

    Article  CAS  Google Scholar 

  9. Schroll, C. et al. Light-induced activation of distinct modulatory neurons triggers appetitive or aversive learning in Drosophila larvae. Curr. Biol. 16, 1741–1747 (2006)

    Article  CAS  Google Scholar 

  10. Banghart, M., Borges, K., Isacoff, E., Trauner, D. & Kramer, R. H. Light-activated ion channels for remote control of neuronal firing. Nature Neurosci. 7, 1381–1386 (2004)

    Article  CAS  Google Scholar 

  11. Ehrengruber, M. U. et al. Activation of heteromeric G protein-gated inward rectifier K+ channels overexpressed by adenovirus gene transfer inhibits the excitability of hippocampal neurons. Proc. Natl Acad. Sci. USA 94, 7070–7075 (1997)

    Article  ADS  CAS  Google Scholar 

  12. Ibanez-Tallon, I. et al. Tethering naturally occurring peptide toxins for cell-autonomous modulation of ion channels and receptors in vivo. Neuron 43, 305–311 (2004)

    Article  CAS  Google Scholar 

  13. Isles, A. R. et al. Conditional ablation of neurones in transgenic mice. J. Neurobiol. 47, 183–193 (2001)

    Article  CAS  Google Scholar 

  14. Johns, D. C., Marx, R., Mains, R. E., O'Rourke, B. & Marban, E. Inducible genetic suppression of neuronal excitability. J. Neurosci. 19, 1691–1697 (1999)

    Article  CAS  Google Scholar 

  15. Kobayashi, K. et al. Targeted disruption of the tyrosine hydroxylase locus results in severe catecholamine depletion and perinatal lethality in mice. J. Biol. Chem. 270, 27235–27243 (1995)

    Article  CAS  Google Scholar 

  16. Lechner, H. A., Lein, E. S. & Callaway, E. M. A genetic method for selective and quickly reversible silencing of mammalian neurons. J. Neurosci. 22, 5287–5290 (2002)

    Article  CAS  Google Scholar 

  17. Nitabach, M. N., Blau, J. & Holmes, T. C. Electrical silencing of Drosophila pacemaker neurons stops the free-running circadian clock. Cell 109, 485–495 (2002)

    Article  CAS  Google Scholar 

  18. Tan, E. M. et al. Selective and quickly reversible inactivation of mammalian neurons in vivo using the Drosophila allatostatin receptor. Neuron 51, 157–170 (2006)

    Article  CAS  Google Scholar 

  19. Karpova, A. Y., Tervo, D. G., Gray, N. W. & Svoboda, K. Rapid and reversible chemical inactivation of synaptic transmission in genetically targeted neurons. Neuron 48, 727–735 (2005)

    Article  CAS  Google Scholar 

  20. Nagel, G. et al. Channelrhodopsin-1: a light-gated proton channel in green algae. Science 296, 2395–2398 (2002)

    Article  ADS  CAS  Google Scholar 

  21. Kolbe, M., Besir, H., Essen, L. O. & Oesterhelt, D. Structure of the light-driven chloride pump halorhodopsin at 1.8 Å resolution. Science 288, 1390–1396 (2000)

    Article  ADS  CAS  Google Scholar 

  22. Bamberg, E., Tittor, J. & Oesterhelt, D. Light-driven proton or chloride pumping by halorhodopsin. Proc. Natl Acad. Sci. USA 90, 639–643 (1993)

    Article  ADS  CAS  Google Scholar 

  23. Duschl, A., McCloskey, M. A. & Lanyi, J. K. Functional reconstitution of halorhodopsin. Properties of halorhodopsin-containing proteoliposomes. J. Biol. Chem. 263, 17016–17022 (1988)

    CAS  PubMed  Google Scholar 

  24. Hegemann, P., Oesterhelt, D. & Bamberg, E. The transport activity of the light-driven chloride pump halorhoposin is regulated by green and blue light. Biochim. Biophys. Acta 819, 195–205 (1985)

    Article  CAS  Google Scholar 

  25. Alfonso, A., Grundahl, K., Duerr, J. S., Han, H. P. & Rand, J. B. The Caenorhabditis elegans unc-17 gene: a putative vesicular acetylcholine transporter. Science 261, 617–619 (1993)

    Article  ADS  CAS  Google Scholar 

  26. Faumont, S. & Lockery, S. R. The awake behaving worm: simultaneous imaging of neuronal activity and behavior in intact animals at millimeter scale. J. Neurophysiol. 95, 1976–1981 (2006)

    Article  Google Scholar 

  27. Rosenmund, C. et al. Differential control of vesicle priming and short-term plasticity by Munc13 isoforms. Neuron 33, 411–424 (2002)

    Article  CAS  Google Scholar 

  28. Yoon, H., Enquist, L. W. & Dulac, C. Olfactory inputs to hypothalamic neurons controlling reproduction and fertility. Cell 123, 669–682 (2005)

    Article  CAS  Google Scholar 

  29. Lu, J., Sherman, D., Devor, M. & Saper, C. B. A putative flip-flop switch for control of REM sleep. Nature 441, 589–594 (2006)

    Article  ADS  CAS  Google Scholar 

  30. Schoppa, N. E. & Westbrook, G. L. Glomerulus-specific synchronization of mitral cells in the olfactory bulb. Neuron 31, 639–651 (2001)

    Article  CAS  Google Scholar 

  31. Hanks, T. D., Ditterich, J. & Shadlen, M. N. Microstimulation of macaque area LIP affects decision-making in a motion discrimination task. Nature Neurosci. 9, 682–689 (2006)

    Article  CAS  Google Scholar 

  32. Kanold, P. O., Kara, P., Reid, R. C. & Shatz, C. J. Role of subplate neurons in functional maturation of visual cortical columns. Science 301, 521–525 (2003)

    Article  ADS  CAS  Google Scholar 

  33. Jensen, O. & Lisman, J. E. Novel lists of 7 ± 2 known items can be reliably stored in an oscillatory short-term memory network: interaction with long-term memory. Learn. Mem. 3, 257–263 (1996)

    Article  CAS  Google Scholar 

  34. Lima, S. Q. & Miesenbock, G. Remote control of behavior through genetically targeted photostimulation of neurons. Cell 121, 141–152 (2005)

    Article  CAS  Google Scholar 

  35. Shoham, S., O'Connor, D. H., Sarkisov, D. V. & Wang, S. S. Rapid neurotransmitter uncaging in spatially defined patterns. Nature Methods 2, 837–843 (2005)

    Article  CAS  Google Scholar 

  36. Helmchen, F., Fee, M. S., Tank, D. W. & Denk, W. A miniature head-mounted two-photon microscope. high-resolution brain imaging in freely moving animals. Neuron 31, 903–912 (2001)

    Article  CAS  Google Scholar 

  37. Pettit, D. L. & Augustine, G. J. Distribution of functional glutamate and GABA receptors on hippocampal pyramidal cells and interneurons. J. Neurophysiol. 84, 28–38 (2000)

    Article  CAS  Google Scholar 

  38. Volgraf, M. et al. Allosteric control of an ionotropic glutamate receptor with an optical switch. Nature Chem. Biol. 2, 47–52 (2006)

    Article  CAS  Google Scholar 

  39. Chambers, J. J., Banghart, M. R., Trauner, D. & Kramer, R. H. Light-induced depolarization of neurons using a modified Shaker K+ channel and a molecular photoswitch. J. Neurophysiol. 96, 2792–2796 (2006)

  40. Zemelman, B. V., Lee, G. A., Ng, M. & Miesenbock, G. Selective photostimulation of genetically chARGed neurons. Neuron 33, 15–22 (2002)

    Article  CAS  Google Scholar 

  41. Wang, S. S., Khiroug, L. & Augustine, G. J. Quantification of spread of cerebellar long-term depression with chemical two-photon uncaging of glutamate. Proc. Natl Acad. Sci. USA 97, 8635–8640 (2000)

    Article  ADS  CAS  Google Scholar 

  42. Adams, S. R. & Tsien, R. Y. Controlling cell chemistry with caged compounds. Annu. Rev. Physiol. 55, 755–784 (1993)

    Article  CAS  Google Scholar 

  43. Dittgen, T. et al. Lentivirus-based genetic manipulations of cortical neurons and their optical and electrophysiological monitoring in vivo. Proc. Natl Acad. Sci. USA 101, 18206–18211 (2004)

    Article  ADS  CAS  Google Scholar 

  44. MacLean, J. N., Fenstermaker, V., Watson, B. O. & Yuste, R. A visual thalamocortical slice. Nature Methods 3, 129–134 (2006)

    Article  CAS  Google Scholar 

  45. Grygorczyk, R., Hanke-Baier, P., Schwarz, W. & Passow, H. Measurement of erythroid band 3 protein-mediated anion transport in mRNA-injected oocytes of Xenopus laevis. Methods Enzymol. 173, 453–466 (1989)

    Article  CAS  Google Scholar 

  46. Zennou, V. et al. The HIV-1 DNA flap stimulates HIV vector-mediated cell transduction in the brain. Nature Biotechnol. 19, 446–450 (2001)

    Article  CAS  Google Scholar 

  47. Brun, S., Faucon-Biguet, N. & Mallet, J. Optimization of transgene expression at the posttranscriptional level in neural cells: implications for gene therapy. Mol. Ther. 7, 782–789 (2003)

    Article  CAS  Google Scholar 

  48. Sena-Esteves, M., Tebbets, J. C., Steffens, S., Crombleholme, T. & Flake, A. W. Optimized large-scale production of high titre lentivirus vector pseudotypes. J. Virol. Methods 122, 131–139 (2004)

    Article  CAS  Google Scholar 

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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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