Abstract
Optogenetics enables experimental control over neural activity using light. Channelrhodopsin and its variants are typically activated using visible light excitation but can also be activated using infrared two-photon excitation. Two-photon excitation can improve the spatial precision of stimulation in scattering tissue but has several practical limitations that need to be considered before use. Here we describe the methodology and best practices for using two-photon optogenetic stimulation of neurons within the brain of the fruit fly, Drosophila melanogaster, with an emphasis on projection neurons of the antennal lobe.
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References
Yizhar O, Fenno LE, Davidson TJ, Mogri M, Deisseroth K (2011) Optogenetics in neural systems. Neuron 71:9–34
Helmchen F, Denk W (2005) Deep tissue two-photon microscopy. Nat Methods 2:932–940
Rickgauer JP, Tank DW (2009) Two-photon excitation of channelrhodopsin-2 at saturation. Proc Natl Acad Sci U S A 106:15025–15030
Andrasfalvy BK, Zemelman BV, Tang J, Vaziri A (2010) Two-photon single-cell optogenetic control of neuronal activity by sculpted light. Proc Natl Acad Sci U S A 107:11981–11986
Mardinly AR, Oldenburg IA, Pegard NC, Sridharan S, Lyall EH, Chesnov K et al (2018) Precise multimodal optical control of neural ensemble activity. Nat Neurosci 21:881–893
Klapoetke NC, Murata Y, Kim SS, Pulver SR, Birdsey-Benson A, Cho YK et al (2014) Independent optical excitation of distinct neural populations. Nat Methods 11:338–346
Hernandez O, Papagiakoumou E, Tanese D, Fidelin K, Wyart C, Emiliani V (2016) Three-dimensional spatiotemporal focusing of holographic patterns. Nat Commun 7:11928
Zhang Z, Russell LE, Packer AM, Gauld OM, Hausser M (2018) Closed-loop all-optical interrogation of neural circuits in vivo. Nat Methods 15:1037–1040
Kazama H (2015) Systems neuroscience in Drosophila: conceptual and technical advantages. Neuroscience 296:3–14
Ito K, Shinomiya K, Ito M, Armstrong JD, Boyan G, Hartenstein V et al (2014) A systematic nomenclature for the insect brain. Neuron 81:755–765
Gouwens NW, Wilson RI (2009) Signal propagation in Drosophila central neurons. J Neurosci 29:6239–6249
Jenett A, Rubin GM, Ngo TT, Shepherd D, Murphy C, Dionne H et al (2012) A GAL4-driver line resource for Drosophila neurobiology. Cell Rep 2:991–1001
Jeanne JM, Fisek M, Wilson RI (2018) The organization of projections from olfactory glomeruli onto higher-order neurons. Neuron 98:1198–1213
Inada K, Tsuchimoto Y, Kazama H (2017) Origins of cell-type-specific olfactory processing in the drosophila mushroom body circuit. Neuron 95:357–367.e354
Kim SS, Rouault H, Druckmann S, Jayaraman V (2017) Ring attractor dynamics in the Drosophila central brain. Science 356:849–853
Takagi S, Cocanougher BT, Niki S, Miyamoto D, Kohsaka H, Kazama H et al (2017) Divergent connectivity of homologous command-like neurons mediates segment-specific touch responses in Drosophila. Neuron 96:1373–1387.e1376
Hsiao PY, Tsai CL, Chen MC, Lin YY, Yang SD, Chiang AS (2015) Non-invasive manipulation of Drosophila behavior by two-photon excited red-activatable channelrhodopsin. Biomed Opt Express 6:4344–4352
Murthy M, Turner G (2013) Whole-cell in vivo patch-clamp recordings in the Drosophila brain. Cold Spring Harb Protoc 2013:140–148
Pologruto TA, Sabatini BL, Svoboda K (2003) ScanImage: flexible software for operating laser scanning microscopes. Biomed Eng Online 2:13
Lin JY, Knutsen PM, Muller A, Kleinfeld D, Tsien RY (2013) ReaChR: a red-shifted variant of channelrhodopsin enables deep transcranial optogenetic excitation. Nat Neurosci 16:1499–1508
Inagaki HK, Jung Y, Hoopfer ED, Wong AM, Mishra N, Lin JY et al (2014) Optogenetic control of Drosophila using a red-shifted channelrhodopsin reveals experience-dependent influences on courtship. Nat Methods 11:325–332
Stocker RF, Heimbeck G, Gendre N, de Belle JS (1997) Neuroblast ablation in Drosophila P[GAL4] lines reveals origins of olfactory interneurons. J Neurobiol 32:443–456
Murthy M, Turner G (2013) Dissection of the head cuticle and sheath of living flies for whole-cell patch-clamp recordings in the brain. Cold Spring Harb Protoc 2013:134–139
Brady J (1965) A simple technique for making very fine, durable dissecting needles by sharpening tungsten wire electrolytically. Bull World Health Organ 32:143–144
Maimon G, Straw AD, Dickinson MH (2010) Active flight increases the gain of visual motion processing in Drosophila. Nat Neurosci 13:393–399
Chaigneau E, Ronzitti E, Gajowa MA, Soler-Llavina GJ, Tanese D, Brureau AY et al (2016) Two-photon holographic stimulation of ReaChR. Front Cell Neurosci 10:234
Acknowledgments
The authors gratefully acknowledge the support of Rachel Wilson in developing and refining this methodology. Kristyn Lizbinski provided critical feedback on the manuscript. This work was supported by NIH grants R01DC008174, F32NS083262, and P30NS072030, Yale University, and the Kavli Institute for Neuroscience at Yale.
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Fişek, M., Jeanne, J.M. (2021). Two-Photon Optogenetic Stimulation of Drosophila Neurons. In: Dempski, R. (eds) Channelrhodopsin. Methods in Molecular Biology, vol 2191. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0830-2_7
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DOI: https://doi.org/10.1007/978-1-0716-0830-2_7
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