Use of Channelrhodopsin for Activation of CNS Neurons

Jonathan P. Britt1, Ross A. McDevitt1, Antonello Bonci1

1 Cellular Neurobiology Research Branch, National Institute on Drug Abuse—Intramural Research Program, National Institutes of Health, Baltimore, Maryland
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 2.16
DOI:  10.1002/0471142301.ns0216s58
Online Posting Date:  January, 2012
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Abstract

Optogenetics—the use of optically activated proteins to control cell function—allows for control of neurons with an unprecedented degree of spatial, temporal, and neurochemical precision. Three protocols are presented in this unit describing the use of channelrhodopsin‐2 (ChR2), a light‐activated cation channel. These protocols emphasize practical issues of working with ChR2, including guidelines for selecting a gene delivery method, light source, and method of tissue implantation, as well as steps for fabricating fiber optic patch cables and chronic implantable optical fibers. The first protocol describes the use of ChR2 in electrophysiological recordings from brain slices. The second and third involve the use of ChR2 in vivo, with light delivered through chronic fiber implants or guide cannula. Curr. Protoc. Neurosci. 58:2.16.1‐2.16.19. © 2012 by John Wiley & Sons, Inc.

Keywords: optogenetics; channelrhodopsin; ChR2; optical; light; laser; LED

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Channelrhodopsin Use in Brain Slice Preparations
  • Basic Protocol 2: Channelrhodopsin Use in Awake Behaving Animals
  • Support Protocol 1: Channelrhodopsin Use with Guide Cannula
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Channelrhodopsin Use in Brain Slice Preparations

  Materials
  • Tissue containing ChR2 proteins
  • Optical fiber (e.g., multimode, 0.22 numerical aperture, visible to near infrared, low OH, 105‐µm core fiber; Thorlabs)
  • Fiber stripping tool (e.g., clad/coat: 125 µm/250 µm fiber stripping tool; Thorlabs)
  • FC fiber optic connector (e.g., FC simplex connector, 900 µm fiber‐multimode, 128 µm; Fiber Instrument Sales)
  • Hemostat, optional
  • Heat‐cure epoxy (e.g., blue dye epoxy; Fiber Instrument Sales)
  • 3‐ml luer‐lock syringe with blunt 22‐G needle
  • Heat gun, optional
  • Diamond wedge scribe (Thorlabs)
  • FC/PC connector polishing disc (Thorlabs)
  • Fiber polishing/lapping film diamond sheets (6 in. × 6 in. sheets with grit sizes of 1, 3, or 6 µm; Thorlabs)
  • 200× Fiber scope (Thorlabs)
  • Blue light laser with fiber coupler (e.g., 10 mW, 473‐nm diode‐pumped solid‐state continuous wave laser system with FC/PC multimode fiber coupler; OEM Laser Systems)
  • Fiber optic power meter (e.g., 400 nm to 1100 nm, 1 nW to 40 mW; Thorlabs)
  • Digitizer or pulse generator
  • Micromanipulator on an electrophysiology rig (see Fig. C)

Basic Protocol 2: Channelrhodopsin Use in Awake Behaving Animals

  Materials
  • Animal to be injected
  • Optical fiber (e.g., multimode, 0.22 numerical aperture, visible to near infrared, low OH, 105 µm core fiber; Thorlabs)
  • Fiber stripping tool (e.g., clad/coat: 125 µm/250 µm fiber stripping tool; Thorlabs)
  • Scissors
  • Hemostat
  • Ferrule (LC 1.25 mm OD multimode ceramic zirconia ferrule with 126 µm OD bore, Precision Fiber Products)
  • Heat‐cured epoxy (e.g., blue dye epoxy, Fiber Instrument Sales)
  • 3‐ml luer‐lock syringe with blunt 25‐G needle
  • Heat gun
  • Diamond wedge scribe (Thorlabs)
  • 1.25‐mm polishing disc (e.g., Universal 1.25 mm Polish Disc; Fiber Instrument Sales)
  • Fiber polishing/lapping film diamond sheets (6 in. × 6 in. sheets with grit sizes of 1, 3, or 6 µm; Thorlabs)
  • 200× Fiber scope (Thorlabs)
  • FC fiber optic connector (e.g., FC simplex connector, 900‐µm fiber‐multimode, 128 µm; Fiber Instrument Sales)
  • FC/PC connector polishing disc (Thorlabs)
  • Furcation tubing (e.g., furcation tubing, 900‐µm o.d., for 250‐µm fiber; Precision Fiber Products)
  • Heat shrink tubing (both 3/32‐in. and 1/8‐in. diameter)
  • Blue light laser with fiber coupler (e.g., 150 mW, 473‐nm diode‐pumped solid‐state continuous wave laser system with FC/PC multimode fiber coupler; OEM Laser Systems)
  • Fiber optic power meter (e.g., 400 nm to 1100 nm, 1 nW to 40 mW; Thorlabs)
  • 1.25‐mm i.d. ceramic split sleeve (Precision Fiber Products)
  • Test chamber
  • Additional reagents and equipment for mouse stereotaxic surgery ( appendix 4A) and virus injections (unit 4.24)

Support Protocol 1: Channelrhodopsin Use with Guide Cannula

  • Cannula connector assembly (C313C/SP without inside tubing; Plastics One)
  • Infusion cannula (C312IS‐4/SP without the stainless steel tubing; Plastics One)
  • Short cannula pedestal (C312GS‐4/SP; Plastics One)
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Figures

Videos

Literature Cited

   Adamantidis, A.R., Zhang, F., Aravanis, A.M., Deisseroth, K.,and de Lecea, L. 2007. Neural substrates of awakening probed with optogenetic control of hypocretin neurons. Nature 450:420‐7424.
   Airan, R.D., Thompson, K.R., Fenno, L.E., Bernstein, H., and Deisseroth, K. 2009. Temporally precise in vivo control of intracellular signalling. Nature 458:1025‐1029.
   Arenkiel, B.R., Peca, J., Davison, I.G., Feliciano, C., Deisseroth, K., Augustine, G.J., Ehlers, M.D., and Feng, G. 2007. In vivo light‐induced activation of neural circuitry in transgenic mice expressing channelrhodopsin‐2. Neuron 54:205‐218.
   Gutierrez, D.V., Mark, M.D., Masseck, O., Maejima, T., Kuckelsberg, D., Hyde, R.A., Krause, M., Kruse, W., and Herlitze, S. 2011. Optogenetic control of motor coordination by Gi/o protein‐coupled vertebrate rhodopsin in cerebellar Purkinje cells. J. Biol. Chem. 286:25848‐25858.
   Lin, J.Y. 2011. A user's guide to channelrhodopsin variants: Features, limitations and future developments. Exp. Physiol. 96:19‐25.
   Saito, T. and Nakatsuji, N. 2001. Efficient gene transfer into the embryonic mouse brain using in vivo electroporation. Dev. Biol. 240:237‐246.
   Yizhar, O., Fenno, L.E., Prigge, M., Schneider, F., Davidson, T.J., O'Shea, D.J., Sohal, W.S., Goshen, I., Finkelstein, J., Paz, J.T., Stehfest, K., Fudim, R., Ramakrishnan, C., Huguenard, J.R., Hegemann, O., and Deisseroth, K. 2011. Neocortical excitation/inhibition balance in information processing and social dysfunction. Nature 477:171‐178.
   Zhao, S., Ting, J.T., Atallah, H.E., Qiu, L., Tan, J., Gloss, B. Augustine, G.J., Deisseroth, K., Luo, M., Graybiel, A.M., and Feng, G. 2011. Cell type‐specific channelrhodopsin‐2 transgenic mice for optogenetic dissection of neural circuitry function. Nat. Methods 8:745‐752.
Internet Resources
  http://genetherapy.unc.edu/services.htm
  Web site for the University of North Carolina at Chapel Hill Gene Therapy Center; offers packaged virus for sale and related services.
  http://www.stanford.edu/group/dlab/
  Web site for the laboratory of Dr. Karl Deisseroth; contains information on ChR2 (and variants) DNA sequences, hardware, and protocols.
  http://syntheticneurobiology.org/
  Web site for the laboratory of Dr. Ed Boyden; contains information on inhibitory optogenetic proteins.
  http://www.stuberlab.org/
  Web site for the laboratory of Dr. Garret Stuber; contains protocols for optical fiber construction and optogenetics hardware.
  http://www.stanford.edu/group/dlab/cgi‐bin/graph/chart.php
  Calculator for the spread of light in brain tissue, courtesy of the laboratory of Dr. Karl Deisseroth.
  http://thorlabs.com/Thorcat/1100/1166‐D02.pdf
  “Guide to Connectorization and Polishing Optical Fibers,” courtesy of Thorlabs.
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