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

  •   FigureFigure 2.16.1 Steps to epoxy optical fiber into an FC/PC connector. (A) The fiber stripping tool clamps onto the optical fiber and strips off the buffer material that surrounds the core and cladding of the fiber. (B) A vise holds the FC/PC connector so epoxy can easily be injected into the ferrule inside the connector. (C) The fiber is threaded through the epoxy‐filled connector. (D) The use of a heat gun will considerably speed up the curing time of the epoxy. (E) The boot of the connector is threaded onto the optical fiber to protect the connection.
  •   FigureFigure 2.16.2 Steps to polish and examine the patch cord. (A) A diamond wedge scribe is used to score the optical fiber so that a light flick to the end of the optical fiber will cause it to break off immediately as it leaves the connector. (B) The connector is inserted into a polishing disc. The connector is gently pressed against the lapping film sheet during the polishing process. (C) The quality of the polishing job is assessed by attaching the FC/PC connector to a magnification scope. (D) The fiber patch cord is connected to the laser coupler that is attached to the laser.
  •   FigureFigure 2.16.3 Testing and using the fiber patch cord. (A) The light beam created from an unpolished fiber. Excess epoxy is covering the ferrule and preventing light to enter the patch cord from the laser coupler. (B) The light beam created from a well‐polished patch cord has a uniform light intensity and crisp circular shape. (C) The bare fiber end of the patch cord is placed in a micromanipulator that is positioned on an electrophysiology rig.
  •   FigureFigure 2.16.4 Steps to epoxy optical fiber into a loose ferrule. (A) When working with a small piece of optical fiber, it is best to strip off the buffer material surrounding the optical fiber while the fiber is still attached to the roll. (B) A small ferrule and piece of optical fiber are used to make an implantable optical fiber. (C) The ferrule is held in a hemostat while epoxy is injected into its center. (D) The piece of optical fiber is threaded through the epoxy‐filled ferrule. (E) The epoxy must be completely hardened before polishing the ferrule.
  •   FigureFigure 2.16.5 Steps to polish and finalize an implantable optical fiber. (A) A diamond wedge scribe is used to score the optical fiber so that a light flick to the end of the optical fiber will cause it to break off immediately as it leaves the ferrule. (B) The fragility of the optical fiber necessitates the use of a hemostat to hold and polish the ferrule. One option to polish a loose ferrule is to use a polishing disc. In this case, use the hemostat to hold and position the ferrule in the polishing disc throughout the polishing process. (C) A second option to polish a loose ferrule is to forgo the polishing disk and simply press the ferrule against the lapping sheets with the help of the hemostat alone. (D) A diamond wedge scribe is used to score the optical fiber at the distance from the ferrule that corresponds to the depth of the targeted brain structure.
  •   FigureFigure 2.16.6 Steps to fabricate a fiber optic patch cable that can attach to an implantable optical fiber. (A) Furcation tubing covers the exposed optical fiber to prevent visualization of the light from becoming a cue to the animal. (B) Heat shrink tubing is used to both strengthen this junction and hold the furcation tubing in place. (C) A zirconia split sleeve is placed over three quarters of the ferrule. The remaining area of the split sleeve will accommodate the loose ferrule that will be implanted in the skull of an animal. (D) Heat shrink tubing is again used to both strengthen this junction and hold the furcation tubing in place. (E) The ferrule of the implantable optical fiber is inserted into the split sleeve on the patch cable. A mark is made on the loose ferrule to indicate how much of it must remain exposed to form a solid connection to the cable. Cement can be applied below this mark when affixing the ferrule to the skull of the animal. (F) Illustration of a completed fiber optic patch cable. Heat shrink is indicated by translucent green sheathing over the connector boot and ferrule + ceramic split sleeve. Fiber optic cable is indicated by thin bright pink line, while furcation tubing is represented by yellow covering running along the length of exposed fiber optic cable. Drawing is not to scale, and should be viewed digitally for optimal clarity.
  •   FigureFigure 2.16.7 Steps to create an optical fiber guide cannula system. (A) The cannula connector assembly is threaded over the furcation tubing on the optical fiber. Bare optical fiber will protrude out of the captive collar as seen on the right side of the image. (B) Pieces of the guide cannula assembly are shown. The cannula pedestal (top left) will be implanted into the skull of the animal following the virus injection during stereotaxic surgery. (C) The infusion cannula is held in a hemostat while a drop of epoxy is injected into it. (D) The epoxy‐filled infusion cannula is slid over the optical fiber until it is flush against the captive collar on the connector assembly. (E) To determine exactly where the optical fiber should be cut, it is necessary to screw the cannula pedestal into the captive collar. The optical fiber should be cut so that it does not protrude out of the cannula pedestal more than 1 mm.
  •   FigureFigure 2.16.8 Accessories used for in vivo applications. (A) A fiber splitter from Precision Fiber Products with bare fiber on all ends. It splits light evenly and is customizable. (B) A fiber optic rotary joint (commutator) from Doric Lenses with FC/PC connections on either end. It has minimal torque and can be easily rotated by mice. (C) A fiber optic patch cable with rotary joint and fiber splitter can be used for bilateral optical stimulation in vivo.

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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