Use of channelrhodopsin for activation of CNS neurons

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.

Figure 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 vice 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.

Figure 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.

Figure 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.

Figure 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.

Figure 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 disc 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.

Figure 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. Heatshink 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.

Figure 7

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.