In vivo Optogenetic Stimulation of the Rodent Central Nervous System

! CAUTION: This protocol involves the use of class 3b lasers and will require proper training and safety guidelines to be followed. Safety goggles must be worn at all times when operating lasers, with alignment procedures presenting an especially high risk. Contact the laser provider to determine the eyewear that will provide maximal attenuation for a given laser. If available, enroll in an institutional laser safety-training course. Never operate a laser without the appropriate safety eyewear and training.

1. Laser Apparatus Set-up

Where appropriate, steps in Section 1 are designated as (A) or (B) to differentiate between single or dual laser systems, respectively.

1. Attach and secure lasers to the breadboard. Breadboards are excellent heat conductors and act as a heat sink to prevent damage to internal laser components with prolonged use.
2. (A) Secure the pre-coupled laser to a 10” x 12” breadboard (or as required) using ¼-20” cap screws and washers (Figure 2A). If breadboard holes do not align with laser mounting holes, use small ‘table clamps’ to secure laser to breadboard.
3. (B) If the 2 lasers to be used have very different beam heights (>~1 cm), use small 4” x 6” breadboards to create a platform for one of the lasers. Attach these boards to the main large 12” x 18” breadboard using ¼-20” cap screws with washers, then attach the laser to the smaller boards using cap screws or table clamps as shown in Figure 2B. Attach the other laser directly to the breadboard using cap screws or a variable height table clamp.
   Critical Step: Breadboards, screws, and optical components can be purchased as imperial or metric so be consistent when purchasing items; the default for this protocol is imperial.
4. (A) Attach a thick-jacketed flat-cleave/physical contact (FC/PC) patch cord to the coupler (referred to as a coupler cord; see Figure 3) that is physically attached to the front of the laser (Figure 2A).
5. (B) Thread the coupler onto a ¾” optical post, then epoxy the joint between the coupler and the top of the post using J-B Kwik, or similar epoxy, to prevent loosening and misalignment during use. Attach the post to the breadboard (as the breadboard holes will not always line up with the required placement of optical components, a post holder, pedestal base adapter, and clamping fork are used to secure the coupler’s optical post in place). Attach a thick-jacketed patch cord (coupler cord) to the back of the coupler.
6. (B) Insert the first steering mirror for the blue laser into the kinematic holder, and attach it to the breadboard using a ¾” optical post. Attach this post to a base adapter and clamping fork. Position the clamping fork and post assembly directly in front of the blue laser with mirror angled at 45° to steer the laser towards the dichroic mirror. Use the grid pattern of holes on the breadboard as a rough alignment guide. Once roughly positioned, use a ¼”-20 cap screw to secure the clamping fork to the breadboard (see Figures 2B,C).
7. (B) Insert the dichroic mirror into a kinematic holder and attach it to a 1” optical post and secure directly to the breadboard. Position the dichroic mirror to the far left of, and in line with, the blue laser mirror. Angle the dichroic mirror at a 45° angle such that blue light reflected off the first mirror is reflected into the coupler, then tighten the screw attaching the kinematic holder to the post (see Figures 2B,C).
8. (B) Attach the first steering mirror for the yellow laser to a ¾” optical post. Attach the optical post to a base adapter and clamping fork. Position the clamping fork and post-ensemble directly in front of the yellow laser and angle the mirror at a 45° angle such that yellow light will be directed towards the second steering mirror. Secure the clamping fork in place with a ¼”-20 cap screw and washer.
9. (B) Attach the second steering mirror for the yellow laser to a 1” optical post. Angle the mirror such that the yellow light beam from the first mirror will be reflected through the dichroic and into the coupler (Figure 2C). Secure the post directly to the breadboard and tighten the mounting screw once the mirror is angled appropriately. Fine mirror adjustments will be made using the kinematic mirror mounts in a later step.
10. (B) Attach a neutral density filter wheel to a ¾” optical post and place the post into a post holder attached to a mounting base. Secure the ensemble to the breadboard between the first and second yellow mirrors using a single ¼”-20 cap screw. This wheel is used to adjust the power of yellow laser light reaching the coupler.
    Tip: Individually tighten all components (such as the screws holding kinematic mirror holders to the top of posts, and the threads holding base adapters to the bottom of posts) before attaching them to the base. Use a shaft of a small hex wrench in the provided through-holes in optical posts to get enough torque. This will prevent components coming loose during use, necessitating realignment.
11. Attach an FC/PC to FC/PC L-bracket adapter to the breadboard.
12. Optional: Secure a 1 x 2 50/50 mini cube fiber splitter directly to the breadboard for simultaneous in vivo stimulation of two or more animals. Additionally, handles can be added to assist in movement of breadboard assembly (as seen in Figure 2A).

2. Laser Coupling (Non-contact Style Coupling)

This section pertains to the dual-laser set-up (Figure 2B). Align the inner blue laser path before aligning the outer yellow laser path.

! CAUTION: Use a low light power coupling (~1 mW) to ensure eye safety. Wear safety goggles to power on the laser and until light intensity is measured and deemed to be safe.

1. Set the switches on the back of the laser to “Curr” (current) and transistor-transistor logic mode (TTL)+ for constant illumination (as opposed to Analog mode). Make sure that the power knob on the front of the driver is set at zero. Turn on the laser by turning on the driver first and then the laser key.
2. Slowly adjust the power knob located on the front of the laser driver so that ~1 mW laser light is being emitted. Wait 10 – 15 min (or as specified by manufacturer) for laser to warm up.
3. Connect the fiber optic cable tester directly to the free end of the coupler patch cord and turn on the cable tester (Figure 3A). Adjust the angle of the coupler so that the red beam travels straight back towards the center of the dichroic mirror. The beam path of the red light emitted from the cable tester is the exact path that the incoming laser light will need to follow in order to be coupled into the laser.
4. Perform a coarse alignment: Use the lateral and horizontal knobs on the kinematic mirrors to steer the beam of laser light into the coupler. The pedestal clamps may need to be loosened to slightly reposition the mirrors and the coupler. The kinematic mounts should still have some travel available for further fine adjustments. Do not be concerned if no blue light is being emitted out of the coupler-attached patch cord at this time.
5. Place a single piece of semi-translucent paper directly in front of the dichroic mirror, in between the dichroic and coupler. There will be both a blue and a red dot on this paper from the laser and the cable tester, respectively. Use paper that is translucent enough to see both the red and blue spots simultaneously from the same side of the paper.
6. Make fine adjustments to the first steering mirror (i.e., the one closer to the laser, not the dichroic) by carefully adjusting the lateral and horizontal knobs to align the center of the red dot with the blue dot.
7. Move the paper back towards the coupler so that it is directly in front of the coupler and adjust the knobs on the second (i.e., dichroic) mirror to align the laser beam with the red beam.
8. Iterate over steps 2.6 & 2.7 until the center of the blue/yellow and red beams are exactly aligned in both positions (i.e., until the red and blue beams are colinear).
9. Remove the cable tester from the coupler cord. Laser light should now be emitted from the end of the coupler patch cord.
10. Determine coupling efficiency by measuring the light power emitted from the fiber tip of the coupler patch cord using a power meter. Use the 500 mW setting on the power meter’s photodiode and change the wavelength setting (λ) to blue (473 nm) or yellow (635 nm) spectrum light depending on the laser being used.
11. Place the fiber tip perpendicular to the photodiode to obtain a power reading. Compare the light power entering the coupler to the light power emitted from the fiber end. A coupling efficiency of >80% is considered very good. Very small further adjustments of the second steering mirror can sometimes slightly improve coupling. In general, when the beam pattern from the end of the coupling fiber is a small, tight, central spot (with no rings surrounding it), coupling efficiency into the fiber core is optimal.
12. Repeat steps 2.1 – 2.11 for yellow laser coupling, except use the two steering mirrors for the yellow laser (see Figure 2C). Do not adjust the position of the dichroic mirror or alignment of the blue laser will be lost.

3. In vivo Optogenetic Stimulation

Ensure that any procedures involving animal use are conducted in accordance with local and national guidelines and approved by the corresponding Institutional Animal Care and Use Committee.

1. Optic fiber set-up (see Figure 3B for identification of the different types of patch cords referred to below). To stimulate a single mouse,connect the coupler patch cord to a thick-jacketed patch cord using the FC/FC L-bracket adapter directly attached to the breadboard (see Figure 4A). To stimulate two animals from one laser, connect the coupler patch cord to two thick-jacketed patch cords using the 1 x 2 50/50 mini cube (see Figure 4B). To stimulate three or more animals, attach the coupler cord to a multimode fiber splitter using the 1 x 2 mini cube already attached to the breadboard (see Figure 4C).
2. Attach a commutator/rotary joint to the free ends of the thick-jacketed patch cord/fiber splitter. Commutators are essential as they allow rotation of the fiber with the rodent’s movement, which prevents accumulation of torque on the patch cord. Too much torque can twist cords, lead to breakage, and interfere with the animal’s natural movement during testing.
3. Attach the animal patch cord to the commutator.
4. Attach a connecting split sleeve to the free metal ferrule end of the animal patch cord (Figure 5). Do not force the sleeve all the way up the ferrule; leave ~0.5 cm of sleeve exposed as this is what connects to the implanted fiber optic affixed to the animal (Figure 6).
   Critical Step: Always purchase sleeves that contain a split to allow expansion of sleeve over implanted fiber ferrule during connection and removal. Too tight of a fit can cause severe trauma to the animal if the implant dislodges from the skull when attempting to disconnect the sleeve from the implant. If this does occur, the animal should be removed from the study and receive immediate veterinarian care. Similarly, before using a new sleeve for the first time, ‘break it in’ by plugging and unplugging a ferrule until it disconnects with the desired amount of force.
   Tip: It is easy to break a fiber while removing a sleeve that is tightly attached to a ferrule. To avoid this, push the ferrule out by inserting a small wooden rod into the open end of the sleeve (the handle of a standard cotton swab is the right size).
5. Connect the blue laser driver to a pulse generator using a BNC cable and turn the pulse generator on.
6. Put appropriate safety goggles on. Set the switches on the back of the laser to “Curr” and “TTL+” mode. Make sure that the power knob on the front of the driver is set at zero andturn the laser on (turn the driver on first and then the laser key).
7. Adjust the power knob on the front of the laser so that 5 – 10 mW is being emitted from the animal patch cord fiber tip as measured using a light power meter. 5 – 10 mW is a general guideline – the exact power intensity required to affect a given volume of tissue should be calculated prior to the start of the experiment, as in Aravanis et al.³
8. Switch the blue laser to “Analog” mode for in vivo stimulation. Note: Yellow DPSS lasers are operated in TTL+ mode for constant illumination. Wait 10-15 min for the laser to warm up.
9. Gently restrain the mouse and connect the split-sleeve on the animal patch cord to the chronic implantable fiber (see Figure 6). Make sure the ends of both fibers make physical contact with one another. Use the split on the connecting sleeve as a window to visualize direct contact between the two. Critical Step: Sometimes debris can collect on the metal ferrule of the animal’s implantable fiber and interfere with proper connection.  In this case, use an ethanol wipe to gently clean the ferrule on the animal’s head prior to attachment. Never force a connecting sleeve over the ferrule as this can cause severe trauma to the animal. If a physical connection between fiber ends can not be made after cleaning, remove the animal from the study.
   Tip: Light leakage can occur at the connection point between the implanted fiber and animal patch cord. Visualization of this light by rodents may present an experimental confound¹⁰. Heat shrink tubing can be attached to patch cords and slid over the connecting point to minimize extraneous light.
10. Allow the mouse to recover for a few minutes prior to the start of behavioral testing.
    Tip: Depending on the behavioral test to be administered, it is best to habituate mice to the connection and tethering process 2 – 3 days prior, as the handling required to connect the animal may induce stress and confound behavioral testing.
11. Place the mouse in the behavioral testing apparatus ensuring that the connector cord is free of snags. Never leave an animal unattended during stimulation. Even with the use of commutators, patch cords do have a tendency to twist during extended periods of time and may interfere with behavioral testing.
12. Use a pulse generator to pulse the blue laser at a predetermined frequency that will activate opsin of choice. For yellow laser use: pulse the yellow laser with external shutters or by simply blocking the beam path with an opaque, non-reflective, non-flammable object.

4. Post In vivo Stimulation Considerations

This section is not intended to be a complete protocol but is offered as guidance for additional procedures that should be considered following in vivo optogenetic stimulation.

1. Upon completion of an experiment, confirm viral and fiber placement histologically for accurate interpretation of behavioral results. Euthanize animal according to institutional guidelines and perfuse the animal with ice-cold phosphate-buffered saline (PBS) and 4% (w/v) paraformaldehyde in PBS.
2. Remove implanted fiber optic by firmly grasping the exposed metal ferrule with pliers or hemostats. Pull up in one smooth, yet swift, motion. It is important to test the integrity of the implanted fiber by measuring the light output at the end of each experiment.
3. Post-fix the brains in paraformaldehyde for at least 24 – 48 hr before sectioning through the region of interest. (If using a freezing microtome, incubate brains in a 30% sucrose solution for several days before sectioning). Perform immunohistochemistry using standard protocols for detection of the appropriate opsin-tagged fluorophores, i.e., green florescent protein (GFP), enhanced yellow florescent protein (eYFP) or mCherry.
4. Check site of opsin expression and fiber implant under a microscope and visually confirm appropriate placement of virus injection and implant based on chosen coordinates.