The overall goal of this procedure is to successfully perform in vivo optogenetic stimulation of the rodent central nervous system in an awake, freely moving animal. This is accomplished by first setting up and configuring the appropriate laser system for performing optogenetic stimulation. Next, fiber optics compatible with behavioral testing are set up for delivering laser light to an implanted fiber optic and into the opsin expressing brain region of interest.
Then the animal is tethered to the laser system for optogenetic stimulation and is placed into a behavioral testing apparatus. Finally, experimenter defined parameters of light stimulation to turn neurons on or off are delivered while the animal performs a behavioral task. Ultimately, in vivo, optogenetic stimulation is used to permit real-time control of defined populations of neurons or neural circuits in awake, behaving animals to determine their functional role in a given behavior of interest.
The main advantage of this technique over existing methods is that it permits the real-time control of genetically and spatially defined populations of neurons in awake, behaving animals with the temporal resolution required to ally link neural activity patterns to complex behavioral states. This protocol involves a use of class three B lasers and requires that proper training and safety guidelines be followed. Safety goggles must be worn at all times when operating lasers with alignment procedures presenting an especially high risk following the setup of the laser apparatus according to the text protocol, carry out non-contact style laser coupling of the inner blue laser by first setting the switches on the back of the laser to cur for current and TTL for transistor transistor logic mode for constant illumination on the front of the driver, ensure that the power knob is set to zero.
Then turn on the laser by first turning on the driver, followed by the laser key. To ensure eye safety, slowly adjust the power knob so that approximately one milliwatt of laser light is being emitted. Then allow 10 to 15 minutes for the laser to warm up.
Next, connect the fiber optic cable tester directly to the free end of the coupler patch cord and turn on the cable tester. Then 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.
To perform a course alignment, use the lateral and horizontal knobs on the kinematic mirrors to steer the beam of laser light into the coupler. Do not be concerned if no blue light is being emitted out of the coupler attached patch cord at this time. Now 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 the same side of this paper from the laser and the cable tester respectively. Make fine adjustments to the first steering mirror by carefully adjusting the lateral and horizontal knobs to align the center of the red dot with the blue dot. Move the paper back towards the coupler so that it is directly in front of it, and adjust the knobs on the second dichroic mirror to align the laser beam with the red beam.
Continue to make fine adjustments to the two mirrors until the center of the blue and red beams are exactly aligned. Coline, remove the cable tester from the coupler cord. Laser light should now be admitted from the end of the coupler patch cord.
To determine coupling efficiency, use a light power meter to compare the light power entering the coupler to the light power emitted from the fiber end. A coupling efficiency of greater than 80%is considered very good while maintaining the position of the dichroic mirror. Use the two steering mirrors for the outer yellow laser and follow the alignment procedure just demonstrated for coupling the yellow laser to set up an optic fiber for stimulating a single mouse.
Begin by using the F-C-F-C-L bracket adapter directly attach to the breadboard to connect the coupler patch cord to a thick jacketed patch cord. Attach a commutator rotary joint to the free ends of the thick jacketed patch cord. Then attach the animal patch cord to the commutator.
Attach a connecting split sleeve to the free metal feral end of the animal patch cord without forcing the sleeve all the way up. The feral leave about 0.5 centimeters of sleeve exposed as this is what connects to the fiber optic affixed to the animal with A BNC cable. Connect the blue laser driver to the pulse generator and turn on the pulse generator.
Now put on appropriate safety glasses. Then after making sure the power knob on the front of the driver is set to zero, turn on the laser by first turning on the driver, and then the laser key with the laser set to TT L positive mode. Slowly adjust the knob on the front of the laser and use a light power meter to set a five to 10 milliwatt light power emission from the tip of the animal patch cord.
Refer to the text protocol for additional details regarding this setting. Switch the blue laser to analog mode for in vivo stimulation and wait 10 to 15 minutes for the laser to warm up. Next, gently restrain the mouse and connect the split sleeve on the animal patch cord to the chronic implantable fiber.
Make sure the ends of both fibers Make physical contact with one another by using the split on the connecting sleeve as a window to visualize direct contact between the two. Allow the mouse to recover for a few minutes prior to the start of behavioral testing. Then place the animal in the behavioral testing apparatus, making sure that the connector cord is free of snags.
Use the pulse generator to pulse a blue laser at a predetermined frequency that will activate the option of choice. After completing the experiment histologically confirm viral and fiber placement as outlined in the accompanying text protocol for accurate interpretation of behavioral results. Under a microscope, check the site of opsin expression and fiber implant and visually confirm the appropriate placement of the virus injection and implant based on chosen coordinates.
In this example of behavioral results obtained through in vivo optogenetic stimulation, dopamine neurons in the ventral area or VTA of tyrosine hydroxylase cream mice were transduced either with a stable step function OPSIN or EYFP as a controlled virus and a fiber implant was chronically implanted. Groups of mice were simultaneously stimulated and locomotive behavior was recorded for one hour as demonstrated here. Repeated stimulation of the ops and transduced mouse on the left resulted in a hyperactive phenotype that persisted throughout the stimulation.
While no change in locomotive behavior was seen in the control mouse on the right following behavioral testing, immunohistochemistry was performed to verify accurate viral targeting to VTA dopamine neurons and fiber placement was visually confirmed as shown in this figure Once mastered. This technique can be used to manipulate neural activity in any given brain region of interest in combination with a variety of behavioral tests as a functional readout of neural circuit modulation.
