The current protocol contributes to the study of the brain mechanisms underlying skilled motor behavior. Wireless optogenetics permits the manipulation of specific neurons while subjects perform a task in free movement. In combination with high-speed videography, it is possible to have a detailed analysis of fine motor behavior.
The method could help to understand motor problems in neurodevelopmental and neurodegenerative disorders. These techniques can be used to study brain function in other behavioral paradigms as well. Diana Rodriguez-Munoz, an undergrad student from my lab, will be assisting with experiment.
For the surgical procedures, start by preparing an LED cannula of the desired length according to the dorso-ventral coordinates of the structure-of-interest. To do so, cut the glass fiber to a length longer than the final desired size and grind the fiber tip to the target length with rough sandpaper. Then, polish the fiber tip with fine sandpaper.
For microinjections, fill a pipette with the mineral oil and place the pipette in the microinjector. Ensure the microinjector works correctly by injecting some mineral oil. Next, prepare the anesthetized mouse for the surgery by applying an ophthalmic ointment and removing hair from the scalp with a trimmer and hair removal cream.
Then, wipe the scalp with cotton swabs having 8%povidone iodine and 70%ethanol alternated three times each. After sanitization, place the mouse in the stereotaxic apparatus and secure the head, ensuring that the skull is leveled in the medial-lateral and anterior-posterior axes. Use a scalpel to make a one-centimeter incision through the scalp at the level of the eyes along the sagittal axis.
Then, retract the skin to expose the skull and clean the periosteum with cotton swabs. Clean the cranium surface with saline solution and cotton swabs and resolve any bleeding at the surface using sterile absorbent eye spears. Then, apply a drop of 2.5%hydrogen peroxide with a cotton swab and let it act for a few seconds to make the skull sutures visible and have a better reference.
After a few seconds, clean the area thoroughly with a clean cotton swab. With a glass pipette of 15-micron final tip diameter, locate bregma and lambda to check that the skull is leveled in the anterior-posterior axis. Then, paint a reference point in the scalp above the selected coordinates with a marker.
In the reference point, perform a one-millimeter diameter craniotomy applying gentle pressure to the skull with a rotary tool at a low to medium speed with a small, round dental drill bit. Once done, move the capillary toward the selected anterior-posterior, or AP, and medial-lateral, or ML, coordinates. Load the capillary with 300 to 400 nanoliters of the CreE-dependent adeno-associated virus, or AAV, such as AAV1, D-flox channelrhodopsin mCherry to express channelrhodopsin in the region-of-interest.
An AAV can be used as a control to express the reporter protein. After ensuring that the tip is not clogged, introduce the glass pipette in the brain at the dorso-ventral minus 3.35 millimeter coordinates in the dorsal-lateral striatum and inject 200 nanoliters of the virus using an automatic injector at a rate of 23 nanoliters per second. Wait for 10 minutes post-injection before withdrawing the glass pipette slowly to avoid spillage.
Then, clean and dry any residues with cotton swabs. Next, attach the glass LED cannula to the stereotaxic arm and calibrate the coordinates using bregma as a reference. Then, insert the cannula slowly to avoid tissue damage and place it 100 micrometers above the injection site.
Once the LED cannula is in place, add a drop of 100 microliters of tissue adhesive at the edge of the craniotomy. Use a sterile brush to apply a freshly prepared dental cement mixture around the cannula connector little by little, building layers until the skull is covered and the connector is securely attached to the skull, leaving the pins completely free. When the cement dries completely, close the skin around the implant using tissue adhesive.
Then, place the mouse in a recovery cage over a heating pad at 33 degrees Celsius while monitoring for the signs of discomfort or pain. Record the behavior of the animal with a regular camera and capture 30 to 60 frames per second from the front of the chamber. A mirror can be placed under the training chamber at a 45-degree angle to monitor the animal's posture.
When mice start reaching the pellet, turn the LED cannula manually with the remote controller to have a continuous stimulation for the time the behavior is performed for no longer than two seconds. The stimulation device triggers an LED of 470 nanometers with intensity at the tip of one milliwatt per millimeter square. The fine motor behavior of the animal under optogenetic manipulations was studied with the reach-to-grasp task.
The mice learned to execute the task in a couple of days and achieved more than 55%accuracy, reaching a plateau after five days of training. The trajectory of movements was tracked with high-speed videography, making it possible to analyze kinematics. The quantifiable assessment of parameters such as distance traveled, velocity, acceleration, endpoint, and trajectory was performed.
In the missed trials, the mice started the grasping movement further away from the pellet than the hit trials. Additionally, errors were significantly associated with the mouse posture, shown by differences in the body angle during hit and missed trials. The contralateral activation of D1 dopamine expressing spiny projection neurons, or SPNs, reduced the grasping success compared to the control conditions.
During an optogenetic stimulation, the paw trajectory increased in the travel distance, leading to the incapability to target the pellet and an increase in initial error type one. Principle component analysis, or PCA, showed that all trials trajectories during contralateral D1 SPNs activation separated in a cluster with almost no overlap with the control cluster, indicating a low similarity. Activation of the DSPNs in the ipsilateral side led to an increase in trajectory dispersion shown by PCA analysis.
Hence, the ipsilateral D1 SPNs activation modified the reaching trajectory without changing the behavioral outcome. When performing this protocol, be careful to place the cannula correctly so it doesn't detach during training sessions. This method can be used in other behavioral paradigms to study motor behavior, sensory processing, learning, and memory.
Wireless optogenetics will allow to study naturalistic behavior and its neurobiological underpinnings in truly free-moving subjects.
