The overall goal of this imaging procedure is to combine F-MRI with optogenetic stimulation for cell-type-specific mapping of functional neural circuits and their dynamics across the whole living brain. This method can help answer key questions in the field of neuroscience such as determining the role of specific brain circuit elements in driving global brain activity. The main advantage of this technique is that is allows for cell-type-specific stimulation and relatively high spatial resolution readout of global brain activity.
Demonstrating this procedure will be Jia Liu and Zhongnan Fang, graduate students in my laboratory. After anesthetizing the animal as described in the text protocol, shave the head with an electric razor and perform a triple surgical scrub on the skin using Betadine and a 70%ethanol rinse. Next, immobilize the animal's skull in a stereotaxic apparatus.
Use a scalpel to make a 15 to 20 millimeter mid-line scalp incision. Retract the scalp using surgical hemostats attached to the periosteum. Identify Lambda and Bregma locations on the skull, then position the drill bit over the region of interest, or ROI.
Drill a small craniotomy over the ROI with a dental drill, taking care not to puncture the brain. Slowly insert a needle attached to the microliter syringe through the craniotomy into the ROI in the brain. Then use a microsyringe pump controller to inject two microliters of the vector solution into the ROI.
After the injection is complete, wait 10 minutes, then slowly remove the syringe at a rate of 0.5 millimeters per minute. After the injection, dry the surface of the skull. Confirm coordinates for the ROI and insert the ferrule implant to the target depth at a rate of 0.5 millimeters per minute.
Finally, mount the ferrule implant to the skull using dental cement. After the dental cement has solidified, seal the incision with sutures around the dental cement cap. Begin by connecting the fiberoptic patch cable to a laser light source, and measure the output at the tip of the patch cable's barrel with a power meter.
Adjust the appropriate power level to produce the desired output at the tip of the fiberoptic cable, which is implanted inside the brain. Prevent light leakage from the implant by covering the eyes of the animal. Then, place the coil over the head of the animal.
Use a ferrule sleeve to cup-hold the fiberoptic cable to the ferrule implant. Insert the cradle with the animal into the bore of the scanner. Monitor breathing rate and tidal CO2 and body temperature throughout the experiment by adjusting the artificial ventilator to keep physiological values within proper limits.
Connect BNC cables from the triggering port of the MRI scanner to the function generator. Select a positioning sequence and click scan'in the operation window. Click continue'to image the animal's head location.
If the brain is not at the isocenter, adjust the animal head location and repeat the positioning scan until the brain is at the isocenter. Acquire a high resolution anatomical image to check on overall integrity of the brain, and to confirm the location of the optical fiber implant. Then, select a T2 weighted sequence.
Adjust the number of slices, and then click scan'to acquire the T2 weighted high-resolution coronal anatomical images. Finally, select a multi-slice gradient recalled echo-sequence, then click continue'to acquire the functional image. This protocol uses optogenetic F-MRI to stimulate the motor cortex in an animal model.
This activation map shows activated voxels in the motor cortex and thalamus, indicating long-range synaptic connections between these regions. Here, the bold signal is shown for active voxels in the motor cortex and thalamus during optogenetic stimulation of the motor cortex. The thalamic hemodynamic response function shows a delayed response relative to the response in the motor cortex after stimulation.
After watching this video, you should have a good understanding of how to perform implantation surgeries and optogenetic functional magnetic resonance imaging. Once mastered, this imaging procedure can be completed in two hours. While attempting this procedure, it is very important to monitor the animal and maintain its physiology within the normal limits.
Following this procedure, complimentary methods can be used, such as electrophysiology to investigate the temporal dynamics of neuroactivity, or immunohistochemistry to validate the expression and specificity of the opsin. The development of this technique paved the way for neuroscientists to investigate functional connectivity in an intact, living brain. Don't forget that working with an MRI scanner can be extremely hazardous.
Precautions such as keeping magnetic equipment sufficiently far away from the scanner should always be taken while following this procedure.
